Dengue is one of the most aggressively expanding mosquito-transmitted viruses. The human burden approaches 400 million infections annually. Complex transmission dynamics pose challenges for predicting location, timing, and magnitude of risk; thus, models are needed to guide prevention strategies and policy development locally and globally. Weather regulates transmission-potential via its effects on vector dynamics. An important gap in understanding risk and roadblock in model development is an empirical perspective clarifying how weather impacts transmission in diverse ecological settings. We sought to determine if location, timing, and potential-intensity of transmission are systematically defined by weather.We developed a high-resolution empirical profile of the local weather-disease connection across Peru, a country with considerable ecological diversity. Applying 2-dimensional weather-space that pairs temperature versus humidity, we mapped local transmission-potential in weather-space by week during 1994-2012. A binary classification-tree was developed to test whether weather data could classify 1828 Peruvian districts as positive/negative for transmission and into ranks of transmission-potential with respect to observed disease. We show that transmission-potential is regulated by temperature-humidity coupling, enabling epidemics in a limited area of weather-space. Duration within a specific temperature range defines transmission-potential that is amplified exponentially in higher humidity. Dengue-positive districts were identified by mean temperature >22°C for 7+ weeks and minimum temperature >14°C for 33+ weeks annually with 95% sensitivity and specificity. In elevated-risk locations, seasonal peak-incidence occurred when mean temperature was 26-29°C, coincident with humidity at its local maximum; highest incidence when humidity >80%. We profile transmission-potential in weather-space for temperature-humidity ranging 0-38°C and 5-100% at 1°C x 2% resolution.Local duration in limited areas of temperature-humidity weather-space identifies potential locations, timing, and magnitude of transmission. The weather-space profile of transmission-potential provides needed data that define a systematic and highly-sensitive weather-disease connection, demonstrating separate but coupled roles of temperature and humidity. New insights regarding natural regulation of human-mosquito transmission across diverse ecological settings advance our understanding of risk locally and globally for dengue and other mosquito-borne diseases and support advances in public health policy/operations, providing an evidence-base for modeling, predicting risk, and surveillance-prevention planning. Aedes aegypti mosquitoes, the primary vector of dengue, slight changes in different components of weather have important effects on population dynamics, lifespan, biting-frequency, virus incubation period and capacity to transmit the virus, thus inducing changes in transmission probability. These complicated dynamics produce a weather-disease connection that is not well-defined for different ecological settings. Understanding this connection is important to critical elements of policy development and operational control of dengue such as predicting risk, developing human-vector transmission models, and planning surveillance-intervention strategies locally and globally. The empirical profile of the weather-disease connection for dengue developed in this study provides a needed understanding of how temperature and humidity work together in regulating human-mosquito transmission. The observed likelihood of low to epidemic-level transmission was highly sensitive to local seasonal duration in limited areas of this two-dimensional weather-space. Data presented represent a resource for estimating where and when transmission-potential supports epidemics of varying magnitude. This high-resolution weather-disease profile for dengue reveals systematic relationships that are informative for mosquito-borne diseases in general and discussions of consequences of global warming.Timing and spatial-extent of diseases such as dengue and malaria that result from transmission between humans and mosquitoes are regulated by weather in complicated ways. For  Aedes aegypti [The escalating geographic scope and disease burden associated with dengue viruses (DENVs) over the past 50 years are serious global health concerns. Recent estimates of the global burden are nearly 400 million infections and 500,000 hospitalizations annually ,2. Weath aegypti ,19,20. F aegypti . Each of aegypti –37. SyntTemperature and humidity are emerging as weather components with greatest impact on DENV transmission, yet the separate and combined effects of this coupling across a range of ecological conditions remain ill-defined ,4,7,11. We present a high-resolution empirical view of the interplay between weather and DENV transmission across Peru, a richly diverse ecological setting where transmission is intense and escalating –40. DuriWe developed a binary classification tree to elucidate the location, timing and intensity of DENV transmission in relation to local daily weather patterns across Peru during 1994–2012. We explored evidence of transmission indirectly by examining patterns in reported cases and considered the concept of transmission potential by investigating the maximum levels of incidence observed in relation to specific weather conditions. We investigated three hypotheses: Weather determines (1) local potential for DENV transmission; i.e. local weather conditions must meet/exceed specific thresholds for a defined minimum duration annually in order to support vector dynamics necessary for transmission; (2) local potential for epidemic magnitude; and (3) the seasonal timing of local dengue epidemics. Weather was conceived as a 2-dimensional “weather-space” composed of temperature vs. humidity, inclusive of the full range of conditions observed locally across Peru, within which DENV dynamics are observed  and combined with birthrate, death rate, and migration rate data provided by INEI for the same time span. A dynamic population model was developed from these data to represent population dynamics by district-week throughout Peru for 1994–2012.www.ncdc.noaa.gov. Weekly mean, min, and max values for temperature and humidity represent means of daily values. Only conservative corrections to the weekly weather data were applied in order to avoid introducing an artificial or subjective bias. Extraneous outliers, exceeding 3 standard deviations from the mean per weather component/location, were corrected by linear interpolation. 33 such weekly corrections were applied across all time series for all locations. Short gaps with missing data for up to 11 consecutive weeks were filled by linear interpolation. A spatial 1 kilometer grid of western South America was generated for each weather component by day using triangulation with cubic interpolation between weather station control points for 2005–2012. District-week observations were derived by averaging over grid points that fell within a district polygon within a given week. Resulting values were randomly checked for consistency with public daily weather reports for cities across Peru provided by the Peruvian government. In all checks, comparison of mean temperature for the same location and time of year were within 1–2°C and were considered consistent. Map summaries of the temperature and humidity range across Peru developed from gridded data are shown in Weather, including mean, minimum (min), and maximum (max) temperature and mean, min, and max humidity were assessed for all Peruvian districts by week for 2005–2012 and for the 6 highest incidence areas of Peru by week for 1994–2012. Weather data per district-week for mean, min, and max temperature and humidity were developed from daily records of weather stations (see map in A binary classification tree was developed to accept weather data by district-week (2005–2012) as inputs and predict districts in which DENV transmission did and did not occur during this time period (Level 1) and distinguish between districts with varying intensity of transmission (Level 2). In each classification step, each Peruvian district was classified into one of two groups based on weather inputs. In Level 1, districts were classified as positive or negative for dengue transmission during 2005–2012. Each weather component  was tested individually and in all permutations of pairs as classification inputs. Possible thresholds per weather component for predicting transmission were tested based on the full range of weather conditions observed across Peru, 2005–2012 with stepping intervals of 0.5°C for temperature and 2% for humidity. At each threshold of each weather component tested, the mean duration in weeks spent above that threshold annually was assessed for each district. Thus each classification test consisted of a configuration that specified (1) the components of weather to be used as inputs, (2) the test threshold for each weather component and 3) the minimum duration of time (1 to 52 weeks) above each respective threshold annually. Each test configuration produced a complete classification of districts. If a district satisfied the test of minimum duration per year above the respective threshold per weather component for all weather components in the test configuration, it was classified as dengue positive, otherwise it was classified as dengue negative. Preliminary tests indicated that max temperature and max humidity were least informative for classification (consistently lower sensitivity and specificity then other measures) and were eliminated, thus reducing the number of test configurations to more than 2 trillion. Pattern recognition was applied to dynamically vary the stepping interval and reduce the number of tests to a tractable level computationally. Previously a suppressive effect of max temperature above 34°C was observed in Thailand [ the miniThe true status of each district was assessed from cumulative dengue cases for 2005–2012. Districts with no cases during this period were considered negative and districts with 10 or more cases were considered positive. Districts with less than 10 total cases during the 8 year period were considered uncertain with respect to true DENV transmission status and not included in the classification. We observed multiple occurrences of 1–6 total cases reported in southern Peru where temperatures were extremely cold and dengue transmission was highly unlikely. Such low case counts could easily be associated with a few travelers who became infected in a different location and are unlikely to reflect ongoing transmission dynamics in the residence location. Considering there were likely to be similar instances that were less apparent, we chose to address this issue in a conservative approach by identifying a low threshold  in which the true status of a district would be considered ambiguous. This choice was guided by examining the distribution of case counts and eliminating a small cluster in the lower tail of the distribution that was likely due to a combination of infections from travelers and low reporting in remote rural districts. 80% of the excluded districts had 4 or fewer total cases during 2005–2012. The spatial distribution of excluded districts appeared random throughout all of Peru and was not focused in any particular climate zone. It included remote rural areas in the Amazon Basin where transmission is likely and southern and coastal areas where travel related infections are likely. Scaling the threshold for exclusion criteria to larger case counts in more populated areas would have been a more aggressive approach and would have excluded many true positive districts. 1557 districts were classified of which 186 were considered true-positives and 1371 were considered true-negatives for DENV transmission. Classification results from all test configurations were evaluated to identify the weather conditions that produced the lowest overall misclassification rate and the highest level of both sensitivity and specificity in classifying 1557 districts. Sensitivity represents the percent of true-positive districts that were classified as positive. Specificity represents the percent of true-negative districts that were classified as negative. Thus testing of a very low mean temperature threshold would identify most districts as positive, resulting in high sensitivity but poor specificity; and testing a high mean temperature threshold would identify most districts as negative resulting in poor sensitivity but high specificity. The best classification criteria was chosen such that both sensitivity and specificity were high. Binary classification algorithms were developed in Matlab and are summarized in In level 2 of the classification tree, true positive districts were categorized into 5 groups according to 2005–2012 cumulative incidence rate per 1000 population; low to high: group 1: >0 to 1, group 2: 1–5, group 3: 5–20, group 4: 20–60, group 5: 60–130. These categories are linear on a log-scale and reflect the exponential increase in incidence associated with longer seasonal duration of transmission. Binary classification tests were run in a similar manner to the protocol described above for level 1. In level 2, the goal was to test the ability to differentiate between high and low transmission groups based on weather inputs. Mean and min temperature and mean humidity were used individually and in combinations as weather inputs for tests of each pairing of groups. Min humidity was dropped from test configurations because it was consistently less informative than mean humidity in classification outcomes, i.e. it followed a similar pattern to mean humidity but with consistently lower specificity and sensitivity in test results.Statistical analyses were performed to further investigate the relationship between local weather patterns and DENV transmission patterns across districts, with a focus on quantifying transmission-potential under different weather conditions. Potential magnitude of virus transmission is a complex measure since locations associated with high levels of transmission do not always experience high transmission when weather is optimal due to other regulating factors. Considering such factors fluctuate over time, two indicators of transmission-potential were assessed: (1) cumulative incidence rate by district and (2) maximum dengue impact per district-week across the temperature-humidity weather-space of Peru. Linear regression was used to examine the association between (a) annual temperature and humidity ranges versus cumulative incidence rate by district and (b) mean annual duration above specific temperature and humidity thresholds versus cumulative incidence rate by district, 2005–2012. The natural log of cumulative incidence rate was used because the increase in incidence as temperatures rise within the optimal range and as humidity rises amidst temperatures optimal for transmission is exponential in nature. As a surrogate for transmission-potential, maximum dengue impact was measured as the mean of the top 1% of local weekly incidence rates incurred per 1°C temperature and 2% humidity weather interval across Peruvian weather-space. Profiles of maximum dengue impact across weather-space were developed graphically in Matlab and are presented as 2-dimensional weather-space grids for mean, min, and max temperature-humidity.Six areas of Peru were identified as locations of highest cumulative dengue incidence rates during 1994–2012. These areas include Maynas and Alto Amazonas provinces of Loreto department, and Ucayali, Madre de Dios, Tumbes, and Piura departments. Weather and DENV transmission dynamics were assessed by week during 1994–2012 for the 6 areas in order to better understand the role of weather in development of large epidemics. For the 6 locations combined and for each location individually, frequency (where time is spent), cumulative incidence rate, and maximum dengue impact profiles were developed in Matlab.Nadir, Onset, and Peak. Peak was defined as the first week during the seasonal cycle in which the local seasonal maximum case count occurs. Peak marks the end of accelerating case counts and the beginning of seasonal decline. Nadir was defined as the last occurrence between consecutive seasonal Peaks in which the case count is at a seasonal low. Onset, identified using a statistical algorithm, follows seasonal Nadir and marks the time-point when potential epidemic development begins. Nadir, Onset, and Peak from each seasonal cycle were used to compare the timing of transmission change-points across seasonal cycles of varying magnitude in each location and were quantified using linear regression. A more detailed description is provided in In each of the 6 locations, each seasonal DENV transmission cycle during 1994–2012 was evaluated to identify systematic change-points in the number of weekly cases. Three change-points were identified within each seasonal transmission cycle in each location: Peru encompasses a diverse dengue history spatially, including 1828 districts that range from districts with no reported dengue cases to districts with repeated large epidemics. Median size of districts (subunit of departments/provinces) is 208 sq. km. and 4360 people. The goal of level 1 of the binary classification tree , producing the largest epidemics when humidity >80% for >6 weeks per year or >75% for >19 weeks per year, while concurrently mean temperature >25°C. Note that incidence rate is presented in a log scale in Annual temperature range per district was a predictor of cumulative incidence magnitude. Cumulative incidence rate was strongly associated with upper and lower limits of annual range of mean and minimum temperature across districts using linear regression, scenario . Weatherscenario . For meaA profile of dengue impact across weather-space captures the highly focal nature of transmission with respect to the range of weather conditions observed in Peru . MaximumThe extreme sensitivity of incidence magnitude to the coupling of temperature and humidity was evident from more detailed examination of the highest incidence areas of Peru. We identified 6 areas of highest cumulative dengue incidence: Maynas and Alto Amazonas provinces and Ucayali, Madre de Dios, Tumbes, and Piura departments (referred to as areas of “elevated-risk”). See Weather-space profiles of cumulative incidence and maximum impact were developed for each of the elevated-risk areas individually Figs  and 6. Drd and 4th quartiles in ranking of seasonal transmission cycles per location; see Weather patterns varied across Peru and the timing of seasonal highs in temperature and humidity varied spatially. Locally, the presence of dengue disease was associated with a minimum annual duration above 22°C mean temperature as demonstrated by the binary classification tests., Moderate outbreaks to large epidemics were associated with local seasonal timing in which mean temperature ranged 25–29°C with highest incidence when humidity was concurrently >80%. In elevated-risk locations, week of seasonal peak incidence locally occurred when mean temperature was 26–29°C and minimum temperature was >21°C for 96% of moderate-large seasonal transmission cycles Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigMaps show the mean annual upper and lower limits for min temperature, mean temperature and mean humidity.(TIF)Click here for additional data file.S4 FigThe diagram summarizes level 1 of the classification tree using weather to classify Peruvian districts as dengue positive or negative for 2005–2012. Classification tests were based on minimum duration in which weather components exceeded specific thresholds annually per district. Best performing classification test provided specificity 0.95 and sensitivity 0.95 in identifying dengue positive/negative districts. See (TIF)Click here for additional data file.S5 FigLevel 2 follows the same logic applied to subgroups of dengue-positive districts.(TIF)Click here for additional data file.S6 FigSeasonal transmission cycles across the 6 locations were organized into 4 quartiles  according to seasonal incidence rates per location (see bottom panel). The relationship between timing of seasonal change-points Nadir, Onset, Peak and seasonal incidence rate magnitude per location is presented in the top 2 rows. Linear regression was performed on seasonal observations of timing vs magnitude and is summarized above by incidence quartiles. Areas with the strongest weather induced seasonal forcing  show a significant earlier occurrence of Nadir and Onset change-points in larger epidemics. Areas with less seasonal forcing,  do not show a timing effect for larger epidemics.(TIF)Click here for additional data file.S7 FigOptimal weather conditions for dengue transmission extend from Peru north and east across the Amazon Basin indicating an ecological DENV transmission link with neighboring countries.(TIF)Click here for additional data file.S8 FigWeather time series per district is shown for min temperature, mean temperature, and mean humidity for districts in lowest, median, and highest incidence rate groups among dengue-positive districts.(TIF)Click here for additional data file.S9 FigUpper panel represents all of Peru. Top row: Maximum dengue impact observed per weather interval is shown for all district-weeks across Peru, 2005–2007. Second row: The frequency distribution of weather intervals (where time is spent per week) annually is shown for all districts across Peru. Lower panel represents the 6 highest dengue incidence regions of Peru . Third row: Maximum dengue impact observed per weather interval is shown for all district-weeks across the 6 areas combined. Bottom row: The frequency distribution of weather intervals (where time is spent per week) annually is shown for all districts across the 6 highest dengue incidence locations. The shaded grey area indicates the weather-space for all of Peru. Grid interval is 1°C temperature and 2% humidity.(TIF)Click here for additional data file.S10 FigUpper panel represents all of Peru. Top row: Maximum dengue impact observed per weather interval is shown for all district-weeks across Peru, 2008–2010. Second row: The frequency distribution of weather intervals (where time is spent per week) annually is shown for all districts across Peru. Lower panel represents the 6 highest dengue incidence regions of Peru . Third row: Maximum dengue impact observed per weather interval is shown for all district-weeks across the 6 areas combined. Bottom row: The frequency distribution of weather intervals (where time is spent per week) annually is shown for all districts across the 6 highest dengue incidence locations. The shaded grey area indicates the weather-space for all of Peru. Grid interval is 1°C temperature and 2% humidity.(TIF)Click here for additional data file.S11 FigUpper panel represents all of Peru. Top row: Maximum dengue impact observed per weather interval is shown for all district-weeks across Peru, 2010–2012. Second row: The frequency distribution of weather intervals (where time is spent per week) annually is shown for all districts across Peru. Lower panel represents the 6 highest dengue incidence regions of Peru . Third row: Maximum dengue impact observed per weather interval is shown for all district-weeks across the 6 areas combined. Bottom row: The frequency distribution of weather intervals (where time is spent per week) annually is shown for all districts across the 6 highest dengue incidence locations. The shaded grey area indicates the weather-space for all of Peru. Grid interval is 1°C temperature and 2% humidity.(TIF)Click here for additional data file.S1 TextAdditional information is provided to describe methods and results related to analysis of the structure of seasonal dengue transmission cycles and timing of large epidemics.(DOCX)Click here for additional data file.


Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same region. BRCA2, as well as a recurrent missense variant in HOXB13 confer moderately increased disease risks; however, these explain only a limited fraction of the overall heritability (Prostate cancer (PrCa) is one of the most commonly diagnosed cancers and leading causes of cancer-related deaths for men in developed countries. An increased incidence of PrCa among first-degree relatives of patients, together with results from twin studies, provides strong evidence for a heritable component to PrCa . In recetability .MYC, which is associated with many forms of cancer including PrCa). However, in most cases, it is unclear whether these independent signals modulate risk through a common or separate functional mechanism, since the causal variants themselves remain unresolved. A key first step towards identifying the precise causal variants and functional mechanisms that confer risk is to comprehensively evaluate the evidence for association for non-genotyped potentially relevant variants within the region, to refine the original GWAS signal. In principle, re-sequencing associated regions in large case–control series would provide the most thorough data, but this approach is currently prohibitively expensive for routine application. However, since GWAS signals are expected to be predominantly driven by relatively common variants, large-scale genotyping together with imputation provides a cost-effective approach to evaluate the majority of likely causal variants. To date, only a small number of PrCa susceptibility loci have been fine-mapped. In analyses conducted by the PRACTICAL Consortium, for which the largest set of PrCa samples and genotype data are available, we previously identified for the KLK3 locus at Chr19q13 a more strongly associated missense coding variant that has been demonstrated to alter protein function . We used genotype data for 25 723 cases and 26 274 controls of European ancestry from two UK GWAS studies and from the 32 studies in the PRACTICAL Consortium genotyped using the iCOGS array. After imputation to a 1000 Genomes reference panel, data were available for 283 910 SNPs across these 64 regions. For 23 of the 64 regions the iCOGS array contained a dense panel of markers that included almost all variants correlated with the original GWAS hit, thereby facilitating particularly high-resolution interrogation of these loci.P < 5 × 10−8). For four of these variants, the association with PrCa had previously been reported only in East Asian populations , which may indicate that these variants modulate risk through a mechanism predominate among individuals with specific genetic backgrounds, or alternatively require further confirmation in Europeans through additional larger studies. For another variant that did not replicate at genome-wide significance in this study, rs1571801, the previously reported association with PrCa achieved statistical significance only in relation to aggressive disease (P = 5.1 × 10−4); this could reflect the fact that our sample panel was not enriched for aggressive disease. A further four regions had previously been identified in studies by the PRACTICAL Consortium; however, in each case, a larger sample size was available in the original study or consequent replication set than for this fine-mapping analysis. For these four SNPs , and the failure to replicate at genome-wide significance most likely reflects the smaller sample size. For the remaining six regions where the original index SNP did not reach genome-wide significance, our recent meta-analysis that included additional datasets , P-valur2 > 0.7 (LD list = 2202 SNPs). This best candidate list comprised 1623 SNPs across the 55 regions studied, with between 1 and 93 SNPs per association signal and a median of 13 candidate variants . These best candidate SNPs were annotated for overlap with functional elements in PrCa cell lines. For this analysis, bio-features were annotated according to the methodology used in Hazelett et al.. We also analyzed the best candidate SNP list for potential eQTLs using TCGA data available for prostate tissue and a much larger set of EuroBATS data for three different tissue types . To aid in the interpretation of data from this study, we developed Locus Explorer, a Shiny R application which allows the interactive graphical illustration of all the regions we have fine-mapped and which can be accessed at https://github.com/oncogenetics/LocusExplorer (manuscript in preparation). In the Locus Explorer plots for this study, we have included LD structure, statistical association data, functional annotation, gene transcripts and eQTL data; however, this application is customizable as required.For each of the 75 independent association signals identified across the 55 regions analyzed, we have selected a set of the most promising correlated candidate causal variants. Since a greater density of variants are interrogated during fine-mapping than through the ‘tag SNP’ approach used in the discovery phase of GWAS, the causal variants responsible for PrCa risk at each region would generally be expected to associate with PrCa at a similar level to the refined lead SNP, as well as exhibiting relatively high levels of linkage disequilibrium (LD). Consequently, we selected our ‘best candidate’ variants that most warrant further interrogation for functionality and PrCa causality for each association signal using an overlap between two criteria: likelihood ratio of ≥1/1000 relative to the refined lead SNP (‘1000 worse’ list = 6537 SNPs) and correlation with the lead SNP at LD tt et al.. Of our r2 > 0.7) or moderate LD (r2 = 0.3–0.7) with the original GWAS tag SNP; however, for five regions, the new lead SNP was only in weak LD with the original signal (r2 < 0.3). In 10 regions, the originally reported variant was excluded from our set of the best candidate SNPs to further investigate for possible causal functional effects. A good illustration of the refinement of the association signal within simple regions is at ChrXq12 where the original signal, rs5919432, was situated 71 kb 3′ of the androgen receptor (AR) gene. This has now been replaced by rs4446868 which is intronic to AR and in strong LD with a number of variants within the coding sequence, while the original tag SNP did not remain among our list of the best candidate functional variants . It may also be possible to further prioritize the 46 selected candidate SNPs at this region based on statistical and functional evidence; a cluster of five variants were more strongly associated than the remainder and these flank a known AR-binding site within intron 2 that has been reported to function as an enhancer in LNCaP ; both are intronic in FOXP4 and in LD with variants overlapping a number of bio-features. Our top European hit on Chr9q31 near RAD23B, rs1771718, is not correlated (r2 = 0.03) with the index SNP reported in the Chinese population and itself overlaps a DNase1 hypersensitivity site in the LNCaP PrCa cell line. Based on TCGA data, this signal is also an eQTL for RAD23B in normal prostate tissue but not tumor (Table Two of the simple regions had been reported previously as PrCa susceptibility variants only in East Asian populations (rs1983891 at Chr6p21 and rs817826 at Chr9q31); in this study, we have demonstrated for the first time that these loci are also associated with PrCa risk in individuals with European ethnicity. The lead SNP describing the association signal for Europeans at Chr6p21, rs6458228, is strongly correlated with the Japanese hit . At ITGA6 and in complete LD with the original index SNP rs12621278 (r2 = 1). This signal represents a tight cluster of four potentially causative variants. The novel additional independent lead SNP, rs12151618, is weakly correlated with this signal (r2 < 0.07) and situated 5 kb upstream of PDK1 (pyruvate dehydrogenase kinase isozyme 1) in the promoter region. PDK1 is believed to be upregulated by Myc and HIF-1 to facilitate cell survival and proliferation under hypoxia, and is reported to be commonly overexpressed in cancer cells . rs111770284 is intronic in ANO7 , overlaps a DNase1 site in LNCaP and implicates a set of four correlated variants as putative functional candidates. ANO7 is an androgen regulated gene and its expression appears to be prostate specific  and is correlated to two variants that overlap regulatory elements in LNCaP. It is also notable that the original hit rs3771570 was excluded from our list of best candidate causal SNPs after fine-mapping of this region.Two independent signals were also identified at Chr2q37, where the previously reported hit rs3771570 was intronic in specific ,24, withspecific . The secRAD51B but are not correlated with each other or with rs7141529. This region also harbors two independent risk signals for breast cancer, one of which is additionally associated with breast cancer in males; however, there is no correlation between these variants and any of the three PrCa risk SNPs .At Chr14q24, the original signal rs7141529 remained the most significantly associated variant after stepwise logistic regression; however, two further additional independent signals described by rs2189517 and rs17105852 were identified . In addition to these refinements of the original signals, we have identified a previously unknown third independent association described by rs2229295, which lies within the 3′UTR of HNF1B and may itself represent a strong candidate causal variant worthy of further investigation . Several of these eQTLs have been reported previously , implementing a mapping strategy for 19 of these regions which selected candidate causal variants ranging from 1 to 33 SNPs (C10orf32) showed evidence for ubiquitous eQTL association in all four tissue types interrogated. There were nine signals across seven regions which had at least one concordant eQTL between the TCGA PRAD tumor and any tissue type in EuroBATS. Of the eQTL signals which are confirmed in both TCGA and at least one EuroBATS tissue, five have been described before using the TCGA dataset   as robust candidates for functional follow-up, especially due to the large sample set used in the EuroBATS project . TTLL12 is a particularly interesting candidate susceptibility gene as its expression has been shown to increase during cancer progression and metastasis  . We thertastasis . In addiP = 2.01 × 10−20, Supplementary Material, Fig. S4). This enrichment was even stronger when only enhancer elements were included in the analysis (P = 1.67 × 10−27).To evaluate whether the refinement of previously identified PrCa loci that we have achieved in this study reflects a greater enrichment towards variants with annotated functionality, we assessed our list of 1623 best candidate SNPs for overlap with bio-features and compared this against the full set of imputed SNPs within the 55 regions that were significant in this analysis (243 627 SNPs). Using the hypergeometric test, we observed a significant enrichment in overlap with bio-features for the variants in our best candidate SNP list . Twenty-three of these regions were fine-mapped at very high resolution on the iCOGS chip. Nine previously reported GWAS signals were not replicated at genome-wide significance in this study due to either decreased power in comparison with the original studies or having only previously been associated with PrCa susceptibility in a non-European population.HNF1B. For the remaining 11 complex regions, this study provides the first evidence for the presence of multiple independent PrCa susceptibility variants in close genomic proximity to one another. An alternative explanation for the observation of multiple apparently independent association signals would be that both could in fact be moderately correlated with a single untyped causal variant, despite exhibiting limited LD between themselves ; however, our analysis helped to further refine these five regions and identified one additional previously unknown hit at Chr17q12 within the promoter region of emselves . We belir2 > 0.7) with the lead SNP based on 1 KG EU data. The intersection of these two selection criteria generates a list of 1623 variants, which we would expect to retain and be enriched for the causal functional variants. However, we cannot exclude the possibility that in some instances the causal variant(s) may be in lower LD with the novel index variants, particularly in the regions that were less densely genotyped. In addition, our imputed association data may be underpowered to detect any instances where rare causal variants give rise to the association signal through a ‘synthetic association’  has examined 69 risk regions among a multi-ethnic sample panel comprising European (8600 cases and 6946 controls), African (5327 cases and 5136 controls), Asian (2563 cases and 4391 controls) and Latino (1034 cases and 1046 controls) ethnicities. After performing a meta-analysis for marginal tests across multiple populations 12 regions were not significant, while a single novel, more significantly associated lead SNP was identified at 32 of the 57 significant regions (56.1%). In comparison, across the 55 regions that achieved genome-wide significance in our study, the original GWAS tag SNP was replaced with a more significantly associated SNP at 47 (85.4%). For the 46 significant regions that overlapped between these two studies’ final datasets, 32 of the refined SNPs identified by Han et al. were among our list of best candidate SNPs (69.5%). Within these overlapping regions, we identified 12 novel independent signals, 9 of which were nominally significant in the multi-ethnic fine-mapping but none were included in their top order or putatively functional SNPs. Comparing final putative functional candidates for the 46 overlapping regions, 29 (63.0%) of these regions have at least one overlapping functional candidate SNP. More notably, 11 of these 46 regions (23.9%) have a single overlapping functional candidate SNP, which should therefore be assigned high priority as potential causal candidates for future experimental follow-up. Overall, the comparisons between these two approaches highlight the increased power provided by the larger sample set available within our study; in particular in respect to the identification of multiple independent association signals within already known regions, but also demonstrate that the ability to incorporate multiple ethnic populations may further improve the efficiency of fine-mapping. This suggests that large meta-analysis based fine-mapping studies comprising individuals of diverse ancestries may represent the most robust strategy for imputation-based fine-mapping where such data are available.A concurrent PrCa fine-mapping study by Han In conclusion, we have demonstrated the importance of genotyping and imputation-based fine-mapping through the discovery of 12 additional independent PrCa associations within known GWAS regions and by refining the vast majority of the previously reported signals. Before fine-mapping efforts were employed, potential causal SNPs at each susceptibility locus were selected primarily on the basis of high correlation to the reported GWAS tag SNP  or overlet al., Hum. Mol. Genet., submitted). See Eeles et al. . Only the 40 659 iCOGS samples were used for the FRR estimation to avoid inflation from the Stage 1 GWAS.Analyses were based on data from the iCOGS array, a custom array comprising ∼200 000 SNPs designed to study susceptibility to prostate (PRACTICAL Consortium), breast and ovarian cancers , togethes et al.  for detaUK GWAS Stages 1 and 2 were genotyped using the Illumina Infinium HumanHap550 and a custom Illumina iSELECT array, respectively ,40.iCOGS genotyping was conducted using a custom Illumina Infinium array (iCOGS) in seven centers, of which five were utilized for PRACTICAL. Genotypes were called using Illumina's proprietary GenCall algorithm. In addition to SNPs selected for replication of GWAS, the iCOGS array included dense sets of SNPs surrounding susceptibility variants known at the time of design. For PrCa, we included 23 such regions (ccge.medschl.cam.ac.uk/research/consortia/icogs/). To select markers for comprehensive interrogation of these densely genotyped regions, we identified all known SNPs from the March 2010 release of the 1000 Genomes Project with MAF > 0.02 in Europeans and selected all SNPs that were correlated with the published GWAS SNPs (at r2 > 0.1); together with a set of SNPs designed to tag all remaining variants at r2 > 0.9. Approximately 14 000 SNPs were successfully designed across these regions.The primary genotype data were used to impute genotypes for ∼17 M SNPs/indels using the 1000 Genomes Project (March 2012 release) as a reference panel and IMPUTE V2 . ImputatP ≤ 10−5 level. Performing this comparison revealed that an association P = 10−5 was sufficiently strict for the avoidance of false-positive results in the discovery of secondary signals within a region after adjusting for the top signal variant.To establish a suitable significance threshold, we examined 59 known breast cancer regions with no known association with PrCa prior to this analysis. Regions were defined as a ±500 kb boundary around the published breast cancer GWAS SNPs. We interrogated our meta-analysis results (iCOGS and Stages 1 and 2 UK GWAS) in order to establish the likelihood that SNPs within these breast cancer regions would be associated with PrCa by chance at P ≤ 10−5 from the fine-mapping dataset  and performed a stepwise logistic regression on this set of SNPs for each region. For regions in which the initial analysis indicated more than one independent SNP, a second round stepwise logistic regression was performed to confirm the presence of additional independent SNPs, after adjusting for the best signal in the region. SNPs with P ≤ 10−5 from the adjusted results were included in this analysis, alongside the top hit. For regions found to contain multiple independently associated SNPs, haplotype-specific odds ratios were estimated based on the independent SNPs identified through the stepwise logistic regression analysis, using the Haplo.Stats package (http://cran.r-project.org/web/packages/haplo.stats/index.html)  to trim the list where there was a second signal that did not reach the genome-wide significance level (5 × 10−8).To identify independent association signals within a region, we selected all significant SNPs with ex.html) . SNPs wiex.html) . This liThe contribution of SNPs to the familial risk of PrCa, under a multiplicative model, was computed using the formulak. For a single SNP, k, j for the multi-locus region k and where  to be 2 ,47, and https://github.com/oncogenetics/LocusExplorer).We developed Locus Explorer, a Shiny web application for R, to generate the locus plots shown in this manuscript. Locus Explorer can be run locally using R studio with the necessary packages installed. The code is available on GitHub. The application is interactive and enables selecting and zooming on the locus, selecting annotation tracks and downloading the resulting plots and the data used for plotting. Data for plotting are stored in SQLite database which is accessed using R scripts. Plots are displayed mainly using RStudio, sqldf, ggplot and ggbio –51. At tWe used a number of publicly available prostate epithelia and PrCa ENCODE datasets of chromatin features to identify putative regulatory regions at each risk locus ,52. The P-values obtained for each risk variant were corrected for the number of transcripts in the interval using Benjamini–Hochberg method. Significant associations were defined as a false discovery rate < 0.05.The genotypes of the variants in the best candidate SNP list in 145 prostate tumor samples and 45 normal tissue samples were downloaded from TCGA database (Feb 2013). All samples were verified as Caucasian descendants. If a variant was not represented in the TCGA data, the genotypes were imputed using IMPUTE2. A cis-eQTL analysis was performed for these variants and any transcript within a 1 Mb interval (500 kb on either side). The abundance of the transcripts is adjusted for somatic copy number changes and CpG methylation changes using methods described previously. The nominal N = 814), skin (N = 716) and adipose tissue (N = 766) derived simultaneously from a subset of well-phenotyped healthy female twins , Cancer Research UK Grants C5047/A7357, C1287/A10118, C5047/A3354, C5047/A10692, C16913/A6135 and The National Institute of Health (NIH) Cancer Post-Cancer GWAS initiative grant: No. 1 U19 CA 148537-01 (the GAME-ON initiative). Funding to pay the Open Access publication charges for this article was provided by the Institute of Cancer Research.This work was supported by


Drosophila model of HD, expression of mutant Huntingtin (Htt) protein with expanded polyQ leads to formation of inclusion bodies (IBs), increase in cellular toxicity, progression of motor disabilities and reduced viability. Multiple cellular events such as oxidative stress, mitochondrial dysfunction, inflammation and transcriptional dysregulation are reported to contribute to pathology, however, till date there are no disease-modifying treatments with least side effects. Therefore, we investigated effect of the phytochemical curcumin on HD pathogenesis. Curcumin, a phytochemical and commonly used ingredient in Asian food has a wide spectrum of anti-oxidant, anti-inflammatory and anti-fibrilogenic properties. In this study, we provide evidence that curcumin significantly ameliorates disease symptoms in a Drosophila model of HD by suppressing cell death and can be a key to halting the progression of Huntington’s disease with least side effects.Huntington’s disease (HD) is a progressive, dominantly inherited neurological disorder caused by an abnormal expansion of polyglutamine (polyQ) repeat within the Huntingtin (Htt) protein with no disease modifying treatments. In a  Huntingtin (Htt) is a large 350 kDa protein which is expressed ubiquitously and normally carries a repeat of ~8–25 glutamines in its N-terminal portion2Currently, there is no cure or effective therapy for HD or other neurodegenerative diseases, but pivotal studies suggest tight correlation between cell death and neurodegenerative diseases. Therefore, identification of new therapeutic strategies aimed to modulate cell death for preventing or suppressing disease progression may define the desired outcome. One of the major drawbacks of currently available treatments for polyQ diseases is that they result in multiple side effects and only a few of them can alleviate disease symptoms. Therefore, we focused on testing a phytochemical ‘curcumin’ that could counteract multiple pathways specially cell death involved in Huntington’s disease without any serious side effects.A comprehensive understanding of the molecular aspects and pathophysiology of polyQ diseases is necessary for finding potential therapeutic drugs or treatments which can slow down disease progression or delay disease onset. Although the mechanisms through which aggregation-prone mutant proteins cause neurodegeneration remain unresolved, there are well-established links demonstrating their roles in dysregulation of multiple cellular processes and functions such as transcription, cell signaling, mitochondrial function, oxidative stress, inflammation and apoptosis456Curcumin -1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5-dione), a polyphenolic compound with an outstanding safety profile is the major bioactive component of turmeric, a spice commonly used in Indian and Asian food and medicine. Multiple therapeutic beneficial effects of curcumin, which could be linked to its ability to act as a strong antioxidant, anti-inflammatory phytochemical and anti-protein aggregation effect have been reported during the last 10 years. It has been extensively used in the treatment of several medical conditions like cystic fibrosis, cancer, gastric ulcers, liver diseases and arthritis. Curcumin is also reported as a preventive measure for prevention and treatment of Alzheimer’s disease (AD)89Drosophila model of polyQ diseases to test the efficacy of curcumin in alleviating disease symptoms. Transgenic Drosophila has proven to be an excellent model system to study neurodegenerative diseases because it replicates most of the features of the disease condition, for instance, late-onset, decreased lifespan, neurodegeneration, motor dysfunction and progressive accumulation of aggregates in cytoplasm and neurites41314In this study, we used Drosophila16The exon1 fragment of mutant Htt with 93 glutamine residues (Httex1p Q93) as well as expanded polyQ peptides alone are intrinsically cytotoxic and cause early adult death and neurodegeneration in Drosophila expressing Httex1p Q93 and Q48 peptides. We also found that curcumin ameliorates extensive degeneration and external dysmorphology of eye caused by expression of Httex1p Q93 in all cells of the Drosophila eye. Our results indicate that in addition to reducing neuronal loss, curcumin improved polyQ induced motor neuronal dysfunction. Moreover, dietary curcumin did not affect the integrity of photoreceptor cells of adult eye and motor neuron function of transgenic Drosophila expressing the first exon of Htt with a normal-range repeat of 20 glutamine residues (Httex1p Q20).Here, we show that curcumin suppresses degeneration of photoreceptor neurons, indicative of disrupted internal eye architecture, in transgenic Since apoptosis has been linked to neuronal death in polyQ diseases including HD318+mW.hs = GawB} elavC155 and eye-specific gmr driver w; P [w + mC = GAL4-ninaE.GMR] 12. Appropriate crosses were carried out in order to obtain desired genotypes. Curcumin (Sigma Aldrich) was mixed in fly food at desired concentration  for growing experimental larvae or flies while controls were reared in normal food devoid of drug at 25 °C. DMSO (Sigma Aldrich) was supplemented in equal amounts in all the food conditions.The polyglutamine expressing transgenic lines used in the present study were w; P {UAS-Httex1p Q20}, w [*] P {w [+mC] = UAS-Q48.myc/flag} and w; P {UAS-Httex1p Q93}4F117Yeast paste with blue colour  and curcumin at desired concentration (as above) was placed centrally on a petri dish (100 mm × 10 mm) plated with 3.3% (wt/vol) agar. Two groups of 10 feeding early third-instar larvae for each condition were transferred to blue yeast paste and allowed to feed for 2 h. After feeding, each group of larvae were washed in distilled water and dried. Larvae were then homogenised in PBS, centrifuged at 35,000 rpm for 10 min and absorbance at 625 nm was read.The apparatus was a petri dish of 100 mm × 10 mm dimensions composed of 3.3% agar. A track of 2 mm wide, 30 mm long and 5 mm deep was created in the petri dish. The distance covered in 30 s by each wandering third-instar larva placed in the track was recorded. A total of 10 larvae per condition were monitored for two trials.The locomotor ability of flies was monitored by their ability to climb up a vertical glass tube . A group of 10 flies were gently tapped to the bottom of the tube. The number of flies that could climb a height of 10 cm within 15 s was recorded. For each condition, 2 groups of 10 flies each were tested in the marked tube for a total of 6 trials.This assay allows rapid visualization of rhabdomere arrangement in the ommatidia of the compound eye which is a direct measurement of the number of surviving photoreceptor neurons. The decapitated head of adult fly was mounted on a microscope slide and the eyes were analyzed under Nikon Eclipse (Ni-E) microscope with 50X oil objective. A total of 6 eyes, a minimum of 200 ommatidia were scored per condition and the number of visible rhabdomeres was counted for each ommatidium.gmr-GAL4 were grown at 25 °C in normal food and food containing 5 or 10 μM curcumin. The external surface of fly eye was examined at days 1, 7 and 14 post-eclosion with a Nikon SMZ745T microscope, and a total of 30 flies were analyzed for each condition. Photographs of at least 5 fly eyes per condition were taken using a Nikon SMZ1500 microscope.Flies expressing Httex1p Q93 driven by Acridine orange (AO) staining was performed to observe apoptotic cells in the eye imaginal discs of third-instar larvae. Larvae were dissected in PBS and eye discs were incubated in 5 μg/ml acridine orange (Himedia) solution for 3 min followed by rinsing in PBS. At least 5 discs for each condition were then photographed immediately using a Nikon Eclipse (Ni-E) fluorescent microscope and analyzed with NIS-elements AR software.Statistics were performed in IBM SPSS statistical package (version 22). Normality of data was assessed by Shapiro-Wilk tests and equal variance using Levene’s test. For normal distribution, statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. If data were not normally distributed, a Kruskal-Wallis test was performed followed by Mann-Whitney U test using Bonferroni correction to adjust the probability.elav-GAL4 driver. In other words, normal as well as diseased larvae fed curcumin at different doses did not show differential feeding behaviour.The vital sense of taste allows animals to prefer palatable and avoid non-nutritious or toxic food. In an attempt to establish a foundation for drug feeding experiments, subsequent interpretation and analysis of results, we monitored larval feeding behaviour. We allowed feeding larvae 2-hour accessibility to a blue-dyed assay plate supplemented with curcumin at various doses and the amount of food consumed was then estimated spectrophotometrically. We found that all concentrations of curcumin including the higher ones had no effect on food intake of larvae expressing normal-length Httex1p Q20  and expaNeurodegeneration is most readily observed in the fly compound eye composed of approximately 750 repeating units, called ommatidia, each containing eight photoreceptor neurons. These photoreceptor cells produce a stack of tightly packed microvilli that form a photosensitive structure called the rhabdomere. A popular assay of neurodegeneration, commonly called the pseudopupil method, involves counting the photoreceptor neurons of the fly compound eye. These photoreceptor neurons produce a repeating trapezoidal arrangement of 7 visible rhabdomeres in each ommatidium13elav-GAL4. Httex1p Q20 flies grown in normal or curcumin-supplemented food exhibited the seven characteristically arranged rhabdomeres in each ommatidial unit irrespective of different doses  of the drug administered  under the control of nistered . Rearingnistered  or only nistered  suppressDrosophila eye development provides a sensitive assay for studying the effects of polyQ expansion on cell differentiation and viability of both neuronal and non-neuronal cells. The cells that are predestined to form the eye grow during the larval period as uncommitted cell types. A morphogenetic wave passes across the eye during the third-instar stage and as the wave passes, cells become committed to particular fates in the eyeelav and gmr-GAL4 are two widely used drivers for neurodegeneration studies. The elav promoter expresses GAL4 in all cells of the nervous system beginning from embryogenesis and continues into adult lifegmr promoter is expressed in the differentiating photoreceptor neurons and pigment cells in the third instar eye imaginal discelav promoter can lead to neurodegeneration but it does not affect external morphology. However, gmr-mediated expression of the mutant proteins causes massive degeneration of the eye and overt external dysmorphology162425The gmr-GAL4 driver to express Httex1p Q93 transgene in the fly compound eye. Our results clearly indicate that administration of 10 μM curcumin from early larval stage can mitigate Httex1p Q93 induced cytotoxicity characterised by visible external morphological defects such as rough eye phenotype with necrotic lesions and loss of pigmentation  has no toxic effect on its own13To quantify locomotor dysfunction, Httex1p Q93 and Q48 larvae were fed increasing concentrations of curcumin and a simple measure of locomotor function called the ‘crawling assay’ was performed. We found that feeding curcumin improved the crawling ability of both Httex1p Q93 and Q48 larvae, in a concentration-dependent manner. Marked improvement in larval crawling behaviour was observed at 10 μM dose of curcumin ,b.For addressing motor function of adults, the climbing ability of flies grown on food supplemented with different doses of curcumin was evaluated at days 1, 3 and 7 post-eclosion. Curcumin showed no detectable effect on motor ability of Httex1p Q20 flies reared on curcumin-containing food since larval stage . On the Apoptosis, or programmed cell death, is an intrinsic and highly organized form of cell death which is common in various biological processes and pathological conditions. Aberrant apoptosis in the central nervous system has been implicated in the pathogenesis of a broad array of neurological disorders, including polyQ diseases19283301928elav-GAL4-driven Httex1p Q93 eye discs from third-instar larvae fed normal or curcumin-supplemented food  or expanded polyQ peptide (Q48) alone which is free of any disease gene context. Rearing on curcumin-supplemented diet improved different neurodegeneration phenotypes, viz. progressive degeneration in the eye, eye dysmorphology, organisation of rhabdomeres, and impaired motor function in both Httex1p Q93 and Q48 transgenic Drosophila. Since polyQ diseases share common pathogenic features, curcumin is a promising agent for the prevention or treatment of polyQ disorders including HD. We demonstrate that dietary curcumin administered since larval stage or only during adult period ameliorates compromised functioning of neurons. Our results indicate that curcumin suppresses neurodegeneration when it is being inflicted and markedly retard further neuronal degeneration. Continuous synthesis of expanded polyQ protein and neuronal damage during the non-feeding pupal period may have reduced the extent of recovery in eye morphology and motor function.The prevalence of neurodegenerative diseases among people living in the Asian subcontinent, where people regularly consume spices, is lower than in western countries. Research over the last 10 years has shown that phytochemicals derived from several spices target inflammatory pathways and may be effective in treating neurodegenerative diseasesDrosophila expressing expanded Httex1p Q93 protein fragment driven by the pan-neuronal elav-GAL4. We found a substantial reduction in polyQ-induced cell death in third-instar eye discs due to administration of curcumin which has been implicated in alleviating cytotoxicity, oxidative stress and apoptotic cell death in neurological disorders32333435The identification of genetic and environmental factors responsible for neurodegenerative diseases has augmented evidences for a common pathway of neuronal death viz. apoptosis that involves oxidative stress, perturbed calcium homeostasis, mitochondrial dysfunction and caspase activationPolyQ pathology is complex and may affect multiple cellular events. Therefore, administration of therapeutic agents with least side effects that target multiple cellular processes and functions are postulated to achieve better relief from pathology than the mono-targeted agents. Phytochemical curcumin is known to cross the blood-brain barrier, has an excellent safety profile, number of pleiotropic properties and therefore, has tremendous potential for neuroprotective efficacy. Our findings strongly suggest that curcumin ameliorates polyQ-induced compromised neuronal function, neurodegeneration, and cytotoxicity and can be a promising candidate for the treatment of polyQ disease patients with least side effects.How to cite this article: Chongtham, A. and Agrawal, N. Curcumin modulates cell death and is protective in Huntington′s disease model. Sci. Rep.6, 18736; doi: 10.1038/srep18736 (2016).


Parvovirus B19 virus commonly causes subclinical infection, but it can prove fatal to the fetus during pregnancy and cause severe anemia in an adult with hemolytic diseases. We present the case of a woman with autoimmune hemolytic anemia who was diagnosed with parvovirus B19-induced transient aplastic crisis during her second trimester of pregnancy and faced the high risk of both fetal and maternal complications related to this specific viral infection. To the best of our knowledge, the experience of successful intravenous immunoglobulin treatment for B19 virus infection during pregnancy, as in our case, is limited.A 28-year-old and 20-week pregnant Chinese woman with genetically confirmed alpha-thalassemia trait was diagnosed with cold antibody autoimmune hemolytic anemia and suffered from transient aplastic crisis caused by B19 virus infection. She received intravenous immunoglobulin treatment to reduce the risk of hydrops fetalis. Her peripheral blood reticulocyte percentage recovered, but anemia persisted, so she underwent several courses of high dose intravenous dexamethasone for controlling her underlying hemolytic problem. Finally, her hemoglobin levels remained stable with no need of erythrocyte transfusion, and a healthy baby boy was naturally delivered.Parvovirus B19 virus infection should be considered when a sudden exacerbation of anemia occurs in a patient with hemolytic disease, and the possible fetal complications caused by maternal B19 virus infection during pregnancy should not be ignored. Close monitoring and adequate management can keep both mother and fetus safe. Most parvovirus B19 virus (B19 virus for short in this report) infection causes asymptomatic or only mild illness, such as erythema infectiosum, polyarthropathy syndrome or transient reticulocytopenia in healthy adults or children, but can sometimes be responsible for life-threatening diseases. The cytopathic effect of B19 virus in patients with rapid erythrocytes production results in profuse extinction of proerythroblasts. Failure of differentiation from proerythroblast into later stage erythroid precursors leads to transient aplastic crisis (TAC) . Here weThis 28-year-old and 20-week pregnant Chinese woman with genetically confirmed alpha-thalassemia trait and a current obstetric record of gravida 3, para 0, artificial abortion 1 and ectopic pregnancy 1 (G3P0AA1E1 by Gravida/para/abortus (GPA) system) presented to our emergency unit (EU) due to headache, chills, fever and general soreness for one day. At our EU, her physical examination revealed a fever up to 38.5°C, mild tachypnea with a respiratory rate up to 21 breaths/min and bilateral lower back knocking pain. Her obstetric ultrasound revealed 20 weeks gestational age, normal placenta location and a fetal heart beat between 140 and 150/min. No vaginal bleeding was noted.3 with an absolute neutrophil count (ANC) of 1780/mm3, hemoglobin 5.5g/dL, mean corpuscular volume (MCV) 119.4fl and platelet count 116,000/mm3. Aggregation of erythrocytes  and severe peripheral blood pancytopenia: white blood cell (WBC) count 2200/mmHer initial bone marrow cytology showed myeloid hyperplasia and only very few erythroid precursors with erythroblasts in abnormal megaloblastic change Figure , some ofTAC caused by B19 virus infection in a pregnant woman with cold antibody autoimmune hemolytic anemia (AIHA) was diagnosed. She received intravenous immunoglobulin (IVIG) injection , 0.4gm/Kg/day for five days, for eradication of viremia and prophylaxis of occurrence of hydrops fetalis. Although her peripheral blood reticulocyte percentage increased dramatically after IVIG treatment Figure , her hemWhether our patient’s B19 virus in her second trimester pregnancy was a new infection or a reactivation of a persisting previous infection cannot be surely answered, since the persistence of infection in the bone marrow has been reported in immunocompetent individuals several years after primary infection . B19 virMost patients with TAC had a good prognosis after erythrocyte transfusion and adequate monitoring until their bone marrow recovered, while IVIG is recommended for patients at risk of severe complications or with immunodeficiency ,4,5. RapIn our patient, a 20-week pregnant woman with alpha-thalassemia trait and autoimmune hemolytic anemia, B19 virus infection not only caused TAC in the mother, but could also have led to severe anemia, hydrops and even death of the fetus, as cautioned by experts when maternal infection occurred between 17 and 24 weeks of gestation . TherefoAlthough corticosteroid treatment in the first trimester is well-known to increase the risk of fetal orofacial clefts, and the safety of mother and fetus receiving corticosteroids for treating idiopathic thrombocytopenic purpura in the third trimester of pregnancy, even with high dose intravenous methylprednisolone, was previously confirmed , the expB19 virus infection in a pregnant woman with increased erythropoietic demand from alpha-thalassemia trait and cold AIHA resulted in a TAC in the mother and put the fetus at risk of development of hydrops fetalis. Prompt IVIG injection eradicated the virus rapidly, resolved TAC in the mother and prevented further dangerous fetal infection. Supportive erythrocytes transfusion and intensive high dose dexamethasone treatment maintained hemoglobin at stable levels. Safe delivery of a healthy baby was happily achieved.Both my husband and I felt satisfactorily about the rapid recovery of my hematopoietic function and the safe delivery of a healthy baby boy. We would like to express our great appreciation to our doctors for their efficient and correct diagnosis and prompt management. Although the rather expensive intravenous immunoglobulin was not reimbursed for transient aplastic crisis according to the general health insurance policy in our country and we had to pay for it by ourselves, we thought it was worthwhile since nothing was more important than our health.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Hence, individuals living in natural disaster prone areas or have received early warning for an impending disaster may consider owning/be provided with a pre-emergency nutritional pack for self-sufficiency. In addition, collapsed structures may entrap victims in confined spaces with limited oxygen supply and faces the danger of hypercapnia, so a hypothetical example of a 20 year old healthy male in 14 day entrapment was used to demonstrate the dietary, CO2 output and CO2 scrubbing requirements. A commercially available 20 g protein bar and lithium hydroxide (LiOH) powder were used in the calculations to show how the content of such a pre-emergency pack can be tailored.Populations affected by natural disasters usually rely heavily on search and rescue operations and relief supplies to sustain their road to recovery. It is observed that few survivors are recovered after 2 weeks2 emission of an adult.Calculations based on published sources of dietary requirements and CO2 output from the entrapped 20 year old healthy male weighing 70 kg through 14 days in a 10 m3 space. The CO2 scrubbing capacity of this pre-emergency pack should also scrub any CO2 produced through respiration by the victim during this period of entrapment. Any dietary and/or CO2 scrubbing alternatives can be evaluated against these requirements and selected into the pre-emergency pack.The dietary requirements in Table Prevention is better than cure in most, if not all, harmful situations. This novel pre-emergency survival pack is a convenient "first aid kit" for natural disasters. A carefully designed pre-emergency pack can be easily adapted to the unique conditions of various disasters and enhance the survivability of a victim entrapped under debris at a site of natural disaster. Early distribution of pre-emergency survival packs will also ensure isolated rural dwellers to be self-sufficient, during the aftermath of a natural disaster, while awaiting arrival of relief supplies.


Two lineages, epithelial, and myoepithelial cells are the main cell populations in the normal mammary gland and in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures obtained from fresh breast tissue are a powerful tool to study more reliably new aspects of mammary gland biology, including normal and pathological conditions. Nevertheless, the methods described to date have some technical problems in terms of cell viability and yield, which hamper work with primary mammary cells. Therefore, there is a need to optimize technology for the proper isolation of epithelial and myoepithelial cells. For this reason, we compared four methods in an effort to improve the isolation and primary cell culture of different cell populations of human mammary epithelium. The samples were obtained from healthy tissue of patients who had undergone mammoplasty or mastectomy surgery. We based our approaches on previously described methods, and incorporated additional steps to ameliorate technical efficiency and increase cell survival. We determined cell growth and viability by phase-contrast images, growth curve analysis and cell yield, and identified cell-lineage specific markers by flow cytometry and immunofluorescence in 3D cell cultures. These techniques allowed us to better evaluate the functional capabilities of these two main mammary lineages, using CD227/K19  and CD10/K14  antigens. Our results show that slow digestion at low enzymatic concentration combined with the differential centrifugation technique is the method that best fits the main goal of the present study: protocol efficiency and cell survival yield. In summary, we propose some guidelines to establish primary mammary epithelial cell lines more efficiently and to provide us with a strong research instrument to better understand the role of different epithelial cell types in the origin of breast cancer. The mammary gland is a tubuloalveolar structure consisting of a branching network of ducts ending in terminal ductal lobular units (TDLUs) that constitute the functional domains of the pre-menopausal breast. These ducts are composed of two continuous layers of primary epithelial lineages: the inner part is comprised of luminal epithelial cells that can produce milk during lactation; the outer layer contains myoepithelial cells that provide the ducts with contractile ability  + PSF   (for the digestion mixture)Sterilized surgical material Sterile petri dishes or metal surface to chop the tissueBlender 1 L sterile glass bottle2 L beakerBSA (Bovine Serum Albumine) (Sigma)Collagenase type IV (Sigma C-5138)Hyaluronidase (Sigma H-3506)Cell culture dishes and tubesPBS  500 μm Filters (Corning)250, 100, 40, and 20 strainers (Millipore)Fibroblasts culture medium: DMEM + 10% Fetal Bovine Serum (FBS) + 1% PSF + Glutamax Epithelial and organoid culture medium: M87A [formula previously described 0.25% Trypsin-EDTA (Life Technologies)The human protocol was approved by each institutional review board. Fresh healthy mammary tissue from women between 20 and 60 years old was obtained from 13 patients who underwent reduction mammoplasty and 2 patients who had a mastectomy after breast cancer in the contralateral breast. The samples were obtained under the approval of the Institutional Review Board of the Hospital Clinic and Clinica Planas in Barcelona.Between 80 and 150 g of fresh mammary tissue was used as starting material for the tissue digestion. Samples had to be handled in sterile conditions. In some cases, samples were kept at 4°C for 24 h before being processed, a step that slightly reduced the efficiency of the methodology. Fatty tissue was manually separated from breast areas that were rich in ducts, using sterile scalpels and forceps , supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 5 μg/mL Fungizone (PSF) to prevent the tissue from drying out. Additionally, breast tissue pieces were chopped and grinded in the presence of supplemented DMEMF12 medium using a food processor, until the biggest tissue chunks had disappeared and the remaining pieces' size was between 1 and 4 mm.The processed tissue was placed in a 1 L sterile glass bottle with a stir bar, in the presence of digestion buffer containing supplemented DMEMF12 medium (1% PSF), 250 U/ml of collagenase Type IV, 1400 U/ml of hyaluronidase and 2 mg/mL Bovine Serum Albumin (BSA)  acinar structures, and the preservation of representative antigens was also evaluated. Briefly, 100,000 cells per well were seeded on top of matrigel in a 24-well plate, as published elsewhere  and CD10  as it has been described that their expression is maintained during cell culture using M87A medium , CD45 and CD146  were located in the edges surrounding the epithelial cells (K19+)  and epithelial (CD227+) cells, was analyzed by flow cytometry  Figure . Moreovey Figure . It was y Figure . This try Figure . Even ifThe qPCR Figure  indicateRegarding cell growth, the general idea inferred from the imaging analysis was confirmed by the study of growth curves. Slow digestion combined with the differential centrifugation technique gave rise to cells that presented higher population doublings per day than cells from the other methods studied. In every case analyzed, the growth curve was exponential. However, cells obtained by slow digestion coupled with differential centrifugation had a steeper slope Figure , which iIn order to understand breast cancer etiology, it is crucial to comprehend the behavior of the cells in the normal human mammary breast gland. For this purpose, it is important to work with primary epithelial cells, since commercially sold cells present genetic alterations that distance them from the cells found in normal breast tissue (Burdall et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting. The majority of ROS found in aerobic cells are generated during normal cellular metabolism . W. WPOLB i lesions b [59]. W lesions . Taken tGiven the dual function of MMR in removing both incorrectly placed bases and oxidatively damaged nucleotides, it is inherently difficult to dissect the separate impact of these two roles upon cancer development. The mutator phenotype is clearly the driving force behind carcinogenesis in many MMR-deficient tumours but does reduced 8-oxoG removal also contribute, either via increased MSI or independently?  et al. tested to what extent oxidative DNA damage played a role in the MMR mutator phenotype by expressing MTH1 in MSH2-deficient MEFs; the resulting decrease in DNA 8-oxoG levels translated into a decrease in the mutator effect [et al. reported that growing the MLH1 deficient CRC cell line HCT116 in the antioxidant ascorbate, both with and without H2O2 treatment, significantly reduced mutation rates and reduced MSI by 30% [2O2 than their MMR-proficient counterparts (HCT116+Chr3) [2O2 [Msh2−/− mice displayed a 22.5-fold increase in tumour incidence [Few studies have examined the specific role of oxidative damage repair by the MMR pathway in relation to tumourigenesis. Colussir effect . Glaab eI by 30% . Convers16+Chr3) ,63. Thisr3) [2O2 . It has r3) [2O2 . Upon trncidence ,65.The evidence thus far suggests that the MMR system may supresses carcinogenesis in the context of oxidative damage by directly repairing ROS induced DNA lesions or acting as a sensor of oxidative damage, thereby activating apoptosis.Clinically, it would seem logical to use anti-oxidant or ROS scavenging agents in the treatment of cancers since oxidative DNA damage has been shown to induce mutagenesis. However, several large studies testing a variety of drugs  have not been able to definitively confirm that this approach is effective . This ar in vitro study examining the ROS inducing agent parthenolide and its soluble analogue dimethylamino parthenolide in triple negative breast cancers has shown that these drugs activate NADPH oxidase leading to the production of superoxide anions, and depletion of glutathione and in vivo studies showed a significant reduction in tumour growth and increased survival [Several conventionally used chemotherapeutic agents are known to increase ROS and oxidative stress in addition to their main mechanism of action, including anthracyclines, platinum agents, epipodophyllotoxins and camptothecins ,68. Thersurvival . The addsurvival .The ROS inducing drug, β-Lapachone is currently being studied in a phase 2 clinical trial (NCT01502800). Pre-clinical studies suggest that β-Lapachone exerts its cytotoxic effect by undergoing futile redox cycles on the detoxifying enzyme NAD(P)H:quinone oxidoreductase (NQO1). NQO1 levels are known to be elevated in several solid tumours and in these cancers, β-Lapachone results in excessive ROS formation . Severalet al. have shown that several conventionally used antibiotics produce ROS as part of their mechanism of action [E. coli were increasingly resistant to treatment with these ROS-inducing antibiotics [We have previously shown that treatment with the drug methotrexate is selectively lethal with MSH2-deficient cell lines, due to an increased susceptibility to oxidative stress . The levf action . Upon Muibiotics . The autConcerns have been raised regarding modulating ROS homeostasis to treat tumours. Although ROS can cause oxidative DNA damage, it is also involved in the regulation of molecules involved in tumour biology and therefore could play a role in inhibiting carcinogenesis. There are therefore worries that antioxidants could promote tumourigenesis . ExcessiAlthough there is certainly evidence suggesting a role for the MMR pathway in the repair of oxidative DNA damage, the precise nature of this pathway is far from being fully clarified. Despite this, we and others have utilised high throughput screens to identify several molecules, drugs and compounds which are synthetically lethal with MMR deficiency as a consequence of increased oxidative DNA damage. These approaches provide a step towards understanding the complex link between the MMR pathway, oxidative damage and cancer as well as identifying promising new treatments and drug targets for MMR-deficient tumours.


Background: Popular beliefs that breakfast is the most important meal of the day are grounded in cross-sectional observations that link breakfast to health, the causal nature of which remains to be explored under real-life conditions.Objective: The aim was to conduct a randomized controlled trial examining causal links between breakfast habits and all components of energy balance in free-living humans.Design: The Bath Breakfast Project is a randomized controlled trial with repeated-measures at baseline and follow-up in a cohort in southwest England aged 21–60 y with dual-energy X-ray absorptiometry–derived fat mass indexes ≤11 kg/m2 in women (n = 21) and ≤7.5 kg/m2 in men (n = 12). Components of energy balance  and 24-h glycemic responses were measured under free-living conditions with random allocation to daily breakfast  or extended fasting  for 6 wk, with baseline and follow-up measures of health markers .Results: Contrary to popular belief, there was no metabolic adaptation to breakfast , with limited subsequent suppression of appetite . Rather, physical activity thermogenesis was markedly higher with breakfast than with fasting . Body mass and adiposity did not differ between treatments at baseline or follow-up and neither did adipose tissue glucose uptake or systemic indexes of cardiovascular health. Continuously measured glycemia was more variable during the afternoon and evening with fasting than with breakfast by the final week of the intervention .Conclusions: Daily breakfast is causally linked to higher physical activity thermogenesis in lean adults, with greater overall dietary energy intake but no change in resting metabolism. Cardiovascular health indexes were unaffected by either of the treatments, but breakfast maintained more stable afternoon and evening glycemia than did fasting. This trial was registered at www.isrctn.org as ISRCTN31521726. EpidAlthough much is known about the acute metabolic response over the hours after a prescribed morning meal –18, longPhysical activity thermogenesis is undoubtedly the most malleable component of energy expenditure, yet the few studies that specifically contrasted the relative presence or absence of regular daily breakfast consumption were either not designed to measure physical activity levels , 26 or w2 for women and ≤7.5 kg/m2 for men , and the procedures followed were in accordance with the protocol approved by the National Health Service South-West 3 Research Ethics Committee (10/H0106/13). This protocol has since been published in full , with tr521726, a for men , for who14C)-d-glucose uptake]. Participants then undertook an oral-glucose-tolerance test, which involved ingesting 75 g of glucose polymer in solution  with 5-mL blood samples drawn every 15 min for 2 h.Baseline demographic and anthropometric characteristics of those who completed the trial are presented in All the above measures were followed up 6 wk later, with free-living assessments of energy intake (estimated from directly weighed food diaries) and energy expenditure  monitored throughout the first and last week of intervention, along with continuous  monitoring of interstitial glucose concentrations via a subcutaneous abdominal catheter  both to document chronic glycemic responses and to verify compliance. Eumenorrheic women provided baseline samples 2 wk before the start of the 6-wk intervention so that follow-up samples could be acquired 3–10 d after the onset of menses . During the 6-wk intervention, participants were randomly assigned  to either a group prescribed an energy intake of ≥700 kcal before 1100 daily, with at least half consumed within 2 h of waking (breakfast group) or a group that extended their overnight fast by abstaining from ingestion of energy-providing nutrients  until 1200 each day (fasting group). The randomization scheme was generated by the principal investigator (JAB) by using a computer-based random-number generator and was stratified according to baseline breakfast habits (block size = 4), with frequent breakfast consumption defined as the ingestion of ≥50 kcal within 2 h of waking on most days of the week. Investigators who enrolled participants (JDR and EAC) were unaware of these details and independently requested group assignments to prevent deciphering of the allocation sequence. Because of the self-administered nature of the treatments, it was impossible to blind participants to group allocation and neither was it possible to blind investigators for many outcomes that either required direct interaction with unblinded participants  or where treatment allocation is immediately evident in the data . These same investigators then also shared responsibility for completing various aspects of tissue and data analysis. The intervention was applied under free-living conditions, and all other lifestyle choices were allowed to vary naturally. Compliance was confirmed via self-report and verified via continuous glucose monitoring; one participant was excluded before follow-up for noncompliance, and 4 withdrew before baseline measurements, but none attributed their withdrawal to treatment allocation ; data reported herein are therefore only for those individuals for whom baseline and follow-up measurements were available .t tests for contrasts within and between groups, respectively. Secondary outcomes included regulatory/mechanistic data and markers of cardiovascular health and metabolic control at baseline and follow-up, for which treatment × time interactions were explored by using a mixed-model ANOVA with baseline breakfast habits included as a covariate. Most variables in this experiment therefore involved a single comparison between 2-level scores and so were not adjusted for multiple comparisons across the different variables reported here. However, in cases in which multiple comparisons were made within a given variable , a Holm-Bonferroni stepwise adjustment was applied to prevent inflation of type I error rate . This overall treatment effect is apparent in P = 0.01). Once classified according to accepted thresholds for the intensity of physical activities , the breakfast group consistently engaged in more “light”-intensity physical activity during the morning than did the fasting group (P = 0.03). There was no difference between treatment groups in daily recordings of median (range) waking times [breakfast compared with fasting group: 0727 (0625–0837) compared with 0708 (0630–0834) h] or sleeping times [breakfast compared with fasting group: 2246 (2119–2329) compared with 2247 (2132–2334) h], such that mean sleep duration was very similar between the breakfast group (483 ± 39 min/night) and the fasting group (476 ± 44 min/night).The major component of energy balance that responded to treatment was physical activity thermogenesis . The left side of P = 0.01).On the basis of established constants for the thermogenic effect of each macronutrient reportedly ingested according to food diaries , a smallP = 0.007). The right side of P = 0.004), particularly in the form of sugar . Analysis of the timing, type, and quantity of foods consumed at given eating occasions showed that overall daily eating patterns were unaffected by the relative consumption or omission of a daily breakfast. Specifically, once unrestricted food intake was permitted , the breakfast and fasting groups exhibited a similar frequency of consumption occasions defined as “meals”  and as “snacks” , with no difference in the macronutrient composition of these defined eating occasions  and thyroxine (free-T4) closely matched between treatments at baseline and follow-up  with no treatment × time interaction. Similarly, mean glucose concentrations during the morning did not respond differently between treatments over time but were significantly higher with breakfast (5.4 ± 0.5 mmol/L) than with fasting (5.1 ± 0.5 mmol/L) when considered across both weeks .Subcutaneous glucose was monitored continuously throughout the first and last week of intervention. Peak glucose concentrations from waking until 1200 were consistently higher with breakfast (7.6 ± 1.2 mmol/L) than with fasting (6.5 ± 1.0 mmol/L) throughout the first and last week . Conversely, the fasting group exhibited greater glucose variability across weeks 1 and 6 from 1200 onward , with a nonsignificant tendency to increase over the course of intervention  such that the greatest group difference was apparent during week 6 . Beyond the above effects, continuous glucose monitoring showed similar peak, mean, and variability values between groups during sleep, from 1200 until sleep, and over the full 24-h period.The CV (SD corrected for mean) is the preferred method for expressing glucose variability (accumulated hyper-/hypoglycemic episodes) when continuously monitored glucose data are available for individual patients . This meHere we provide the first report, to our knowledge, of all components of energy balance with regular daily breakfast relative to extended morning fasting measured under otherwise free-living conditions. Contrary to popular belief, there was no metabolic adaptation  to 6 wk of daily breakfast nor any meaningful suppression of energy intake later in the day. Rather, the major factor that compensated for the prescribed difference in morning eating patterns was that regular daily breakfast resulted in significantly higher physical activity thermogenesis than occurred with extended morning fasting. This difference in the energy expended via daily physical activities was partially attributable to a significant difference in light-intensity activities performed during the morning and has never previously been objectively measured. Although fasting until 1200 every day for 6 wk presented no negative cardiovascular effects as reflected by the concentration of blood metabolites under fed and fasted conditions, there was a 10% increase in adipose tissue insulin sensitivity within the breakfast group. Conversely, those in the fasting group exhibited no such effect on adipose tissue insulin sensitivity  but instead experienced progressively more variable interstitial glucose concentrations during the afternoon/evening, with significantly less tightly regulated glucose control than the breakfast group by the final week of the intervention. Further research is required to establish whether similar responses would be apparent in more overweight, less healthy, and/or less physically active individuals.Because physical activity thermogenesis differed significantly between treatments in light-intensity activities, this suggests that breakfast may have affected spontaneous behaviors as opposed to conscious decisions to participate in structured physical activity or exercise. This reasoning may explain why attempts to explore the causal effects of breakfast on energy expenditure by using physical activity records/diaries would not be sensitive to such an effect. Nonetheless, cross-sectional studies have detected slightly higher self-reported overall physical activity levels among breakfast eaters , and sevP = 0.04) or last  week of intervention. This indicates that the effect of morning eating patterns on physical activity levels may be direct and immediate rather than secondary to accumulated physiologic adaptations with sustained exposure to the presence or absence of daily breakfast. One possible immediate mechanism is that an increase in glucose availability may directly signal the transition from the fasted to fed state and thus enable nonessential physical activity energy expenditure above basal levels  value, this effect would be apparent in the first week as well as in the last. The group difference in afternoon-evening glucose variability may instead therefore be explained by the Staub-Traugott  effect, which describes how an initial meal can reduce the acute glycemic response to subsequent feedings within hours of the first . The pre1) it is highly unlikely that randomization of a group this size would generate such a marked and consistent difference in this variable ; 2) this difference was most consistently observed during the morning, thus coincident with the time of day at which treatments were applied; and 3) all of the other laboratory-based variables that could be measured acutely before intervention were equivalent between groups . Given the already intensive nature of this trial [including measures beyond those reported here ], additiThis study was designed to examine mechanisms linking daily breakfast and components of energy balance in free-living humans and not the long-term impact of breakfast habits on weight change. Therefore, whereas we detected no significant difference in weight loss between treatments , longer-We conclude that daily breakfast is causally linked to higher physical activity thermogenesis in lean adults, with limited evidence of any dietary compensation later in the day nor any change in resting metabolism. Blood metabolites indicative of cardiovascular health and metabolic control were not significantly affected by breakfast or fasting and neither was there any difference in adipose tissue glucose uptake between these treatments. However, regular daily breakfast maintained more stable glucose responses during the afternoon and evening.