Most longitudinal studies showed increased relative mortality in individuals with type 2 diabetes mellitus until now. As a result of major changes in treatment regimes over the past years, with more stringent goals for metabolic control and cardiovascular risk management, improvement of life expectancy should be expected. In our study, we aimed to assess present-day life expectancy of type 2 diabetes patients in an ongoing cohort study.1c 7.0%. During a median follow-up of 5.4 years, 165 patients died (78 from cardiovascular causes), and 17 patients were lost to follow-up. There were no differences in life expectancy in subjects with type 2 diabetes compared to life expectancy in the general population. In multivariate Cox regression analyses, concentrating on the endpoints ‘all-cause’ and cardiovascular mortality, a history of cardiovascular disease: hazard ratio (HR) 1.71  1.23–2.37), and HR 2.59 (95% CI 1.56–4.28); and albuminuria: HR 1.72 (95% CI 1.26–2.35), and HR 1.83 (95% CI 1.17–2.89), respectively, were significant predictors, whereas smoking, HbA1c, systolic blood pressure and diabetes duration were not.We included 973 primary care type 2 diabetes patients in a prospective cohort study, who were all participating in a shared care project in The Netherlands. Vital status was assessed from May 2001 till May 2007. Main outcome measurement was life expectancy assessed by transforming actual survival time to standardised survival time allowing adjustment for the baseline mortality rate of the general population. At baseline, mean age was 66 years, mean HbAThis study shows a normal life expectancy in a cohort of subjects with type 2 diabetes patients in primary care when compared to the general population. A history of cardiovascular disease and albuminuria, however, increased the risk of a reduction of life expectancy. These results show that, in a shared care environment, a normal life expectancy is achievable in type 2 diabetes patients. The incidence and prevalence of diabetes mellitus has risen worldwide during the past few decades. Recently published data from the Framingham Heart Study showed an absolute increase in the incidence of diabetes of ∼2.5% yearly during the 1990s compared to the 1970s Most published data were extracted from representative national cohorts in North-America or the United Kingdom. Some reports showed a decline in mortality rates amongst diabetic men only, whereas women showed an increase or no change in mortality rates at all Our aim was to investigate present-day life expectancy in a type 2 diabetes population treated in primary care, with additional support both for patients and health care professionals in a shared care setting in a Western European country.In 2001, 973 type 2 diabetes patients participated in a cross-sectional study with measurements of skin advanced glycation endproduct (AGE) accumulation as described previously Patients with a cognitive disability or terminal disease were not included in the ZODIAC study because of their inability to undergo educational programs. Furthermore, patients who were physically unable to visit the diabetes specialist nurse at the outpatient clinic were not enclosed in the present cohort.This study was approved by the local ethical committee of the Isala Clinics, Zwolle, The Netherlands and all patients gave written informed consent.Methods of clinical data collection and laboratory assessments have been described in detail elsewhere Mortality was registered from the date of inclusion until May 2007. Death was certified according to the following procedure. In addition to the list of deceased patients reported in the files of the scheduled annual follow-up visits, survival status of the patients was obtained from the local hospital information system and verified with the GPs. Date of death was collected likewise. None of the GPs had involvement or interest in study outcome. Causes of death were coded according to ICD–9 and categorised as: neoplasms (140–239), diseases of the cardiovascular system (390–459), diseases of the respiratory system (460–519), diseases of the digestive system (520–579), diseases of the genitourinary system (580–629), injury and poisoning (800–999). Sudden death, with symptoms present for less than one hour, was encoded in the category of coronary heart disease. For the in-hospital deaths, the medical records were retrieved. For the out-of-hospital deceased patients, the assigned causes of death by the GPs were obtained from the medical records of the GPs. The coded causes of death were combined to all-cause mortality  and cardiovascular mortality (390–459 or sudden death).irrespective of age and sex. Eliminating the effects of age and sex excludes the influence of disease-specific risk of age and sex in the standardised analysis. P values <0.05 were considered to be statistically significant. Clinical and laboratory variables with an expected effect on mortality risk were first analysed in a univariate analysis, and secondly, in a multivariate model. Detailed analyses were performed specifically for two end-points: all-cause mortality and cardiovascular mortality.Life expectancy analysis was performed primarily by the use of ‘standardised survival time’ (SST), which is a novel approach to survival analysis 1c 7.0%). 1c of 7.5%.Characteristics of the 973 type 2 diabetes patients at baseline (2001) are shown in The proportion of prescribed lipid-lowering drugs, renin-angiotensin system (RAS) inhibitors and antiplatelet therapy at baseline, is shown at the end of 1c did not reach significance in both multivariate models.This study shows a normal median overall life expectancy in a defined cohort of type 2 diabetes patients treated in a primary care setting, during a follow-up period from 2001 till 2007. This finding strongly suggests that current available treatment strategies may eventually lead to a life expectancy equal to the general population in this subset of type 2 diabetes patients. Secondly, in this type 2 diabetes study population, patients with a history of cardiovascular disease and/or the presence of albuminuria still had an increased risk to die before their median life expectancy was reached. The differences in effects of all items of cardiovascular disease on ‘survival in years’ and SST, could be explained by the age correction enclosed in the SST – method. As the prevalence of cardiovascular diseases is increasing with increasing age, SST does have definite advantages compared to the ‘classical survival time’ in identifying premature mortality.Finally, we still found an increased proportion of deaths due to cardiovascular disease compared to the general population (47% versus 31%). This is in agreement with established observations of increased cardiovascular disease in diabetes, and also with the fact that the presence of classical cardiovascular risk factors still is most intimately related to life expectancy reduction The United Kingdom Prospective Diabetes Study reported a 5 years reduction of life expectancy for males aged 45 to 50 years at diagnosis of diabetes when compared to the general United Kingdom population A large study of the non institutionalised United States population, which was conducted between 1971 and 1993, showed a median reduced life expectancy of 8 years for the diabetic population aged 55–64 years, and a 4 years reduction for the diabetic population aged 65–74 years A previous study in the first ZODIAC-cohort (1998) reported an annual mortality rate of 4.8% between 1998 and 2000 (the first three years of the shared care project), definitely higher than the mortality rate in the present analysis (∼3%), which was performed over the subsequent years within this shared care environment More than half of our population received either a statin, RAS-inhibitor or aspirin at baseline. At follow-up, this proportion had increased to at least 80%. Widespread treatment of the traditional cardiovascular risk factors resulted in vastly improved blood pressure readings and lipid levels. This could also be the explanation for disappearance of systolic blood pressure from the model to predict mortality. Recent studies, although maybe underpowered, addressed the importance of statins and blood pressure lowering drugs in patients with type 2 diabetes mellitus, showing a reduction in cardiovascular events with these lipid-lowering drugs compared to placebo 1c had also no effect on life expectancy in uni- and multivariate analysis. This may possibly be explained by the low number of patients with poor glycemic control (only 7% had a HbA1c >9%). Alternatively, other mechanisms could be involved in the development of diabetes related complications. E.g., we recently reported increased levels of advanced glycation endproducts (AGEs) rather than HbA1c in the same study group, to be related to chronic complications HbAThere are some limitations to our study regarding the possible general applicability of the results. Diabetic patients who were referred to the secondary care in the past, mainly for reasons of poor metabolic control or comorbidity, were not included in this study and almost certainly will have a reduced life expectancy. Also, there has been a selection bias by excluding diabetic patients with a very short life expectancy , as described in the methods section. Still, the selection comprised a considerable subset of the total population known with type 2 diabetes see , and 40%Despite this apparent selection bias, we still are able to conclude that we defined a large subset of patients with a life expectancy comparable to that of the general population of the same age and sex. In The Netherlands, the large majority (70–80%) of type 2 diabetes patients is treated in primary care or in a shared-care setting. Therefore, this study population could be representative for the majority of type 2 diabetes patients in The Netherlands, and probably also for a larger part of type 2 diabetes patients in other countries with structured diabetes care.Our choice to compare life expectancy of this type 2 diabetes cohort to the general population can be criticised, since the general population also includes people with diabetes, cardiovascular disease, cancer, and other life shortening diseases. We nevertheless preferred to choose the general population instead of a non-diabetic control group, since one of the aims of caregivers in medical practice is to regain a life expectancy for their patients equal to the general population when life expectancy is reduced due to a specific disease.To visualise whether a life expectancy equal to the general population had been achieved, we used SST. Traditional survival analysis focuses more on ‘mortality’ within a certain follow-up time, but with this more conventional method it is not clear whether it is ‘normal mortality’ or ‘excess mortality’. Using SST, the mortality rate is adjusted for the median survival of subjects in the general population of the same age and sex. In this way, we eliminate the effect of age and sex, by excluding the influence of disease-specific risk of age and sex in the standardised analysis. Excess mortality or a reduced life expectancy will be identified more easily in that way. We consider the results of this study to be relevant for clinical practice, because they offer a hopeful perspective of a definitely improved life expectancy in type 2 diabetes patients. We suggest that those results are also (partly) due to the fact that these patients were and are participating in a care system promoting adherence to evidence-based guidelines and to a system emphasizing close cooperation between health care providers focusing on this patient group.In summary, this study shows a normal life expectancy in a large subset of type 2 diabetes patients treated in a primary care setting compared to the general population. The presence of previous cardiovascular disease and albuminuria, however, is still associated with a markedly reduced life expectancy.


Moderate alcohol use is generally associated with lower ischaemic heart disease (IHD) mortality but it is difficult to ascertain whether this is due to attributes of moderate alcohol users or the properties of alcohol itself. Evidence from populations with different patterns of alcohol use and IHD can provide crucial evidence. We assessed the association of moderate alcohol use with IHD mortality in older Chinese people from Hong Kong.We used Cox regression to determine whether moderate alcohol use was associated with IHD mortality in a prospective, population-based cohort study of all 56167 attendees, aged 65 years or over, from July 1998 to December 2000 at all 18 Elderly Health Centers operated by the Department of Health in Hong Kong.After a median follow-up of 4.2 years, there were 406  deaths from IHD in 54,090 subjects (96.3% successful follow-up). Moderate alcohol use in men was not associated with IHD mortality adjusted only for age  or additionally adjusted for socio-economic status and lifestyle. Almost all women were occasional drinkers and their current alcohol use was not significantly associated with IHD mortality .Moderate alcohol use had no effect on IHD mortality in older Chinese men. Lack of replication of the usual protective effect of moderate alcohol use in a setting with a different pattern of alcohol use and IHD could be due to chance or could suggest that the protective effect of alcohol on IHD does not extend to all populations. Many observational studies have shown that moderate alcohol use is associated with lower mortality from ischemic heart disease (IHD) Evidence from settings with different social patterns of alcohol use and ischaemic heart disease can potentially make a contribution. Compared with many western countries, alcohol use is less prevalent and more restrained in southern Chinese, so the characteristics of moderate users may be different. In Hong Kong, only 55% of men and 19% of women drink alcohol with an average daily intake amongst alcohol users of 9 grams ethanol in men and 2 grams in women, and the main alcoholic beverage is beer Details of the methods have been reported elsewhere Alcohol use was initially recorded in four categories i.e. never-drinker, ex-drinker, social drinker and regular drinker. Social drinkers are people do not usually drink alcohol, but who may drink alcohol at social gatherings on special festive occasions, such as at Chinese New Year or a wedding. Regular drinkers were further asked if they drank more than 3 units per day (men) or 2 units per day (women). A unit was defined as 10 grams of ethanol, and the interviewer was provided with a list for converting common alcoholic drinks into units, for example a 335 ml can of beer at 5% is 1.4 units. On this basis regular drinkers were categorized as moderate or excessive. Moderate drinkers were defined as regular drinkers of 3 or fewer units per day in men and 2 or fewer units per day in women. Excessive drinkers were defined as regular drinkers of more than 3 units per day in men and 2 units per day in women. Self-reports of alcohol use are prone to mis-reporting; as a check, we examined the association between alcohol use and esophageal cancer.th Revisions before 2001 and 10th Revision in and after 2001. Most Hong Kong residents die in hospital, enabling accurate ascertainment of cause of death, which we have used previously in similar studies Vital status was ascertained from death registration, special outpatient and hospitalization databases in Hong Kong by record linkage using the unique Hong Kong identity card number. The last date of follow-up or censor date was December 31, 2003. The subjects not found dead or alive by record linkage were followed up by telephone interview from November 2004 to January 2005, after which only 3.5% (1951) remained untraced. Adjusted for age, socio-economic status, lifestyle habits, untraced men were no different in alcohol use from traced men, but untraced women were significantly more likely to be social or excessive drinkers (odds ratio of being lost to follow-up 1.25 (95% CI 1.03 to 1.50) and 3.24 (95% CI 1.38 to 7.60) for social drinking and excessive drinking women respectively compared with never drinking women). Among the 54216 traced subjects, 3884 had died by December 31, 2003. Causes of death obtained for 3825 were routinely coded by the Department of Health according to the International Classification of Disease – 9The primary outcome was cause-specific mortality from the IHD (ICD-9 410-4 or ICD-10 I20-5); a secondary outcome was cause specific mortality from cancer of the esophagus (ICD-9 150 or ICD10 C15).Chi-square tests were used to compare proportions of subject's characteristics across alcohol use groups. The Cox proportional hazards model was used to estimate the hazard ratios (HR) and the 95% confidence intervals (CI) for IHD and esophagus cancer mortality by alcohol use group adjusted in model 1 for age and in model 2 additionally for all other baseline potential confounders  and smoking status), categorized as in There were 18,750 men and 37,417 women in the study. After excluding 1951 clients with no follow-up information and 126 clients with missing, relevant baseline data, there were 95.5%  with follow-up and complete baseline data, including 3819 deaths. There were 188 and 218 deaths from IHD in men and women respectively and 24 and 10 from cancer of the esophagus in men and women. Men were much more likely to use alcohol than women . Never-dIn men there was no association between occasional or moderate alcohol use and death from IHD with hazard ratios of almost exactly unity  in both In a large prospective study of older Chinese, we found no overall association between moderate alcohol use and lower mortality from IHD in either men or women, which is somewhat different from the two previous prospective studies of alcohol and IHD mortality in Chinese men. However, one of these studies from four small communities in Shanghai found moderate alcohol use associated both with a 36% reduction in IHD mortality and with a non-significant reduction in mortality from causes where alcohol usually has no effect or monotonically increases risk, There are several possible explanations for the differences between our study and a large body of evidence showing that moderate alcohol use protects against IHD mortality. First, the period of follow up was relatively short, with correspondingly few deaths, but also less error due to change in alcohol use. Second, the participants were volunteer attendees at a primary care facility, who might be generally healthier with less need of the potential protection offered by alcohol. Third, we distinguished between life-long never-drinkers, ex-drinkers, occasional  and moderate drinkers, so that the reference group of never-drinkers was not contaminated by “sick quitters” or people who had never used alcohol because of ill-health. Fourth, the association of alcohol use with IHD may vary with age, however most previous studies have found benefits in older people Nevertheless, despite these limitations it is possible that moderate alcohol use may have less effect on IHD mortality in some settings, perhaps because of role of co-factor, such as folate intake, In a population-based prospective study of older Chinese people with or without adjustment for multiple confounders, there was no association between moderate alcohol use and lower IHD mortality. This could be a chance association, or it could indicate that moderate alcohol use may not be as protective against IHD some sub-populations, for reasons which need to be clarified.


P=0.93). However, patients with a positive QDA value did have a progression-free survival at 1 year of 3% and overall survival at 2 years of 17%, against 22 and 36% for patients with a negative QDA value . Breast cancer patients with metastatic disease have a significantly worse progression-free and overall survival when circulating tumour cells can be detected in their peripheral blood.We investigated the prognostic significance of circulating breast cancer cells in peripheral blood detected by quantitative RT–PCR of marker genes in patients with advanced breast cancer. Blood samples from 94 breast cancer patients with metastatic disease (M1) were examined for circulating tumour cells by studying the mRNA expression of CK19, p1B, PS2 and EGP2 by real-time PCR. Using a score function, developed for predicting circulating tumour cells by quadratic discriminant analysis (QDA), the four expression levels were combined into a single discriminant value. Tumour cells were present in 24 out of 94 (31%) of the patients. In 77% (72 out of 94) of the patients distant metastatic disease was localised in the bone. In 36% (26 out of 72) of the patients with bone metastases at the time of blood sampling, a positive QDA for the four genes was found, in contrast to only 14% (three out of 22) without bone involvement. Overall survival rates by Kaplan–Meier revealed no prognostic effect for the presence of bone metastases ( Metastases in breast cancer may arise through either lymphatic spread or through hematogenous dissemination. While the local treatment of breast cancer is quite successful and modern treatment can adequately deal with local lymph node metastases, a substantial proportion of breast cancer patients ultimately die from metastases to distant organs or tissues. Micrometastatic disease may be cured by adjuvant systemic therapy and the ability to detect it reliably would thus be a significant advance. We have recently shown that the tendency for hematogenous spread can already be shown in small tumours, using messenger-RNA (mRNA) expression profiling .RNA-based methods have been used to detect the presence of mRNA species in RNA isolations from peripheral blood mononuclear cells that are characteristic of epithelial cells and that may originate from circulating tumour cells. The main problem of RNA-based assays continues to be the almost universally present background signal  count, platelets, LDH, CEA and CA15.3. In addition, the presence of either progressive or responding disease at the time of blood sampling was noted. The dates for first treatment progression and death were also extracted from the charts.For this study, nine of the 103 patients had to be excluded because they were not under further treatment at the NKI and had been lost in follow-up, leaving 94 patients for the analysis.The QDA score function as defined earlier  or date of death from any cause (event). Progression-free survival was calculated from the date of blood sampling to the date of last follow-up (censored) or date of progression/death from any cause (event). Patients, in whom evidence of progression was present at the date of blood sample, were assigned a progression-free survival of zero months. (Progression-free) survival curves were calculated using the method of Kaplan–Meier and compared using the likelihood ratio test based on proportional hazard regression analysis.Proportional-hazard regression analysis was used to adjust the association between QDA and survival or progression-free survival for confounding by any other metastastic site  or form of treatment .The median follow-up from the date of blood sampling for all the 94 patients included is 15 months (range 0.4 – 53) and for the 17 patients still alive 45 months (12 – 53).Ninety-four breast cancer patients with metastatic disease were studied for the prognostic significance of positive breast cancer marker gene expression in their peripheral blood. All the patients had metastatic disease, most of them had metastases at several locations. Twelve patients did not receive any systemic treatment at the time of blood sampling, 36 patients received chemotherapy, 39 hormonal treatment and seven received both . A positive QDA value was also associated with liver metastases. Thirteen of the 27 patients (48%) with liver metastases had a positive QDA value, versus 16 of the 67 patients (24%) without liver metastases . Logistic regression analysis revealed that these associations are independent of each other. The results of the circulating breast cancer cell assay were further correlated with each of the quantitative data extracted from the patient charts. There was no apparent relation with age, the time since primary diagnosis, levels of LDH or tumour markers, WBC counts or platelet count or with the presence of disease progression at the time of sampling.A positive test for circulating tumour cells was  found in patients with bone metastases  that progression-free survival is worse for patients with a positive QDA value from the four marker genes .Thirty-three percent of the patients had progressive disease at the date of the blood sampling (31 out of 94). After 6 months, 33% were still alive without progression, after 12 months 16%, after 2 years 10% and after 4 years 5%. There is evidence (P=0.93).The poor prognosis observed in patients with circulating tumour cells is not simply because of the more frequent presence of bone metastases: as expected, bone metastases were not a particularly negative prognostic feature for breast cancer patients with stage IV disease . Our test for circulating tumour cells was, however, not designed to predict progression-free survival, but rather to establish a biological correlate.Our results show that the test has a clear biological correlate and may truly represent the presence of circulating breast cancer cells. It is tempting to speculate that particularly tumour cells lodging in the bone marrow, that are responsible for bone metastases, may spill over into the blood and circulate through the body. It could also be that our patients with bone metastases and liver metastases had a larger total tumour volume than the other patients. It is not possible to determine this retrospectively. In fact, a multivariate analysis for survival that did take the site of metastasis and form of therapy into account, resulted in an increase of the We could, however, exclude the possibility that the interval between the date of the first metastasis detected and the time, at which the blood sample was drawn, influenced the QDA value  breast cancer. In this situation, prognostic information is essential to appropriately select patients for adjuvant therapy, such as hormone treatment and/or chemotherapy. We have recently shown, using expression profiling that even in early, lymph node-negative tumours, a group of patients can be identified who have a poor prognosis (


We analyzed the transcriptome under normal conditions as well as with forced parthenocarpic development in RP75/59, emasculating the flowers 2 days before anthesis. This analysis helps to understand the fruit set in tomato.Tomato fruit set is a key process that has a great economic impact on crop production. We employed the Affymetrix GeneChip Tomato Genome Array to compare the transcriptome of a non-parthenocarpic line, UC82, with that of the parthenocarpic line RP75/59 (Differentially expressed genes were extracted with maSigPro, which is designed for the analysis of single and multiseries time course microarray experiments. 2842 genes showed changes throughout normal carpel development and fruit set. Most of them showed a change of expression at or after anthesis. The main differences between lines were concentrated at the anthesis stage. We found 758 genes differentially expressed in parthenocarpic fruit set. Among these genes we detected cell cycle-related genes that were still activated at anthesis in the parthenocarpic line, which shows the lack of arrest in the parthenocarpic line at anthesis. Key genes for the synthesis of gibberellins and ethylene, which were up-regulated in the parthenocarpic line were also detected.Comparisons between array experiments determined that anthesis was the most different stage and the key point at which most of the genes were modulated. In the parthenocarpic line, anthesis seemed to be a short transitional stage to fruit set. In this line, the high GAs contends leads to the development of a parthenocarpic fruit, and ethylene may mimic pollination signals, inducing auxin synthesis in the ovary and the development of a jelly fruit. Fruit development and ripening are key processes for crop production, tomato has been widely used as a model for the regulation of these processes . Tomato Fruit development can be divided into several phases . The firFruit set is affected by multiple environmental conditions, such as light, humidity and temperature which must be within a certain range to allow fruits to develop. A better understanding of the developmental and environmental factors that control fruit set would lead to an optimization of growing conditions that might improve crop production.Aux/IAA transcription factor IAA9. Plants with IAA9 inhibited present auxin related growth alterations as well as fruit development triggered before fertilization, giving rise to parthenocarpy [TM29, a tomato SEPALLATA homologue, develop parthenocarpic fruits and produce aberrant flowers with morphogenetic alterations in the organs of the inner three whorls [arf 8 (auxin response factor 8) and tomato plants carrying ARF8 transgenic constructions also develop parthenocarpic fruits [Besides the influence of these external factors in the control of fruit set the existence of a hormonal control is also obvious and has been demonstrated by various studies reviewed by Ozga  and Srive whorls . Arabidoc fruits ,12.Although natural and artificial mutants have demonstrated the existence of a genetic control of fruit set, little is known about how it works. Parthenocarpic fruit development is a trait of great interest as it provides an ideal framework for studying the factors affecting fruit set in addition to improving fruit set in harsh conditions.pat, pat-2 and pat3/pat4 [pat mutant has been widely analyzed, although it presents pleiotropic effects that affect not only fruit set but also flower morphology, with abnormal stamen and ovule development [pat-2, a single recessive gene with no pleiotropic effects, is responsible for the parthenocarpy in the "Severianin" cultivar [pat-3/pat-4 system (RP75/59) was described in a progeny from a cross between Atom × Bubjekosko. Studies of RP75/59 have finally led to the acceptance of a genetic model with two genes, pat-3 and pat-4 [pat gene has been mapped [There are three main sources of parthenocarpic growth in tomato: at3/pat4 -15. Theselopment . The patcultivar . The patnd pat-4 ,19. GAs nd pat-4 ,20,21. Un mapped .pat3/pat4 mutant), to identify the genes modulated throughout carpel development and fruit set and to determine the differences between parthenocarpic and normal fruit set. We have identified changes in cell division genes that imply cell cycle alterations in the parthenocarpic line. In addition, differences in several hormone-related genes are relevant and asses the importance of GAs for parthenocarpic development and a new role for ethylene in this process.As of this work, no global analysis of gene expression during parthenocarpic fruit set has been published for tomato. Most of the studies related to this crop have been focused on later stages of fruit development and ripening -25, and Carpel development in tomato arrests at anthesis and is not resumed until pollination and successful fertilization. However, the facultative parthenocarpic line RP75/59 sets fruits in absence of pollination.To study carpel development, fruit set and parthenocarpic development, we compared the non-parthenocarpic UC82 and RP75/59 transcriptomes. UC82 was selected as the normal development control due to its high percentage of fruit set, which is higher than 90%, and its phenotypic resemblance to RP75/59. In order to analyze the carpel development and fruit set of both lines, flowers were collected at four time points: flower bud, flower bud to pre-anthesis, anthesis and 3DPA (days post anthesis).PCNA (proliferation cell nuclear antigen), a cell division marker, was tested by quantitative PCR (QPCR) to monitor the developmental arrest at anthesis and the restart that takes place when fruit sets . To analyze the different stages of development, we discarded the constant genes in order to avoid background noise and clustered the samples according to gene expression by UPGMA.Figure . ReplicaTo identify processes altered in parthenocarpic carpel development in tomato, we compared the transcriptome of the non-partenocarpic UC-82 line with that of the partenocarpic RP75/59 line. Differentially expressed genes were extracted with maSigPro , which i2842 differentially expressed genes were associated to the TIME variable   of the differentially expressed genes. Even though Affymetrix provides an annotation of the arrays, we found it incomplete as only a thousand probes had GO terms assigned. To improve the functional analysis of the genes, we re-annotated the array using the blast2GO package Addition. At the Using the FatiGO program  we extraTo identify other processes that may be involved in fruit set, we analized the GO terms whose frequency was greater than 2%. In the TIME differentially expressed genes Figure , we founAs RP75/59 can produce both seeded and seedless fruits. To improve the differential analysis, we forced parthenocarpic development in RP75/59 by emasculating the flowers 2 days before the anthesis to prevent natural pollination. Only UC82 flowers, and not RP75/59 flowers were pollinated at anthesis. The transcriptomes of the emasculated and non-emasculated flowers were quite similar Figure . We focuWe detected the genes whose expression changed between emasculated anthesis and emasculated 3DPA. Three new variables were defined for the emasculated stages: eTIME (for those genes that changed between anthesis and 3DPA in UC-82), eTIME RP75/59 (for those genes that changed between anthesis and 3DPA in a different way in RP75/59 from that in UC-82) and eUC-82vsRP75/59 (for those genes whose expression was different between the two lines) Figure . We seleTo explore the expression changes, we grouped these genes into 5 clusters Figure . There wTo identify the biological processes involved in parthenocarpic fruit set, we analyzed the GO terms that label the differentially expressed genes. We found mainly the same terms as in the analysis of the TIME variable and three new terms: DNA replication (which was present in TIME RP75/59 and RP75/59vsUC82), RNA processing and amino acid derivate biosynthetic process Figure .We also extracted the GO terms that were over- or underrepresented in the differentially expressed genes associated with the variables eTIME RP75/59 and eRP75/59vsUC82 with respect to the rest of the array using the Fatigo program Table . We founArray results were validated by QPCR, PCNA and 10 genes out of the differentially expressed along carpel development (TIME) were tested in the 6 stages analyzed . In the QPCR we used actin gene as reference, the fold change between RP75/59 and UC-82 was calculated and the result was log 2 transformed to made the data comparable with the microarray. In spite of the differences between both methods, the correlation was 0.88 , which maybe caused by the cell cycle stop that takes place at anthesis. Additional file In order to evaluate the differences in cell cycle that maybe caused by parthenocarpic development, we also checked differentially modulated genes in parthenocarpic fruit set with respect to normal fruit set (variables eTIME RP75/59 and eRP75/59vsUC82) Table . All of Hormones play a key role in all of the development processes. Here we focused on the hormone related genes to determine which ones were involved in tomato carpel development, fruit set and to find differences between normal fruit set and parthenocarpy. We analyzed the genes regulated during normal carpel development and fruit set (variable TIME)  in two tomato lines (a control and a facultative parthenocarpic line). We identified 2842 modulated genes in the control line (UC82). When we clustered the modulated genes into 15 groups by their expression pattern, we observed that the differences between the two lines were mainly due to expression level, and that it was at anthesis where we found the greatest differences. These differences of expression were also detected when we clustered the experiments. Flower bud and bud to preanthesis were clustered together and then grouped with 3DPA, while all of the anthesis samples were clustered in a different group, thereby demonstrating the special nature of this stage.With our new annotation of the GeneChip Tomato Array we analyzed the frequency of the different GO terms of the modulated genes during UC82 carpel development and fruit set with respect to the rest of the genes present in the microarray. The cell cycle genes were regulated throughout this process, as carpel cells are divide at flower bud and stop at anthesis until pollination and fertilization, which leads to fruit set when the cell division restarts . We alsoOur aim was to study parthenocarpic fruit set and to analyze the differences with respect to normal fruit set that could be the cause or consequence of the ability to set parthenocarpic fruits; we focused our study on the most different stage and forced parthenocarpy to improve the analysis.When we analyzed the gene expression we detected 758 genes differentially expressed in parthenocarpic fruit set with respect to normal fruit set. The expression patterns of these genes showed that the differences between the parthenocarpic line and the control line at anthesis and 3DPA were mainly related to level expression. Moreover, the most significant differences between lines were detected at anthesis, just as in the non-emasculated samples. This suggests that parthenocarpic and normal fruit set follow similar paths and that the main differences are concentrated at the anthesis stage.To detect the processes involved in parthenocarpic fruit set, we analyzed the GO terms of the differentially expressed genes. The GO terms were the same ones as in normal development, normal and parthenocarpic fruit set were very similar, and just a few changes confer the ability to set parthenocarpic fruits.CYCLIN D3:2, that are known to be repressed at the anthesis stage in tomato and activated at 2DPA in pollinated ovaries [Flower bud is a fast-growing stage in carpel development that changes at anthesis when the ovary is static, waiting for pollination and fertilization signals that will be followed by cell division during 7–10 days . However ovaries ,32, werepat mutant at anthesis, ovaries were bigger before pollination [pat-2 genes, the ovary weight was significantly higher with regard to near isogenic non-parthenocarpic lines [These cell cycle activation is in concordance with previous works that reported an increase in ovary size at anthesis in other parthenocarpic lines. In the lination . In partic lines .In spite of these differences, RP75/59 fruits at 3DPA followed the same developmental paths as the UC82 ones, as all of the genes activated in the control line in response to pollination were also activated in the parthenocarpic fruit set. In contrast, parthenocarpic fruits induced by GAs application did not show this activation of all of the cell cycle related genes .The five classic hormones gibberellins, auxins, ethylene, citokinins and abcisicacid have long been known to be involved in the different developmental phases of fruits ,6. Here pat-3/pat-4 system might be independent of cytokinins action.We did not find any cytokinin related genes differentially modulated in parthenocarpic fruit set, which suggest that parthenocapy in the Most of the ABA related genes were activated at normal anthesis. In the parthenocarpic line, genes related to ABA showed fewer differences between anthesis and 3DPA. ABA may keep the carpel in a state of temporary dormancy at anthesis which changes to an active state upon pollination and fruit set. However, in the parthenocarpic line the anthesis is not in such state of temporal dormancy, as there were cell cycle processes active.GA 3β-hydroxylase 2 was highly expressed in anthesis, GA20-oxidase 1 was inhibited at anthesis, GA20-oxidase 2 was activated at flower bud and GA20-oxidase 3 was activated at 3DPA and flower bud. When we analyzed the genes related to GAs differentially expressed in the parthenocarpic line (pat3/pat4) with respect to the non-parthenocarpic line, we found that GA20-oxidase 1 was not differentially expressed and GA20-oxidase 2 had small differences, whereas GA20-oxidase 3 expression was constitutively expressed in the parthenocarpic line, even at anthesis. These results showed that the parthenocarpy in pat3/pat4 is mediated by an overexpression of a GA20-oxidase 3 in the carpel, as in the pat mutant where the GA20-oxidase 1 was constitutively expressed in the ovaries [pat-3/pat-4 fruits had normal development in the locular tissue, meaning that the alteration of GAs production is not sufficient to explain this phenotype.GAs are known to be involved in the natural parthenocarpy of tomato fruits . In the  ovaries . However ovaries ,38, and pat-3/pat-4, which had filled locules, can develop pseudoembryos, as has been described in pat-2 tomato [Auxins, such as gibberellins, are known to be involved in fruit set . The app2 tomato , and tho2 tomato .IAA10 was activated only at 3DPA in the parthenocarpic line. A GH3 like protein was more activated at both stages of the non-parthenocarpic line. In addition, IAA2 and AXR2|IAA7 expression was highly activated at 3DPA with respect to anthesis, but in the parthenocarpic line this activation was clearly lower only at 3DPA in emasculated flowers. It is possible that IAA2 and AXR2|IAA7 needed pollination signals to be activated. IAA2 has been described as only being activated after pollination and not after GA3 treatment [On the other hand, there were some auxin responsive proteins whose expression was altered in the parthenocarpic line. reatment . These dEthylene plays a key role throughout fruit development and ripening in climacteric fruits and has been broadly studied ,1,6. In When we analyzed the expression of ethylene related genes, we found that most of the genes were inhibited at 3DPA in parthenocarpic and non-parthenocarpic carpels. This decrease in ethylene biosynthesis and signaling genes was also observed by Vriezen  in the oACO5 (1-aminocyclopropane-1-carboxylic acid oxidase 5) expression was clearly activated at anthesis in the parthenocarpic line with respect to the control in emasculated ovaries, and it was also more expressed in all of the stages tested in the parthenocarpic line. ACO (1-aminocyclopropane-1-carboxylic acid oxidase) is the last enzyme in ethylene biosynthesis and is considered to be a key point of regulation [dgt tomato mutant, where the differential expression of subsets of the IAA and ACS genes and the alterations in fruit morphology suggest that early stages of fruit development in tomato are regulated by auxin and ethylene [We investigated the differences in ethylene related genes between the carpels of the two lines. We found that gulation . The temethylene .pat3/pat4 the altered synthesis of ethylene might mimic pollination signals and may be involved in the induction of auxins synthesis and the activation of fruit set, which in normal anthesis is in a state of temporary dormancy.In pat-3/pat-4 system. We detected 758 genes differently modulated in parthenocarpic fruit set. These differences in gene expression were concentrated at anthesis, the key step. The most significant differences were found in cell cycle related genes. Cell cycle was not stopped at anthesis in the parthenocarpic line, contrary to normal development where carpels remain in a state of temporary dormancy, waiting for pollination signals. This dormancy state does not exist in RP75/59 anthesis is a transitional state to fruit set. We also checked the hormones related genes; GA and ethylene synthesis key genes were activated in the parthenocarpic line, and some aux/IAA gene expression was also altered despite the lack of differences in the auxin metabolism. In the parthenocarpic line the high expresion of GA20-oxidase 3 leads to the development of the parthenocarpic fruit ever in the absence of fertilization. Ethylene may mimic pollination signals, activating auxin synthesis and a response like that of normal fruit set. This leads to the production of pseudoembryos and fruits with normal locule development. Future work will elucidate the exact role of ethylene in fruit set and its relationship to auxin activation.Transcriptomic analysis of tomato carpel development and fruit set provides a resource for future study of tomato carpel development. We identified 2842 genes regulated throughout these processes and identified the hormone related gene involved. Comparison between array experiments determined that anthesis was the most different stage and the key point at which most of the genes were modulated. We also studied the alterations of gene expression in the parthenocarpic fruit set of the Tomato lines UC82 and RP75/59, a strongly facultative parthenocarpic tomato line ,18; planThe percentage of fruit set when the flowers were self-pollinated was greater than 90% in both lines. When the flowers were emasculated but not pollinated, the percentage of fruit set was greater than 90% in RP75/59 , whereas no fruit was set in the UC82 plants.Flowers were collected at four different developmental stages and under two conditions. Time stages were flower bud , flower bud to pre-anthesis , anthesis, and 3DPA (days post anthesis). Anthesis and 3DPA flowers were collected under two different conditions, emasculated 2 days before anthesis (UC82 flowers were hand-pollinated at anthesis) and non-emasculated.Flowers at all these stages were collected during three independent weeks to have biological replicates, the carpels were extracted, frozen in liquid nitrogen and stored at -80°C.Total RNA was extracted with TRI Reagent  following the manufacturer's instructions. The RNA was purified with the RNeasy plant mini kit . For the quantitative PCR (QPCR), first strand cDNA was synthesized from 1 μg of total RNA with oligo d(T) primer, using Expand Reverse Transcriptase . We used actin (TIGR acc. TC171374) as reference, PCNA (proliferating cell nuclear antigen EMBL acc. AJ515747.1) as control of the cellular division and 10 of the differential expressed genes according to the array analysis .S. lycopersicum probe sets to interrogate over 9,200 S. lycopersicum transcripts. Sequence information for this array was selected from public data sources including Lycopersicon esculentum UniGene Build #20  and GenBank mRNAs up to November 5, 2004. More information can be found at the Affymetrix home page [cDNA synthesis and cRNA production and fragmentation for the microarray hybridization were carried out as described in the Expression Analysis Technical Manual . We employed the Affymetrix GeneChip Tomato Genome Array designed specifically for monitoring gene expression in tomato. The comprehensive array consists of over 10,000 ome page . The GenRaw data with no background subtraction were analyzed with the affy package  from bioDifferentially expressed genes were extracted with the maSigPro package  from bioTo cluster the samples, we discarded the constant genes in order to avoid background noise. We made a hierarchical cluster with Euclidean distance by UPGMA method (bootstrap 100 replicates).To create the gene clusters, we employed the K-means method  with PeaThe GeneChip Tomato Genome Array was re-annotated using the Blast2GO package , which aLP obtained the experimental data and participated in the microarray analysis. JC designed the study and experiments and participated in the microarray analysis. JMB did the bioinformatic analysis to obtain the microarray annotation. LP and FN prepared the manuscript. All authors read and approved the final manuscript.TIME variable genes. Differentially expressed genes throughout normal carpel development and fruit set.Click here for fileTIME RP75/59 and UC-82vsRP75/59 variables genes. Differentially expressed genes throughout carpel development and fruit set in the parthenocarpic line with respect to the non parthenocarpic one.Click here for fileTentative annotation of Affymetrix tomato GeneChip. Array probe set were annotated to the higher GO level possible in each case.Click here for fileeTIME RP75/59 and eUC-82vsRP75/59 variables genes. Differentially expressed genes in parthenocarpic fruit set with respect to the non-parthenocarpic one.Click here for fileCell division and cycle genes. Cell cycle and cell division genes that changed during normal carpel development and fruit set (TIME).Click here for fileTIME variable genes related to hormones. Hormone related genes modulated along normal carpel development and fruit set.Click here for fileQPCR primers. Primers employed in the microarray verification.Click here for file


Rapid displacement across multiple time zones results in a conflict between the new cycle of light and dark and the previously entrained program of the internal circadian clock, a phenomenon known as jet lag. In humans, jet lag is often characterized by malaise, appetite loss, fatigue, disturbed sleep and performance deficit, the consequences of which are of particular concern to athletes hoping to perform optimally at an international destination. As a species renowned for its capacity for athletic performance, the consequences of jet lag are also relevant for the horse. However, the duration and severity of jet lag related circadian disruption is presently unknown in this species. We investigated the rates of re-entrainment of serum melatonin and core body temperature (BT) rhythms following an abrupt 6-h phase advance of the LD cycle in the horse.Six healthy, 2 yr old mares entrained to a 12 h light/12 h dark (LD 12:12) natural photoperiod were housed in a light-proofed barn under a lighting schedule that mimicked the external LD cycle. Following baseline sampling on Day 0, an advance shift of the LD cycle was accomplished by ending the subsequent dark period 6 h early. Blood sampling for serum melatonin analysis and BT readings were taken at 3-h intervals for 24 h on alternate days for 11 days. Disturbances to the subsequent melatonin and BT 24-h rhythms were assessed using repeated measures ANOVA and analysis of Cosine curve fitting parameters.We demonstrate that the equine melatonin rhythm re-entrains rapidly to a 6-h phase advance of an LD12:12 photocycle. The phase shift in melatonin was fully complete on the first day of the new schedule and rhythm phase and waveform were stable thereafter. In comparison, the advance in the BT rhythm was achieved by the third day, however BT rhythm waveform, especially its mesor, was altered for many days following the LD shift.Aside from the temperature rhythm disruption, rapid resynchronization of the melatonin rhythm suggests that the central circadian pacemaker of the horse may possess a particularly robust entrainment response. The consequences for athletic performance remain unknown. The suprachiasmatic nucleus (SCN) of the hypothalamus is the location of the master mammalian pacemaker that controls circadian rhythms of diverse physiological and behavioral phenomena, including rest-activity cycles, hormone secretion and body temperature. The circadian system provides endogenous timekeeping mechanisms that allow organisms to optimize survival by adaptively anticipating periods of activity and entraining physiological function to the solar day . AccordiIn humans, disruption of the circadian timing system occurs in response to rotational shift work and transmeridian travel. Rapid displacement across multiple time zones results in a mismatch between the previously entrained program of the internal circadian clock and the new cycle of light and dark (LD), a phenomenon known as jet lag ,4. The mBecause it is not possible to directly monitor the functional timing of the endogenous circadian clock, marker rhythms that measure clock output are commonly used to assess circadian phase position. Two common, useful and physiologically important markers of circadian phase have been the circadian rhythms of blood melatonin (MT) and core body temperature (BT). The 24-h melatonin profile provides a robust marker of circadian phase in humans ,13 and hThe primary aim of this study was to evaluate the rates of re-entrainment of the equine MT and BT rhythms following an abrupt 6-h phase advance of the LD cycle, mimicking an eastward transmeridian journey across 6 time zones. We demonstrate that these two markers of circadian phase resynchronize significantly more rapidly than in humans and rodents in response to a similar phase shift, suggesting a more rapid and robust entrainment response of the central pacemaker in the horse.Equus caballus) of mixed light horse breed were used in this study. Animals were maintained outdoors under conditions of natural photoperiod prior to the experiment, which was conducted at the time of year (mid-September) corresponding to an approximate 12 h light/12 h dark (LD12:12) natural photoperiod . Five days prior to the experiment, mares were housed in individual stalls in a light-proofed barn under a lighting schedule that mimicked the external photoperiod but with abrupt L/D and D/L transitions (LD 12:12). During the hours of light, stalls were lit by two 200 W light bulbs that produced an average light intensity of 350 lux at eye level. Access to water was ad libitum and feed was provided 4 times a day to prevent a conspicuous 24-h temporal cue [Six healthy 2 year old mares  on Day 0, and ending ~24 h later (ZT 0) blood samples were collected and rectal BT readings were taken by digital thermometer at 3-h intervals (9 samples). Blood samples were allowed to clot and kept overnight at 4°C. The next day, samples were centrifuged and the serum harvested and stored at -20°C until assayed for MT. Throughout the experiment, sampling during the hours of darkness was conducted with the aid of a dim red light from handheld flashlights. Mares were well accustomed to the sampling procedure and were only minimally disturbed by the presence of the sample collector. At each time point, samples were collected from mares in the same order with approximately 3 min intervals between animals. After experiencing one additional 12 h photoperiod at the usual time, an advance shift of the LD cycle was accomplished by ending the dark period 6 h early, resulting in lights on from 1:30 AM to 1:30 PM (ZT 18 became new ZT 0) starting on experiment Day 1 and continuing through Day 11. Beginning at lights on (1:30 AM/new ZT 0), blood sampling and BT readings were again taken at 3-h intervals for 24 h (ZT 0 to ZT 0) on alternate days for 11 days. The mares were consistently sampled in the same order at each sampling time with approximately 3 min intervals between animals. Sampling at ZT 0 was conducted immediately following lights-on and at ZT 12, immediately following lights-off.Melatonin was measured by a commercial RIA kit  as described previously . BrieflyMT and BT time series data were analysed using a computer program based on the least squares cosine fit method of Nelson et al. . For eacIndividual cosine fits for both melatonin and BT profiles were significant for all mares on baseline Day 0 Figure . The acrTwo-way repeated measures ANOVA of the MT data on Day 0 vs. Days 1–11 referenced to prevailing ZT times revealed no significant Day × Time interaction on any day post-shift Figure . This isFollowing the 6 h phase advance of the LD cycle, there was no significant Day × Time interaction effect on the BT rhythm on Day 0 vs. Days 1, 3 and 5 as determined by two-way repeated measures ANOVA Figure . On thesThis is the first experiment of its kind to investigate the re-entrainment of two crucial circadian rhythm phase markers in the horse to an abrupt 6-h advance of the LD cycle. The null hypothesis was that following the 6-h phase shift of the LD cycle there would be no change in the phase of the MT and BT rhythms in relation to the LD cycle. Rejection of this null hypothesis would follow with evidence of acrophase times (ZT) on Days 1 through 11 that differ significantly from the corresponding Day 0 baseline acrophase. As no such differences could be demonstrated, there is, based on cosine analysis, no compelling evidence in the present study for phase-shift related transients in the entrainment of these two equine rhythms to a 6-h phase advance of an LD12:12 cycle. This surprising outcome is discussed further below in the context of human jet lag and in association with a more detailed consideration of the present data.In humans, jet lag symptoms persist longer following an easterly rather than a westerly flight , theoretOur results indicate that in the horse, the MT rhythm, often regarded as the best marker of SCN pacemaker phase in humans , rapidlyIt is well known that MT production by the pineal gland increases significantly at night in both diurnal and nocturnal species and is a consequence of the high magnitude nocturnal rise in the enzyme serotonin-N-acetyltransferase . The immThe rapid response of MT to the phase-advanced LD cycle can not distinguish true circadian entrainment from potential masking effects. For example, with the present data we can not know to what extent light suppression as opposed to a fully advanced circadian clock programming the MT decline, may have contributed to the precipitous drop in MT from ZT 21 to ZT 24 on all days. However, it is less likely that light masking (melatonin suppression) contributed to the abrupt rise in MT at ZT 12 – ZT 15 post shift. Importantly, in order to fully evaluate the physiological disruption caused by time-zone displacement in the natural environment, it is necessary to determine the effects that phase shifts exert on both the phase and waveform of circadian rhythms in the presence of the shifted LD cycle. Additional experiments will be necessary to reveal any contributions attributable to light masking, as opposed to true phase shifts of underlying circadian oscillators.The unusually high melatonin levels that we observed at ZT 21 on Day 1 of the advanced LD cycle suggest an additive effect whereby the elevated MT production in response to the advanced scotophase overlapped with the previously entrained rise (perhaps not fully shifted) in MT associated with the pre-shift photoperiod. In the context of multiple SCN oscillators underlying the control of melatonin secretion, the increased secretion at ZT 21 could involve some oscillators advancing more than others to result in an increased overlap of oscillator outputs necessary to drive MT secretion occurring transiently at ZT 21. This is perhaps indicative of the combined controlling mechanisms of both the endogenous clock (SCN) and the prevailing LD cycle on MT secretion in response to a phase advance, as was similarly suggested for the pig .In contrast to the rapid and apparently uncomplicated stable re-entrainment of the equine MT rhythm, re-entrainment of the BT rhythm was slower and not without disturbances in rhythm waveform. The acrophase of the BT rhythm was only partially advanced on Day 1 post-shift, requiring three days to completely adjust to the new LD cycle. However, while the acrophase of the fitted cosine curve completed the shift by Day 3, considerably faster than observations of BT resynchronization from similar studies in rodents ,36, monkAschoff first suggested that phase shifts of the LD cycle lead to decreases in amplitude . The lowThe differences in re-entrainment rates between MT and BT rhythms in the horse likely reflect their independent regulation by different output pathways and different central circadian oscillators perhaps both within and outside the SCN . Neural The recent discovery of two spatially distinct oscillators within the SCN  providesThe difference between horses and other species in terms of adaptability to abrupt shifts in the LD cycle remains to be fully understood. It is interesting that an animal so dependent on circa-annual cues for seasonal breeding and survival of their young might be more rapidly responsive to circadian re-entrainment. However, we have previously demonstrated that clock gene expression in equine peripheral tissues is less tightly regulated than in other species . As a laJet lag and shift work are considered one of the modern world's most prevalent challenges affecting human health and productivity in the work place -51. ConsThe author(s) declare that they have no competing interests.BAM conceived of the study, designed the study, collected and analysed the data and drafted the manuscript. JAE performed the cosinor analysis of data and assisted with figures, data analysis and drafting of the manuscript. DRS assisted in study design and data collection. MMV assisted in study design and data collection. ELK assisted in study design and data collection. BPF assisted in data collection and MT assays. All authors read and approved the final manuscript.


The classical candidate-gene approach has failed to identify novel breast cancer susceptibility genes. Nowadays, massive parallel sequencing technology allows the development of studies unaffordable a few years ago. However, analysis protocols are not yet sufficiently developed to extract all information from the huge amount of data obtained.In this study, we performed high throughput sequencing in two regions located on chromosomes 3 and 6, recently identified by linkage studies by our group as candidate regions for harbouring breast cancer susceptibility genes. In order to enrich for the coding regions of all described genes located in both candidate regions, a hybrid-selection method on tiling microarrays was performed.We developed an analysis pipeline based on SOAP aligner to identify candidate variants with a high real positive confirmation rate (0.89), with which we identified eight variants considered candidates for functional studies. The results suggest that the present strategy might be a valid second step for identifying high penetrance genes. BRCA1 [MIM +113705] or the BRCA2 [MIM +600185] gene account for 25–30% of these cases Breast cancer (BC [MIM #114480]) is the most frequent malignancy among women, with approximately one million new cases per year around the world Massive parallel sequencing technology allows nowadays the development of studies unachievable a few years ago. Despite the fact that the advantages of this technology were evidenced in each of the published studies based on it, the analysis of all data generated by this process remains a hard task to face. The first step in the regular analysis protocol is based on the alignment of millions of short sequences obtained from each run. For that reason, during the past years many computer tools have been developed to improve the accuracy of this process A major success in the use of this technology was the re-sequencing of the whole human genome, published in several studies in the past years In a previous study, we performed a SNP-based linkage analysis in 41 non-BRCA1/2 families and obtained several candidate regions for containing BC susceptibility genes We performed a complete mutational screening via single-end massive parallel sequencing technology on the 128 known genes located on two different chromosomal regions, previously described as candidates for containing a breast cancer susceptibility gene DNA samples from 20 affected individuals, belonging to 9 different non-BRCA1/2 families, and 4 healthy unrelated individuals from the control population were used for the analysis process. More than 102 million reads were obtained from the affected individuals and almost 22.4 million reads were obtained from the control individuals see . The ave2 = 0.013, 2 = 0.69, We calculated the coverage  per individual and obtained an average of 98.25% of candidate bases covered for the affected and the control individuals . We did 2 = 0.96) was found between the global depth and the number of reads aligned to the candidate coding regions  for every base along the candidate coding regions per individual. As expected, a strong correlation  by using a HapMap sample in the exon-capture report We included a first filter see  in our SRegarding indel variants, we followed the same analysis pipeline as with SNP variants. Even though 12 bp gaps were allowed , an average of only 1.15 indels per individual were identified, and this number decreased to 0.50 after comparison to the control pool of data. Moreover, no indel was found to segregate in our family dataset. In order to rule out the possibility that a putative causal indel could be found in one member of a family but not in the other members, we checked the consequences for the remaining indels after the comparison to the control pool data for all 20 individuals from the 9 non-BRCA1/2 families. We observed a total of 8 undescribed indel variants for the 20 individuals. Three of them were located in intronic regions, two were located in 3' UTR regions, and the remaining three indels were homozygous with a global depth  = 1. Thus, no putative truncating indel was found in our individual dataset .The classical candidate-gene approach turned out to be a low-efficiency tool with regard to cost and time when used for the identification of causal genes in genetic diseases, especially when there is a large number of candidate genes (dozens to hundreds). For that reason, we decided to explore the new possibilities that massive parallel sequencing brings for the analysis of hundred of genes in the same reaction. Moreover, in the present study we developed a solid analysis pipeline based on SOAP aligner for variant detection in families with a common genetic disease via high throughput sequencing data. Hybrid selection on tiling microarrays We performed a CGH-like analysis obtaining the log-ratio between the normalised depth data from every affected individual and the control pool to confirm a homogenous distribution of the coverage along the candidate regions. Although the results showed that the distribution was very homogeneous, some differences in coverage were observed mainly in regions flanking the coding candidate regions (low coverage regions), where small and random differences in depth value may produce bigger differences in the index value. These data supported that the capture and sequencing of the candidate regions were successfully fulfilled in the sample set.In order to identify de novo mutations, we consider that the best option is to maintain those sequences that match equally to more than one location, even though it could be a source of alignment errors due to homology between regions. For that reason, we tested two different aligners (SOAP and Mosaik), which allow the possibility of maintaining this kind of sequences. We finally decided to use SOAP v1.0 because its output file kept all the information of the input data, while being able to detect SNPs and short (gaps >1 bp) indel variants in single-end data.The two high throughput sequencing-based mutation-detection studies published in the literature used MAQ software for SNP-calling In the second mutational screening study performed using high-throughput technology and MAQ software, the authors set up a global exome-capture method based on microarrays and a specific analysis pipeline, which was conceptually quite similar to ours, to identify causal variants for a monogenic disease Regarding indel variants, no indel fulfilled the criteria to be considered a candidate variant, neither in our dataset  nor in tIn another study, a complete mutational screening using Sanger sequencing was performed on 718 genes located on chromosome X in probandi from a set of 208 families diagnosed with X-linked mental retardation In summary, we designed an integral analysis pipeline for mutational screening via SOAP v1.0 that resulted in a low false positive rate with a low probability of discarding real positive variants, with which we identified 8 candidate variants that are currently under functional characterization. We consider that the present strategy might be a valid second step for identifying high penetrance genes, specifically when the regions of interest show significant evidence of linkage.All patients provided written informed consent for the collection of samples and subsequent analysis. We obtained ethics approval for this study from the ethics committees at all institutions/hospitals where participants were recruited As stated earlier, in a previous study we genotyped a total of 132 individuals from 41 non-BRCA1/2 families with almost 6,000 SNP markers, and we observed a linkage profile showing several candidate regions. Suggestive linkage signals (HLOD >2.2) were found in two regions located on chromosomes 3 and 6, which span 10.8 and 6.5 Mb, respectively, and we found 6 and 5 families putatively linked to each region In the present study, 10 of these 11 families were selected based on the availability of DNA  to perform a mutational screening via massive parallel sequencing. One family (Family 21) was excluded from the final analysis because the DNA library preparation of one of the members failed. However, putatively truncating mutations  were analysed for the remaining individual of this family see .We also sequenced DNA of 4 healthy individuals from Spanish control population, which were pooled into a single control data file. This pooled-control design presented several advantages, namely reduced repercussion of differential sample DNA degradation, increased sample heterozygosity, and a balancing effect over the number of reads per sample in the final control data, leading to increased data homogeneity.We used the Illumina single-end technique to perform the sequencing process and an exon-capture protocol recently published 5′-GATCT-3′; #2: 5′-ATCGT-3′; #3: 5′-TGTCT3′; #4: 5′-GTGAT-3′) linked to the ligated oligos, in order to be able to pool 3 or 4 individuals per array and to discriminate the sequences obtained from each individual. After ligating the modified adaptors, 150–300 bp fragments were selected by agarose gel electrophoresis and purified. To obtain a suitable amount of product, multiple parallel PCR reactions were carried out per sample.Genomic DNA (2.5 µg) was fragmented by a triple sonication step. To optimise the sequencing process, we used 5-base barcodes , using the whole genome as reference sequence. One of the main advantages of SOAP v1.0 is its ability to identify short insertions and deletions (indels) in single-end data, while maintaining all input information in the output file. We developed a specific SNP caller written in the Perl programming language to extract the information contained in the SOAP output file. The whole analysis pipeline for re-sequencing data is shown in To evaluate the homogeneity of the coverage, we calculated the mean and the median of the depth for stretches of 15 bases along the candidate regions. For each sample, we obtained the index value  and the allele depth  for both the variant and the reference alleles, and also the global depth (number of different sequences that cover a single base). We calculated two different scores based on the same mathematical formula: VX represents the mean quality  or the allele depth  of the variant allele, and RX represents in each case the same parameter but for the reference allele. Scores close to 100 indicate that both alleles are equally represented.where In order to determine the optimal DS threshold for the analysis, we performed a study using the information from a single family . We usedEnsembl database through the PerlAPI tools in silico, how non-synonymous variants would affect the functionality of the respective candidate proteins  of the remaining SNPs after comparison against the control pool data .Finally, we compared the sensitivity of our scores with a published reference, and for that we followed our analysis pipeline using MAQ software in a subset of families applying no threshold for MAQ variant score .Figure S1Correlations. Coverage along the candidate regions was very high (98% on average) and no correlation between coverage and the number of sequences obtained per individual was observed (A), although we observed a logarithmic trend when the number of sequences aligned to the candidate regions was used (B). On the other hand, a strong correlation between the number of sequences aligned to the candidate coding regions and the mean depth was observed in our dataset (C). Failures in the capture step were discarded since high correlations between the global mean and the global median of the depth per individual (D) and between the mean and the median of the depth in putatively altered 15-bp regions for all the individuals (E) were observed (see text for details).(0.13 MB DOCX)Click here for additional data file.Table S1Indel variant filtering process.(0.05 MB DOCX)Click here for additional data file.Table S2Putative truncating variants discarded during the filtering process. A. Summary of the whole list of truncating variants (STOP_GAINED and ESSENTIAL_SPLICE_SITE) in some individuals after comparison to the control pool data. The original list has been filtered (Depth Score > 50) for simplicity, given that variants showing a Depth Score below the threshold are likely to be false positives. As evidenced in the table, none of the variants above the threshold has a high Global Depth value, which paired to the fair Depth Score means that in every case the variant allele was detected in a low proportion in relation to the respective reference allele. Additionally, this table shows that no putative candidate truncating variants were discarded during the filtering process, reassuring that filtering using Depth Score and Global Depth is a stringent but adequate filtering step. In conclusion, the low Global Depth and Depth Scores explain why these variants are likely to be false positive results and they were therefore excluded from the final candidate SNP list . B. The (0.15 MB DOCX)Click here for additional data file.


Daniel Maeusezahl and colleagues conducted a cluster-randomized controlled trial in rural Bolivia of solar drinking water disinfection, and find only moderate compliance with the intervention and no evidence of reduction in diarrhea among children. Solar drinking water disinfection (SODIS) is a low-cost, point-of-use water purification method that has been disseminated globally. Laboratory studies suggest that SODIS is highly efficacious in inactivating waterborne pathogens. Previous field studies provided limited evidence for its effectiveness in reducing diarrhoea.We conducted a cluster-randomized controlled trial in 22 rural communities in Bolivia to evaluate the effect of SODIS in reducing diarrhoea among children under the age of 5 y. A local nongovernmental organisation conducted a standardised interactive SODIS-promotion campaign in 11 communities targeting households, communities, and primary schools. Mothers completed a daily child health diary for 1 y. Within the intervention arm 225 households (376 children) were trained to expose water-filled polyethyleneteraphtalate bottles to sunlight. Eleven communities  served as a control. We recorded 166,971 person-days of observation during the trial representing 79.9% and 78.9% of the total possible person-days of child observation in intervention and control arms, respectively. Mean compliance with SODIS was 32.1%. The reported incidence rate of gastrointestinal illness in children in the intervention arm was 3.6 compared to 4.3 episodes/year at risk in the control arm. The relative rate of diarrhoea adjusted for intracluster correlation was 0.81 . The median length of diarrhoea was 3 d in both groups.Despite an extensive SODIS promotion campaign we found only moderate compliance with the intervention and no strong evidence for a substantive reduction in diarrhoea among children. These results suggest that there is a need for better evidence of how the well-established laboratory efficacy of this home-based water treatment method translates into field effectiveness under various cultural settings and intervention intensities. Further global promotion of SODIS for general use should be undertaken with care until such evidence is available.www.ClinicalTrials.govNCT00731497Please see later in the article for Editors' Summary Thirsty? Well, turn on the tap and have a drink of refreshing, clean, safe water. Unfortunately, more than one billion people around the world don't have this option. Instead of the endless supply of safe drinking water that people living in affluent, developed countries take for granted, more than a third of people living in developing countries only have contaminated water from rivers, lakes, or wells to drink. Because of limited access to safe drinking water, poor sanitation, and poor personal hygiene, 1.8 million people (mainly children under 5 years old) die every year from diarrheal diseases. This death toll could be greatly reduced by lowering the numbers of disease-causing microbes in household drinking water. One promising simple, low-cost, point-of-use water purification method is solar drinking water disinfection (SODIS). In SODIS, recycled transparent plastic drinks bottles containing contaminated water are exposed to full sunlight for 6 hours. During this exposure, ultraviolet radiation from the sun, together with an increase in temperature, inactivates the disease-causing organisms in the water.www.SODIS.ch). However, although SODIS works well under laboratory conditions, very few studies have investigated its ability to reduce the number of cases of diarrhea occurring in a population over a specific time period (the incidence of diarrhea) in the real world. Before any more resources are used to promote SODIS—its effective implementation requires intensive and on-going education—it is important to be sure that SODIS really does reduce the burden of diarrhea in communities in the developing world. In this study, therefore, the researchers undertake a cluster-randomized controlled trial (a study in which groups of people are randomly assigned to receive an intervention or to act as controls) in 22 rural communities in Bolivia to evaluate the ability of SODIS to reduce diarrhea in children under 5 years old.SODIS has been promoted as an effective method to purify household water since 1999, and about 2 million people now use the approach  to receive the intervention—a standardized, interactive SODIS promotion campaign conducted by Project Concern International —and 11 communities  to act as controls. Households in the intervention arm were trained to expose water-filled plastic bottles for at least 6 hours to sunlight using demonstrations, role play, and videos. Mothers in both arms of the trial completed a daily child health diary for a year. Almost 80% of the households self-reported using SODIS at the beginning and end of the study. However, community-based field workers estimated that only 32.1% of households on average used SODIS. Data collected in the child health diaries, which were completed on more than three-quarters of days in both arms of the trial, indicated that the children in the intervention arm had 3.6 episodes of diarrhea per year whereas the children in the control arm had 4.3 episodes of diarrhea per year. The difference in episode numbers was not statistically significant, however. That is, the small difference in the incidence of diarrhea between the arms of the trial may have occurred by chance and may not be related to the intervention.These findings indicate that, despite an intensive campaign to promote SODIS, less than a third of households in the trial routinely treated their water in the recommended manner. Moreover, these findings fail to provide strong evidence of a marked reduction of the incidence of diarrhea among children following implementation of SODIS although some aspects of the study design may have resulted in the efficacy of SODIS being underestimated. Thus, until additional studies of the effectiveness of SODIS in various real world settings have been completed, it may be unwise to extend the global promotion of SODIS for general use any further.http://dx.doi.org/10.1371/journal.pmed.1000125Please access these Web sites via the online version of this summary at PLoS Medicine editors wrote an editorial arguing that water should be a human rightThe http://www.who.int/household_water and about the importance of water, sanitation, and hygiene for health http://www.who.int/water_sanitation_health/en/index.html The World Health Organization provides information about household water treatment and safe storage solar water disinfection The SODIS Reference Center provides detailed information about America (in Spanish and English)The SODIS Foundation in Bolivia provides practical information for the roll-out of solar water disinfection in Latin Project Concern International provides information about its campaign to promote SODIS in Bolivia (in Spanish)peopleThe Water Supply and Sanitation Collaborative Council (WSSCC) is a global multi-stakeholder partnership organization with a goal of advocating to achieve sustainable water supply and sanitation for all  Globally, 1.8 million people die every year from diarrhoeal diseases the vast majority of whom are children under the age of 5 y living in developing countries www.who.int/household_water, www.sodisafricanet.org). SODIS is currently promoted in more than 30 countries worldwide (www.sodis.ch) and in at least seven Latin American countries through the SODIS Foundation including in Bolivia.Recent systematic reviews concluded that interventions to improve the microbial quality of drinking water in households are effective at reducing diarrhoea, which is a principal source of morbidity and mortality among young children in developing countries Despite this widespread promotion, evidence of the effectiveness of SODIS from field studies is limited. The three reported SODIS trials to date implemented the intervention at the household level, two of them in highly controlled settings that ensured very high compliance Because SODIS is a behavioural intervention designed to reduce infectious diarrhoea, disease transmission and its interruption likely have community level dynamics www.fhi.org/training/sp/Retc/). Project staff comprised all project personnel of all project partners. Field staff comprised all personnel working in our laboratories and at our Totora field station including data enumerators and data- and project-management staff, supervisors, and community-based field workers living in the study communities. The trial protocol , was conducted in an ethnically homogeneous Quechua setting in rural Totora District, Cochabamba Department, Bolivia. Our study was part of a comprehensive SODIS roll-out programme in collaboration with Project Concern International, a nongovernmental organisation (NGO). Most of the local residents are farmers, typically living in small compounds of three buildings with mud floors, with five or more persons sleeping in the same room. Our own surveys showed that 15% of homes have a latrine or other sanitary facilities and that most residents defecate in the nearby environment.n = 223). Samples of at least one water source per community were tested for Giardia lamblia and Cryptosporidium parvum. The two parasites were detected in 18/24 and 11/23 water samples, respectively.Drinking water is typically stored in 10-l plastic buckets or open jerry cans of 5–20 l in the household. Baseline assessments of the drinking water quality in the home indicated a median contamination of thermotolerant coliforms (TTC) of 32 TTC/100 ml (interquartile range (IQR) = 3–344; Parasites were detected by using immunomagnetic separation and PCR techniques Twenty-seven of 78 communities in the study area fulfilled the selection criteria . Two communities were excluded because of other ongoing health and hygiene campaigns, and three communities withdrew participation before baseline activities because of a change in political leadership. Community health workers undertook a census and identified households with at least one child <5 y. All children <5 y were enrolled in the participating villages.We pair-matched communities on the incidence of child diarrhoea as measured in an 8-wk baseline survey We explicitly chose community-level randomization because important components of the intervention  would occur at the community level. Randomization below the community level would not reflect the reality of scale-up programme implementation, and we would not have captured the potential community-level reinforcement of the behaviour change. Furthermore, community-level randomization is considered ethically optimal, because participants expect to equally benefit from interventions within their community k) of similar studies, between 0.1–0.25 (as cited by Hayes and Bennett) and a minimum of 10 child-years of observation per cluster k = 0.20 and an alpha level of 0.05. Anticipating a drop-out of at least one cluster per arm and a loss of follow-up of individuals, the final sample size was adjusted to 11 pairs with 30 children per community cluster. We powered the study to detect a 33% reduction in diarrhoea incidence after reviewing the evidence base for point-of-use water treatment at the time of the study's inception in 2002 Sample size was calculated according to methods outlined by Hayes and Bennett http://www.sodis.ch/files/TrainingManual_sm.pdf). Promotion activities were targeted at primary caregivers and all household members (biweekly), whole communities (monthly), and primary schools (three times) by the NGO as part of its regional community development programme. Eleven communities (262 households and 441 children) were randomized to the intervention; 11 communities  served as a control group  bottles. The households were taught through demonstrations, role plays, video, and other approaches to expose the water-filled bottles for at least 6 h to the sun. NGO staff emphasized the importance and benefits of drinking only treated water , explained the germ–disease concept, and promoted hygiene measures such as safe drinking water storage and hand washing as they relate to the understanding of drinking water and the faecal–oral route of transmission of pathogens . During The primary outcome was the IR of diarrhoea among children <5 y, defined as number of diarrhoea episodes per child per year obtained from daily assessment of individual diarrhoea occurrence. We applied the WHO definition for diarrhoea of three or more watery bowel movements or at least one mucoid/bloody stool within 24 h The primary outcome was measured by community-based field workers who were recruited nearby and who lived one per community during data collection periods. The field workers were extensively trained in interviewing and epidemiological observation techniques, data checking, recording, and in general approaches to community motivation. Community-based field workers were randomly rotated between communities every 3 mo. Child morbidity was reported by the closest caregiver using the vernacular term “K'echalera,” which had been established previously to correspond to the WHO definition of diarrhoea Compliance with the SODIS method was measured using four different subjective and objective indicators. Three of the indicators were assessed by field staff independent from the implementing NGO: (i) the number of SODIS-bottles exposed to sunlight and, (ii) the number of bottles ready-to-drink in the living space, and (iii) the personal judgment about families' user-status was provided by community-based field workers living among the families in the intervention arm. Judgement criteria for this main compliance indicator study included observing regular SODIS practice and bottles exposed to sun or ready to drink in the kitchen and being offered SODIS-treated water upon request. The fourth SODIS-use indicator was based on self-reporting and caregivers' knowledge of and attitudes toward the intervention that was assessed at the beginning  and at the end of the 12-mo follow-up period.An intention-to-treat analysis was applied comparing the IR of diarrhoea between children <5 y in intervention and control communities. Diarrhoea prevalence (PR) and severe diarrhoea (SD) were additionally analysed. Generalized linear mixed models (GLMM) were fitted to allow for the hierarchical structure of the study design (pair-matched clusters). In contrast to our original trial protocol we selected the GLMM approach rather than generalized estimating equations (GEE) because recent publications indicated that the latter method requires a larger number of clusters to produce consistent estimates Y by g(E(Y)), the crude and adjusted models were: g(E(Yijk)) = μ+Bi+τj+ξij, and g(E(Yijk)) = μ+Bi+τj+ξij+x'b where Yijk denotes the observed outcome value for the kth individual from a community allocated to the jth intervention, in the ith pair, μ is the general mean, Bi is the random effect of the ith pair ≈N, τj is the fixed effect of the SODIS intervention, and ξij is the random effect of the interaction of the ith pair with the jth intervention applied to the community ≈N (signifying the within-pair cluster variance and used as error term for τj), x is the vector of potential confounding factors, and b the vector of the corresponding regression coefficients.The crude (unadjusted) model included only the design factors and the intervention effect k) were calculated after data collection to validate the degree of clustering and our assumptions for the sample size. ICC and k were estimated from the unscaled variance of the IR's GLMM. To estimate the uncertainty of ICC and k, we obtained the 95% credible region  through an analogous Bayesian hierarchical regression The intracluster correlation coefficient (ICC) and the coefficient of between-cluster variation  and withdrawal (67%). Supervisors reevaluated the outcome during 984 unannounced random home visits, and discrepancies between community-based field workers' and supervisors' records were found for five (0.5%) of all visits.At baseline the households in the different study arms were well balanced on multiple other factors suggesting successful randomisation . The maiThe NGO conducted 210 community events and 4,385 motivational household visits in intervention communities; 3,060 visits occurred in the households with children <5 y followed up and analysed for the study, and 1,325 household visits took place in homes that were not taking part in the study. Study households attended a median of nine community events (IQR = 5–12) and were visited by the SODIS-programme team a median 11 times at home (IQR = 7–18). To ensure a sufficient number of PET bottles, the NGO provided as many SODIS-bottles as required by participants (mean 955 bottles/community).Children in the SODIS-intervention arm reported a total of 808 episodes or a mean of 3.6 per child per year-at-risk . In the p-values>0.1) or resulted in meaningful changes in estimates of ORs. A multivariable model adjusting for age, sex, baseline-existing water treatment practises, and child hand washing was consistent in its estimate of effect . We repeated the analysis by including confounding covariates in the order of occurrence of the variables in k was estimated to be 0.27 with a 95% confidence region of .The ICC was estimated as 0.0009 with a 95% posterior credible region between ; Community-based field workers who were living in the communities throughout the study observed a mean SODIS-user rate of 32.1% in the intervention arm  . The meaNo positive effect of compliance (proportion of weeks of observed SODIS use) on the IRs in the intervention arm was observed. The incidence did not decline with the increase of weeks using SODIS . SeasonaThe median proportion of sunny days with more than 6 h of sunshine was 70.2% and 67.2% in intervention and control communities, respectively, consistent with the technical and climatic conditions necessary for the proper functioning of the ultraviolet SODIS purification process We conducted a community-randomized trial within the operations of an ongoing national SODIS-dissemination programme, which provided an intensive training and repeated reinforcement of the SODIS intervention throughout the study period. In this context of a “natural experiment” we found a RR of 0.81 for the IR of diarrhoea episodes among children assigned to SODIS compared to controls. However, the CI was broad and included unity  and, therefore, we conclude that there is no strong evidence for a substantive reduction in diarrhoea among children in this setting. Subsequently, we discuss the primary outcome in the context of other study findings, and explain why we hypothesize that the true effect—if there is any—might be smaller.First, the estimate for the longitudinal prevalence of diarrhoea was substantially smaller  than the estimate for incidence and there is some evidence that prevalence is a better predictor in terms of mortality and weight gain than incidence C. parvum was lower in the intervention children (5/94 versus 2/103), but other pathogens were found at similar proportions in intervention and control children . In further exploring the occurrence of other illness symptoms we found the prevalence of eye irritations and cough to be lower in the intervention group compared to the control group. This difference could be the result of the hygiene component in the intervention that increased hygiene awareness among the treatment communities. An alternative explanation is that the lack of blinding led to biased (increased) health outcome reporting in the intervention group.Further, analysing the laboratory results from 197 randomly selected stool specimens also did not provide convincing evidence for an intervention effect: the proportion of Due to the nature of the intervention neither participants nor personnel were blinded to treatment assignment. Ideally, blinding to the intervention allocation should apply to the NGO staff administering the SODIS intervention and our enumerators assessing outcomes In our study the lack of blinding may have reduced motivation in the control communities. However, the number of households lost during follow-up and the number of days under observation were almost identical in both arms. Additionally, the control communities knew that they would receive the intervention after study end. Finally, a reduction of diarrhoea frequency of 20% might be insufficient to be well perceived, i.e., have a noticeable impact in a population with a high burden of child diarrhoea and will, thus, not result in a sustainable behavioural change. Faecal contamination in about 60% of the yards indicates a highly contaminated environment with presumably a large potential for transmission pathways other than consuming contaminated water. This simultaneous exposure to a multiplicity of transmission pathways may explain why we found no significant diarrhoea reduction due to SODIS.On the other hand, our result of a 19% reduction in diarrhoeal episodes appears to be roughly consistent with results of the two other SODIS trials both from Maasai cultural settings conducted by Conroy and colleagues among children <6 y and 5–16 y of age. They report a 16% reduction  To our knowledge this is the first community-randomized trial and the largest study so far to assess the effectiveness of the SODIS method under typical social and environmental conditions in a general rural population setting where children drink untreated water.k = 0.27), we would have needed a study 2.5 times larger for a 20% difference to be significant.Our study was sufficiently powered to detect a 33% reduction in the effectiveness of the SODIS intervention, and we accounted for clustered design in our analysis. On the basis of a post hoc sample size calculations using the model-based estimate for the between-cluster variability  with proxies more responsive to actual treatment behaviour . We consider this a restrictive, more conservative definition of SODIS use compared to that in other studies, which recorded reported use It is possible that respondents would like to please field staff and over-report use out of courtesy. Also, observing exposed bottles on the roof may overestimate use , becauseThere are limitations to our study. As in other studies Despite a comprehensive and intensive intervention promotion campaign, we detected no strong evidence for a significant reduction in the IR of diarrhoea in children <5 y in families using SODIS in our trial in a typical setting in rural Bolivia. We believe that clearer understandings of the discrepancy between laboratory and field results , the role of compliance in effectiveness, and a direct comparison of SODIS to alternate drinking water treatment methods are needed before further global promotion of SODIS.Alternative Language Abstract S1Spanish translation of the abstract by MC.(0.03 MB DOC)Click here for additional data file.Figure S1SODIS promotion and implementation scheme Click here for additional data file.Text S1Trial protocol.(0.52 MB PDF)Click here for additional data file.Text S2CONSORT statement checklist.(0.10 MB PDF)Click here for additional data file.