An understanding of the drivers of tree growth at the species level is required to predict likely changes of carbon stocks and biodiversity when environmental conditions change. Especially in species-rich tropical forests, it is largely unknown how species differ in their response of growth to resource availability and individual size. We use a hierarchical Bayesian approach to quantify the impact of light availability and tree diameter on growth of 274 woody species in a 50-ha long-term forest census plot in Barro Colorado Island, Panama. Light reaching each individual tree was estimated from yearly vertical censuses of canopy density. The hierarchical Bayesian approach allowed accounting for different sources of error, such as negative growth observations, and including rare species correctly weighted by their abundance. All species grew faster at higher light. Exponents of a power function relating growth to light were mostly between 0 and 1. This indicates that nearly all species exhibit a decelerating increase of growth with light. In contrast, estimated growth rates at standardized conditions  varied over a 9-fold range and reflect strong growth-strategy differentiation between the species. As a consequence, growth rankings of the species at low (2%) and high light (20%) were highly correlated. Rare species tended to grow faster and showed a greater sensitivity to light than abundant species. Overall, tree size was less important for growth than light and about half the species were predicted to grow faster in diameter when bigger or smaller, respectively. Together light availability and tree diameter only explained on average 12% of the variation in growth rates. Thus, other factors such as soil characteristics, herbivory, or pathogens may contribute considerably to shaping tree growth in the tropics. Growth rates of tropical tree species vary widely between species and in response to resource availability  and individual condition  Light availability is widely believed to be one of the most important environmental factors driving growth of tropical rainforest trees Quantifying growth response to light across highly diverse tropical communities is challenging. Many species occur at low densities As trees grow taller, they experience successively brighter environments In this study, we aim to disentangle the effects of light availability and tree size on growth across >90% of the 300 tree species occurring at Barro Colorado Island (BCI), Panama. Forest census data from the 50-ha Forest Dynamics Plot at BCI provided information on the spatial location, size and diameter growth of nearly 150 000 individual trees of 274 species in two census intervals . Yearly canopy census data that recorded vegetation density in six height layers were used as a proxy of light availability for each individual tree.We used a hierarchical Bayesian approach to quantify the response of growth to light availability and tree size across the entire community, including rare species. Hierarchical models address the differences in sample size between the species by combining probability models for the growth variability within species and the variation between species Here we report (1) the distribution of growth rates at standardized conditions , light and size effects on growth across the community to assess the degree of growth strategy variation between species. For abundant species (≥100 stems) parameter estimates are largely determined by the data on the given species rather than by the community-level information. Therefore, (2) we use parameter estimates of abundant species to evaluate the respective contributions of light availability and tree size to growth rates, and the scope for light gradient partitioning.We analyzed data from a 50-ha forest census plot on BCI, Panama . BCI is a 1567-ha island in the Panama Canal covered with tropical lowland moist forest. The plot consists of 48 ha of undisturbed old-growth forest and 2 ha of secondary forest about 100 years old www.ctfs.si.edu; All free-standing woody stems ≥1 cm diameter at breast height (dbh) were mapped, identified to species and measured in 1981–1983, 1985, and every 5 years thereafter , sdb  =  0.0038 (s.d. = 0.00036), 2SD = 25.6 mm (s.d. = 2.49 mm), with a fraction (f) of 2.76% (s.d. = 0.39%) of the trees being subject to the larger error. Growth calculations involve two dbh measurements, thus the variance of growth error is twice the variance of measurement error. The posterior distributions of error parameters sda, sdb, 2SD and f enter as fixed priors in the hierarchical Bayesian model.To estimate the error of dbh measurements, 1562 randomly chosen trees were measured twice within 30 days. Assuming that trees did not grow between these measurements, we applied a Bayesian model to estimate true dbh for each tree and fit the differences between measured dbh and true dbh with a sum of two normal distributions We used annual canopy census data to produce an index of the amount of light reaching any point in the forest. The censuses were conducted from 1983 to 1996, except for 1994. Thereafter, the canopy census was omitted for several years and then continued applying a different method. Thus, consistent canopy census data are only available for the two census intervals 1985–1990 and 1990–1995, and we restricted our analysis to these two intervals. The canopy census recorded the presence of vegetation in six height intervals, 0–2, 2–5, 5–10, 10–20, 20–30 and ≥30 m every 5-m across the 50 ha. For each tree, we calculated a shade index as a weighted sum of vegetation located above the tree and <20 m away.th, 25th, 50th, 75th and 95th percentiles of the two distributions. This approach made it impossible to estimate the measurement error for the light index. However, the light index performed well in predicting recruit numbers across 263 species at BCI Light measurements were not available for any tree in the study area, i.e. we could not calibrate the shade index directly. Instead, we used 396 direct measurements of relative irradiance at a nearby site on BCI in 1993 Because the hierarchical Bayesian model requires long computation times, we first performed independent regressions of log(growth) as a function of dbh and light to select the best functional relationship. Stems with negative growth were excluded from this analysis. We applied a step-wise procedure to determine the best predictors of log(growth) for 98 species with >100 individuals and used AIC i (ipred) of species j as a power function (linear log-log relationship) given light availability and dbh,To assess the light and size dependence of growth across tree species at BCI, we used a hierarchical Bayesian framework ja, jb and jc describe the mean growth rate, the light and size dependence of growth of species j, respectively.Variation of growth at a given light availability and dbh is modeled using a lognormal distribution (process error)itrue is the estimated true growth rate of tree i. The process error (pσ) is estimated for each species. Using a lognormal distribution, the process error automatically scales with predicted growth i  and is assumed to be subject to measurement error as described aboveData enter our model as the observed annual dbh growth of individual 1SD describing the size-dependent error component and 2SD the size-independent error component affecting f = 2.7% of the observations. Standard deviations have to be adjusted to the time period elapsed between the two dbh measurements of the tree (iint) from which the annual growth rate has been calculated.ja, jb, and jc varied slightly but systematically with abundance across the community. Therefore, we included abundance (jabun) as species-level predictor of parameters of the growth model. Abundance is measured as the number of individuals ≥1 cm dbh in the 50-ha plot that survived the given census interval. Non-hierarchical model runs also revealed that all model parameters were approximately normally distributed at a given abundance. Thus,Preliminary analyses indicated that aσ, bσ, and cσ measure the between-species variation. As we did not have prior knowledge, we used non-informative uniform priors for these hyperparameters:The standard deviations pσ) was assumed to vary lognormally across the community with hyperparameters hµ and hσ. Priors for both parameters wereThe process error  method that is a hybrid of the Metropolis–Hastings algorithm and the Gibbs sampler a with b and c by centering the light and dbh data on approximately median or mean values across all individualsTo speed up the convergence of the Gibbs sampler, we weakened the correlation of ja represents the log of predicted annual growth of a tree with 5 cm dbh that receives 5% light.Median light is 0.045 and mean dbh is 45 mm. Thus, Post-hoc analyses showed that the correlation between model parameters was <|0.57| for all parameter combinations. These correlations were not strong enough to prevent the chains from mixing well and convergence required 100 to 2900 iterations. We used a burn-in period of 5 000 iterations and additional 5 000 iterations were used for analysis. We computed posterior parameter distributions given observed growth, light availability and dbh of each individual. All analyses were carried out using the software package R version 2.11.1 We monitored convergence by running two chains with different initial values and used the Gelman and Rubin's convergence diagnostics and a value of 1.1 to detect convergence aσ, bσ, cσ).From the posterior distributions, we computed the mean, s.d. and 95% credible intervals (CI) of all model parameters. Significance of coefficients was assessed by a 95% CI that did not include zero. For abundant species with >100 individuals, model parameters and CIs are largely determined by the data available for the given species. For less abundant species, parameter estimates are increasingly determined by the species-level prediction based on abundance and CIs are constrained by the between-species variation  to the arithmetic scale ,To report predicted annual mean growth rate at 5 cm dbh and 5% light, we back-transformed a and b at low (N = 10) and high (N = 1000) abundance, and low (5%) and high (20%) light, respectively. For these calculations we used hyperparameters estimated for the census interval 1985–1990. The covariance between a and b was 0.033 and 0.025 for rare and abundant species, respectively.To evaluate between-species variation of growth rates of rare and abundant species at different light levels, we calculated the probability density of median annual growth rate To visualize the effect of light on growth, we calculated mean observed and predicted growth rates in ten light classes corresponding to deciles of light observations. Predicted mean growth rates were calculated at mean observed light level and mean dbh of the individuals in the respective light class. To visualize the effect of dbh on growth, we calculated mean observed and predicted growth rates in different size classes. The number of size classes depended on the abundance of the species. Predicted growth rates were calculated at mean observed light level and mean dbh of the individuals in the respective size class.2) was evaluated asThe percentage of explained variance  assess the conservation of growth rankings among species at different light conditions, we computed average growth for species with ≥100 individuals over the range of most commonly observed light conditions (2–20%) from the parameters estimated for the respective census interval. Average tree dbh at the different light levels was determined from a nonlinear regression predicting log(dbh) using log(light) and light as predictors across all individuals . We alsoFor species with ≥100 individuals, we calculated contributions of light and dbh to growth by comparing growth rates at standardized conditions. For saplings, we calculated the difference between predicted growth at baseline conditions defined as 1 cm dbh and average light availability at 1 cm dbh (2%) and growth when light and dbh are doubled, i.e. 2 cm dbh and 4% light. In this analysis, only species with maximum dbh ≥2 cm were included. For larger trees (only for species with maximum dbh ≥10 cm), we performed similar comparisons using growth at 10 cm dbh and average light availability (18%) as a baseline and comparing it to growth at doubled dbh (20 cm) and doubled light (32%).b>0; All species grew faster at higher light b>0; . This efc), but increases were as common as decreases  was weakly but significantly related to abundance, with rare species tending to grow faster than abundant species  at low light (5%) . This isb<1 or 0<c<1) appear exaggerated. The shade-tolerant understory tree Faramea occidentalis grew slowly and neither light nor dbh explained much of the variation in growth rates. Growth of the shade-tolerant midstory tree Virola sebifera responded strongly to light, whereas the effect of dbh was insignificant. For Prioria copaifera, a shade-tolerant canopy tree, dbh had a large positive impact on growth, while it responded only moderately to light. Growth of the light-demanding pioneer tree Cecropia insignis increased strongly at higher light, and at the same time strongly decreased with dbh. Growth of Protium panamense, a shade-tolerant midstory tree, decreased with dbh and the apparent slight increase of growth at larger dbh was due to the strong positive effect of light. Model fits for the other species are provided as supporting information  varied little across species and between census intervals; hµ was 1.28 in the first census interval and 1.15 in the second, hσ was 0.09 and 0.16 in the two census intervals, respectively. Thus, at predicted growth of 1 mm/yr, the probability of true growth being between 0 and 2 mm/yr was 71%. When predicted growth was 5 mm/yr, the probability that true growth was between 3 and 7 mm/yr was 26%.The process error . Moreover, correlations of growth ranks at 2% and 20% light were high (Spearman's rho ∼ 0.7). The consistency in the response of growth to light across species suggests that reversals in rank order do not have a strong impact on the outcome of competition among species. Largely consistent growth rankings are also reported for seedlings of small numbers of tropical tree species across different light However, we found little evidence for light gradient partitioning in terms of growth rates among species. The majority of species showed a decelerating increase of growth with light  as compared to low light (9-fold). These large differences in growth rates certainly offer opportunities for differentiation of life-history strategies. Fully understanding the importance of differences in growth rates and light response for coexistence, however, requires the application of simulation models that combine information on the response of all demographic rates  to changes in light availability. The parameters we have estimated here provide the basis for such modeling studies.Nothofagus solandri var. cliffortioides, where only small trees <10 cm dbh were sensitive to changes in above-ground competition The fact that an interaction between light and size improved the model only in 43 species (16%) suggests that species respond in a similar way to changes in light availability across all sizes. This conclusion is corroborated by separate analyses for trees smaller and larger than 5 cm dbh where the estimated distribution of light response was very similar (results not shown). This is in contrast to a study on c and biomass scales with diameter as b ∼ d8/35/3+c. Constant biomass growth is achieved when c = −5/3. When c>−5/3, biomass growth increases with diameter, despite possibly decreasing diameter growth. Parameter estimates and credible intervals of c indicate that no species is expected to have c<−5/3. This suggests that tropical trees continue accumulating carbon at increasingly faster rates as they grow While species responded consistently to light, changes of diameter growth with tree size were very variable among species with about half the species growing faster when taller and the other half of the species growing faster when small. Likewise, significant responses were as frequently positive (12%) as negative (8%–12%). However, we caution against a physiological interpretation of size dependence of diameter growth rates. If diameter growth scales with diameter as dg ∼ dCordia alliodora, C. bicolor, Hirtella triandra, Jacaranda copaia, Protium tenuifolium, and Zanthoxylum ekmanii.For the vast majority of species, growth rates were well approximated by the power function which only allows for a monotonic increase or decrease of growth with diameter. Many other studies have allowed for a hump-shaped response of growth to diameter In other species, the model did not capture the saturation of growth rates at large diameters . This is a result of the unbalanced data sets dominated by the many small individuals. Nevertheless, the model fits show that for the vast majority of species, growth rates were well approximated and deviations only affected the largest size classes. To account for unbalanced data sets and unknown functional relationships between predictor variables and growth, semi- or nonparametric approaches could be used. However, semi- or nonparametric approaches are not suitable for our study because they prevent a straightforward comparison among many species.2 was slightly higher than in slow-growing species 2 have been reported. Using tree size and exposed crown area or accounting for above and belowground competition, up to 90% of growth variance of North American tree species could be explained Generally, a large variation of growth rates is observed that is not explained by tree size or light availability The large amounts of unexplained variance can be due to environmental conditions not considered here, e.g. soil texture Uncertainty in our model fits also comes from other sources: error in the light estimate, measurement error of past growth, and sampling error in rare species A problem inherent in highly diverse tropical forests is the low number of individuals per area of the many rare species However, functional characteristics of tree species such as wood density, specific leaf area or maximum height may relate more directly to growth strategies and hold greater promise for a more mechanistic understanding of life-history strategies of tropical tree species Figure S1Diameter-light relationship. (A) Light estimate vs. tree diameter (dbh) for 148 933 trees at Barro Colorado Island, Panama, in 1990. Trees with dbh >1 m are assumed to receive full sunlight and are not shown. (B) Nonlinear regression predicting average log(dbh) in the light range from 2 to 20% (log(dbh)  =  4.547+0.455×log(light)+2.006×light; dbh is in mm). Average log(dbh) is used to estimate average growth across the light range for (PDF)Click here for additional data file.Figure S2Light dependence of growth rates for species with ≥25 individuals in the two census intervals . Light classes correspond to deciles of light availability across all individuals. Predicted mean growth rates were calculated at mean observed light level and mean dbh of the individuals in the respective light class. Observed and predicted growth rates of individual trees are displayed as grey and orange dots, respectively. Mean observed and predicted growth rates in different light classes are displayed as black and red dot, respectively.(PDF)Click here for additional data file.Figure S3Dbh dependence of growth rates for species with ≥25 individuals in the two census intervals . Observed and predicted growth rates of individual trees are displayed as grey and orange dots, respectively. Mean observed and predicted growth rates in different size classes are displayed as black and red dot, respectively. For species with <100 individuals, the dbh range was split into three size classes each containing a third of the individuals. For species with <2000 individuals, four size classes contain 25% of the individuals each. For species with <3000 individuals, six size classes contain 25%, 25%, 25%, 15%, 5% and 5% of the individuals, respectively. For species with <4000 individuals, seven size classes contain 25%, 25%, 25%, 15%, 3.3%, 3.3% and 3.3% of the individuals. For species with ≥4000 individuals, nine size classes contain 25%, 25%, 25%, 15%, 2%, 2%, 2%, 2% and 2% of the individuals. We only plot the size dependence for species with a maximum dbh of >3 cm. Predicted mean growth rates were calculated at mean observed light level and mean dbh of the individuals in the respective size class.(PDF)Click here for additional data file.Table S1Posterior means, lower and upper limits of 95% credible intervals  of the average growth rate at 5 cm dbh and 5% light (a), log(light) dependence (b), log(dbh) dependence (c), and process error (pσ) for two census intervals  and tree species in the 50-ha Forest Dynamics Plot at Barro Colorado Island, Panama. Dashes indicate no living individuals at the beginning of the respective census interval.(XLSX)Click here for additional data file.Table S2Observed and predicted average growth rate for tree species with <25 individuals at Barro Colorado Island, Panama. N is the number of individuals.(XLSX)Click here for additional data file.


TAS2R38, and with differences in fungiform papilla density on the anterior tongue surface. Recently we showed that the PROP non-taster phenotype is strongly associated with the G variant of polymorphism rs2274333 (A/G) of the gene that controls the salivary trophic factor, gustin. The aims of this study were 1) to investigate the role of gustin gene polymorphism rs2274333 (A/G), in PROP sensitivity and fungiform papilla density and morphology, and 2) to investigate the effect of this gustin gene polymorphism on cell proliferation and metabolic activity. Sixty-four subjects were genotyped for both genes by PCR techniques, their PROP sensitivity was assessed by scaling and threshold methods, and their fungiform papilla density, diameter and morphology were determined. In vitro experiments examined cell proliferation and metabolic activity, following treatment with saliva of individuals with and without the gustin gene mutation, and with isolated protein, in the two iso-forms. Gustin and TAS2R38 genotypes were associated with PROP threshold (p=0.0001 and p=0.0042), but bitterness intensity was mostly determined by TAS2R38 genotypes (p<0.000001). Fungiform papillae densities were associated with both genotypes (p<0.014) , but papilla morphology was a function of gustin alone (p<0.0012). Treatment of isolated cells with saliva from individuals with the AA form of gustin or direct application of the active iso-form of gustin protein increased cell proliferation and metabolic activity (p<0.0135). These novel findings suggest that the rs2274333 polymorphism of the gustin gene affects PROP sensitivity by acting on fungiform papilla development and maintenance, and could provide the first mechanistic explanation for why PROP super-tasters are more responsive to a broad range of oral stimuli.Taste sensitivity to PROP varies greatly among individuals and is associated with polymorphisms in the bitter receptor gene  Individual variability in sensitivity to the bitter taste of phenythiocarbamide was first recognized by Fox more than eight decades ago . Since tTAS2R38. Individuals can be divided into three taster groups  based on behavioral testing assessing their PTC/PROP sensitivity. The percentage of non-taster individuals greatly varies among populations: from less than 7% to more than 40%  = 10.502; p=0.00013). Post-hoc comparisons showed that thresholds were statistically higher in individuals with genotype GG of the gustin gene than in the other genotypes , but not different between AA and AG individuals (p>0.05). Although thresholds were variable in those with the GG genotype, thresholds were more than 10-fold higher in these individuals than in the other groups. Main effects ANOVA also showed an association between PROP threshold and TAS2R38 genotypes . Thresholds of individuals with the AVI/AVI genotype were higher than those of individuals with genotypes PAV/PAV and PAV/AVI , that did not differ from each other (p>0.05).PROP threshold values and bitterness intensity ratings (PROP 3.2 mM) of individuals with genotypes AA, AG and GG of the gustin gene and with genotypes PAV/PAV, PAV/AVI and AVI/AVI of TAS2R38 genotypes  and less so with the gustin gene polymorphism . TAS2R38 bitterness ratings of PAV/PAV individuals were statistically higher than those of heterozygous individuals  who in turn gave higher intensity ratings to PROP than individuals with the AVI/AVI genotype . In the case of gustin, post hoc comparisons showed that PROP bitterness was statistically higher in individuals with genotype AA than in those with the other genotypes , but not different between GG and AG individuals (p>0.05).PROP bitterness intensity ratings (3.2 mM) were strongly associated with TAS2R38 (lower graph). Also shown are representative images of the tongue tip stained area where measures were taken. ANOVA calculations showed that fungiform papillae density on the anterior part of the tongue was strongly associated with the gustin gene  and less so with TAS2R38 polymorphisms . In the case of gustin, fungiform papillae density values were lower in individuals with the GG genotype than in those with genotypes AG and AA . Papillae density was not different between AA and AG individuals (p>0.05). In the case of TAS2R38 genotypes, post hoc comparison showed that individuals with the PAV/PAV genotype had a higher fungiform papillae density than those with PAV/AVI and AVI/AVI genotypes ; the density values of the latter two groups were not different from each other (p>0.05).F = 7.5920; p=0.00118), but not with TAS2R38 genotypes . Post-hoc comparisons showed that mean papilla diameter determined in those with genotypes AA and AG were lower than those of homozygous GG individuals   .TAS2R38 genotypes. However, only associations between these features and gustin were statistically significant. In fact, both the SD of papilla diameter . Post-hoc comparisons showed that individuals with the GG genotype had papillae with greater variation in shape (higher SDs in papilla diameter) as well as a higher percentage of distorted papillae than the other genotypes . No differences were found between AA and AG individuals (p>0.05).ANOVA was also used to examine relationships between fungiform papilla morphology and gustin and diameter  and the diameter  dependedTAS2R38 polymorphisms to PROP tasting and papillae density and morphology  than was gustin genotype (6.18% variance).Multiple linear regression was used to assess the relative contributions of gustin and rphology  and 3. AGustin genotypes and age were the only significant contributors to fungiform papillae density with the overall model explaining 30.90% of the variance. Finally, gustin genotype was the only significant contributor to fungiform papillae diameter, SD of papilla diameter and percentage distortion. However, the predictive power of these models were relatively low, explaining 13.2-16.11% of the variance in these measures.in vitro experiments is shown in p=0.0135; Student’s t test)   or control . No differences were found between treatment with saliva of genotypes GG and control (p>0.05).The effect of gustin gene polymorphism rs2274333 (A/G) from the  t test) . ANOVA s0.00001) . Post hoF = 10.463; P < 0.00001) . No differences were found between these last three treatments P > 0.05.ANOVA also showed that the fluorescence emission depended on treatments performed with the two iso-forms of gustin (gustin Ser90 or gustin Gly90) (0.00001) . PairwisTAS2R38 genotypes and the rs2274333(A/G) polymorphism in the gustin gene on PROP tasting, fungiform papillae density and morphology. Results showed that PROP thresholds and bitterness intensity ratings were associated with TAS2R38 and gustin gene genotypes, as reported previously [TAS2R38 genotypes were associated with fungiform papillae density with a stronger effect for gustin than for TAS2R38. However, only gustin was associated with morphological changes in fungiform papillae such as larger size, greater variation in shape and more distortions.One aim of the present study was to determine the effects of eviously . ImportaTAS2R38 genotypes to these same outcomes. Both genes contributed to threshold acuity, however, TAS2R38 polymorphisms made a much greater contribution to PROP bitterness intensity than did gustin. These data confirm the findings of Calò et al. [TAS2R38 genotypes on suprathreshold intensity than threshold sensitivity. The reasons for these differential effects are unclear, but we can speculate that at low stimulus concentrations, that are further diluted in the oral cavity, both papillae features (as determined by gustin) and the presence of the functional, PAV form of the TAS2R38 receptor are critical for tasting PROP. At higher concentrations, when there is a higher probability that the stimulus molecules arrive at the receptor site, the number of functional (PAV) receptors may be more important for enhancing peripheral nerve signalling than the number of taste cells that are present. This explanation may be overly simplistic as it fails to account for a number of factors that affect taste function such as smoking, damage to taste nerves [Regression modelling permitted us to assess the relative contributions of gustin and ò et al.  showing e nerves ,72 and vTAS2R38 genotypes were associated with papillae number, and PAV homozygous individuals had a higher papillae number with respect to other genotypes. However, in the regression analysis, that looks at multiple variables at the same time, the TAS2R38 genotypes were not significant predictors of papillae number or their other morphological features. It is important to note however, that gustin genotypes predicted only a small percentage of the variance in papillae size, and shape, suggesting that other factors define these morphological characteristics. We did not investigate brain-derived neurotrophic factor (BDNF) which has also been implicated in papillae development and maintenance [Our data showed that ntenance –75, and TAS2R38, who perceived the highest PROP bitterness, had higher papillae densities compared to those who perceived PROP as less intense. Our results complement these earlier observations by also showing that a single A allele in the gustin gene was sufficient to increase papillae density. In addition, we studied for the first time, the relationship between papillae distortion, which seems to be a measure of functionality [TAS2R38 genotypes.Numerous studies have report greater papillae densities in PROP super-tasters compared to those who perceive PROP as less intense ,54–56,76ionality , and genTAS2R38 genotypes and papillae densities. In our previous work [TAS2R38 and the gustin gene had independent effects in modulating PROP phenotype in an ethnically homogeneous population where the majority of PAV homozygotes also carried the AA  form of the gustin rs24743333 polymorphism. In contrast, a majority (55%) of AVI homozygotes carried the GG  form. In the present study, fewer AVI homozygotes (25%) carried the GG form. Nevertheless, the presence of the AA form of gustin was more common in those with at least one PAV allele for TAS2R38. Thus, it is plausible that the higher papillae densities we observed in PAV homozygotes  may better reflect the actions of gustin rather than TAS2R38 genotypes. Future studies will have to confirm this finding. Our results should not lead to the conclusion that TAS2R38 genotypes predict gustin genotypes. The two loci are independent (not in linkage disequilibrium) and, in fact, reside on different chromosomes. Why these two discrete loci appear to have functional overlap in defining PROP tasting and papillae density and morphology is presently unknown. The answer to this question cannot be resolved here and will come from more comprehensive genetic studies.Hayes et al.  reportedous work  we foundTAS2R38 accounted for less variance in the threshold response to PROP than in previous studies [Up to now, only few populations have been tested for variants in the gustin gene, but the allele frequencies in these populations are not known. Variations in the frequency of gustin A and G alleles across populations could produce discrepant findings across studies, and could explain why a genome wide phenotype-genotype association study of PROP threshold failed to detect a relationship with variants in the gustin gene . Both co studies ,80. Thisin vivo studies showed that treatment of cells with saliva from individuals with the AA genotype of gustin resulted in increased cell proliferation and metabolic activity, whereas similar treatment with saliva from individuals with the GG genotype did not. Furthermore, direct treatment of cells with the active iso-form of the protein (gustin90Ser) increased cellular metabolic activity, while treatment with the inactive iso-form (gustin 90 Gly) failed to do so. These novel findings confirm, for the first time, a role for gustin in cell proliferation and maintenance.For more than 40 years, gustin has been described as a trophic factor responsible for the growth and maintenance of taste buds . This rors2274333 (A/G), affects PROP tasting by acting on the density and maintenance of fungiform papillae, and that between the two protein iso-forms that result from this polymorphism, gustin 90Ser exhibits full functional activity, compared to the gustin 90Gly iso-form. In addition, the results of this work, if confirmed in different populations, will provide a mechanistic explanation of why PROP super-taster individuals have a higher density of fungiform papillae than PROP non-tasters, and why they show greater oral responsiveness to a wide range of stimuli that are not mediated via the TAS2R38 bitter taste receptor.In conclusion, our findings in an genetically homogeneous cohort suggest that the gustin (CA6) gene polymorphism,


CENPW, and two variants for ANM, rs897798/BRSK1 and rs769450/APOE, to our African American cohort. Overall, generalization of the majority of previously-identified variants for AM and ANM, including LIN28B and MCM8, was not observed in this African American sample. We identified three novel loci associated with ANM that reached significance after multiple testing correction . Our most significant AM association was upstream of RSF1, a gene implicated in ovarian and breast cancers . While most associations were identified in either AM or ANM, we did identify genes suggestively associated with both: PHACTR1 and ARHGAP42. The lack of generalization coupled with the potentially novel associations identified here emphasize the need for additional genetic discovery efforts for AM and ANM in diverse populations.Age at menarche (AM) and age at natural menopause (ANM) define the boundaries of the reproductive lifespan in women. Their timing is associated with various diseases, including cancer and cardiovascular disease. Genome-wide association studies have identified several genetic variants associated with either AM or ANM in populations of largely European or Asian descent women. The extent to which these associations generalize to diverse populations remains unknown. Therefore, we sought to replicate previously reported AM and ANM findings and to identify novel AM and ANM variants using the Metabochip  in 4,159 and 1,860 African American women, respectively, in the Women’s Health Initiative (WHI) and Atherosclerosis Risk in Communities (ARIC) studies, as part of the Population Architecture using Genomics and Epidemiology (PAGE) Study. We replicated or generalized one previously identified variant for AM, rs1361108/ Age at menarche (AM) and age at natural menopause (ANM) are components of the reproductive lifespan in women. Timing of these reproductive milestones is associated with various diseases and cancers such as type 2 diabetes, cardiovascular disease, endometrial and breast cancers, as well as with fertility issues Both cross-sectional and longitudinal studies have shown an overall decline in age of menarche in US girls from the 1960s to the 1990s Similar to the timing of age at menarche, the age at which natural menopause occurs is affected by multiple factors Genetic and environmental factors that determine AM and ANM have been considered in numerous studies, but many of these studies have conflicting or unreplicated results In this study, we used data from the Metabochip genotyping array to characterize previously identified variants associated with menarche and menopause in African Americans in a combined cohort of African-American women from the Women’s Health Initiative (WHI) and Atherosclerosis Risk in Communities (ARIC) studies .Women participants from two cohorts of the PAGE Study Age at menarche was defined as the age when menstrual periods started in years, with extreme values pooled in groups of 9 years or less and 17 years or older. Age at natural menopause was defined as the age at which cessation of regular menstrual periods due to the body’s natural aging process occurred. In ARIC, women were asked, “Was your menopause natural or the result of surgery or radiation?” Only women who indicated natural menopause were included. Women in WHI who underwent hysterectomy, oophorectomy, or hormone replacement therapy before the onset of natural menopause were excluded. In both studies, women reporting age at natural menopause <40 years were excluded; women reporting age at natural menopause >60 years were censored at age 60. All women included in the present study were post-menopausal.2>0.90 in the CEU HapMap II dataset, plus up to four additional SNPs with r2>0.5 in the YRI HapMapII dataset were also included on the array. Lastly, SNPs selected to fine-map regions of interest related to metabolic traits, copy number variant-tagging SNPs, Major Histocompatibility Complex (MHC) SNPs, SNPs on the X and Y chromosomes, mitochondrial DNA SNPs, and “wildcard” SNPs were also targeted, for a total of approximately 200,000 SNPs. Of these, 161,098 (81.9%) passed quality control filters for tests of Hardy-Weinberg Equilibrium (>1×10−7) and genotyping efficiency . There was no filter for minor allele frequency. The design and performance of this genotyping chip in this African American sample has been described in detail elsewhere Genotyping was performed on the Metabochip, a custom Illumina iSelect genotyping chip designed to genotype SNPs associated with metabolic traits and cardiovascular disease All participants self-reported African American ancestry. To adjust for potential population stratification, we used the principal components method implemented in Eigenstrat Linear regression was performed assuming an additive genetic model to test for associations between individual SNPs and the outcomes of age at menarche in years. We examined two models for menarche: 1) a minimally adjusted model that accounted only for study sites and principal components, and 2) a fully adjusted model that included study site, year of birth, principal components, and body mass index at ascertainment, with the understanding that BMI at ascertainment may be a poor proxy for BMI at age of menarche. Age at menarche was self-reported many years later at time of examination, which has been shown to be fairly accurate The overall goal of the study was to test for SNPs associated with AM and/or ANM using the Metabochip in African Americans from the WHI and ARIC studies. We looked to generalize to our population of African American women genes, gene regions (400 kb upstream and downstream of a gene of interest), and SNPs described in previous GWAS and candidate gene studies associated with AM and ANM. We tested all SNPs in the regions regardless of linkage disequilibrium (LD) with the index SNP, although we only considered a test of association generalized if the tested SNPs were identical to the index SNP or in strong LD with the index variant in HapMap CEU samples. For each candidate gene, we plotted results of single SNP tests of association using LocusZoom and examined regions 400 kb upstream and downstream of the gene/gene region of interest. Tests of association were considered significant for generalization at a liberal threshold of p<0.05. For previously reported variants not genotyped in our study, we identified SNPs in LD with our directly genotyped SNPs −07, after Bonferroni correction . Because this threshold is highly conservative given the correlation among the SNPs on the Metabochip, we also defined an arbitrary suggestive significance level as p<1×10−4 in the discovery phase.In addition to generalization, we sought to discover novel SNP-trait associations using the entire Metabochip. Significance in this discovery phase was defined as p<3.1×10A total of 4,159 and 1,860 African American female participants met the study definitions for AM and ANM, respectively, and both PAGE studies were represented roughly equally . In ARICCYP19A1, CYP17, CYP1B1, FTO, LIN28B, 9q31.2 region, IGF1, TNFSF11, TNFRSF11A, and LHCGRet al. of >87,000 European-descent women from forty-nine studies To generalize previously-associated genetic variants in our African American population, we examined regions/genes previously associated with AM from either published candidate gene studies or GWAS: et al. meta-analysis were directly genotyped or in strong (r2>0.70) LD in the CEU panel of HapMap with those genotyped  , and while the direction of genetic effect was consistent with previous reports, all failed to reach statistical significance in this sample (p>0.30). Four additional SNPs in LD with these LIN28B SNPs were also not significant. At the 9q31 locus, rs7861820 and rs4452860, both located downstream of TMEM38B, had betas opposite to prior reports FSHB were not significantly associated with AM in this African American sample, though rs605765 (β = −0.06) had the same direction of effect and comparable magnitude as rs1782507 (β = −0.07) Overall, 11/21 (52%) SNPs previously identified for AM from earlier studies and 15/42 (36%) from the Elks  = 0.01) . Represe with AM  [35], [3et al. for the ReproGen Consortium et al., we detected an association with rs9385399 (p = 0.01), located downstream of CENPW, which is a perfect proxy (r2 = 1.00) for previously associated variant rs1361108, and the only SNP to generalize to our African American sample. We also identified an association with rs2947411 (p = 0.02) with AM  in CEU panel of HapMap. 1/12 (8%) of the tested SNPs in these regions/genes generalized to this African American sample: rs8113016  and was associated with ANM in our sample (p = 0.03). An intronic APOE variant, rs769450, was associated with ANM (p = 0.03), though the nonsynonymous APOE rs7412 was not (p = 0.55); these SNPs are not in LD with each other (r2 = 0.04). In BRSK1, no previously reported SNPs were genotyped in our study; however, directly genotyped intronic TMEM150B rs4806660 was in very strong LD with intronic BRSK1 rs1172822 (r2 = 0.98). BRSK1 rs1168309, in strong LD with rs2384687 (r2 = 0.85) was not associated with ANM in this African American sample (p = 0.59).Overall, 14/23 (40%) SNPs previously identified for ANM via GWAS and 6/20 SNPs from the Stolk s8113016 . Rs81130et al. as associated with ANM were directly genotyped on the Metabochip. Two of the three genotyped SNPs (rs2303369 and rs2153157) had the same directions of effect, though the magnitudes were smaller. Of the remaining 17 SNPs not directly targeted by the Metabochip, three were in strong LD (HapMap CEU r2 ranging from 0.86 to 0.91) with the SNPs identified by Stolk et al: rs1176133, rs4668368, and rs12593363. For seven SNPs, no proxy SNP could be identified on the Metabochip , no SNPs were significantly associated with AM in either model , which is located upstream of RSF1, a gene encoding a chromatin remodeling protein implicated in ovarian and breast cancers We tested all SNPs genotyped on the Metabochip for an association with AM adjusted for study site and principal components (Model 1) and adjusted for study site, year of birth, principal components, and body mass index (Model 2) . After aer model . The mos−07): LDLR , KCNQ1 , and COL4A3BP . The most significant association was with rs189596789, located approximately 10 kb upstream of the low-density lipoprotein receptor (LDLR) gene, which has been associated with familial hypercholesterolemia LDLR (rs189596789), NOS1AP (rs76078015), DGKB (rs74486449), LYPLAL1 (rs78696400), and CDKAL1 (rs114158228)  . We werePHACTR1 was suggestively associated with AM  to the LIN28B region (28 SNPs).Here we demonstrated the use of the Metabochip genotyping array to identify SNPs associated with AM and ANM in a sample of African American women. Previous GWAS studies for AM and ANM have been performed in primarily European descent populations; generalization to diverse populations has largely been lacking In the discovery phase of our AM analysis, none of our results reached genome-wide significance. However, the ANM analysis yielded three associations that were significant after multiple testing corrections. Broadly, we demonstrate the ability to potentially uncover new variants associated with age at natural menopause in our African American cohort using the Metabochip.LDLR (rs189596789) which encodes a low density lipoprotein receptor implicated in familial cholesterolemia. KCNQ1 (rs79972789) also reached genome wide significance in our ANM analysis. Numerous variants in KCNQ1 have also been implicated in type 2 diabetes in several populations, though none were in linkage disequilibrium with rs79972789 et al. evaluated the role of menopausal hormonal changes with cardiac events in women with mutations in KCNQ1 and congenital long-QT syndrome (LQTS) LPL and CYP4F22, which are associated with type 2 diabetes and lipid metabolism , and TMEM18 (rs2947411), associated with obesity and body mass index Several studies have shown relationships between a woman’s reproductive milestones  and menstrual characteristics and risk for breast cancer, endometrial cancer, and ovarian cancer PHACTR1 and ARHGAP42 SNPs were suggestively significant in both our AM and ANM analyses. PHACTR1 is a phosphatase and actin regulator which has been implicated in coronary artery disease ARHGAP42, a Rho GTPase activating protein, has not yet been evaluated for a role in menarche or menopause. A GWAS identified intronic ARHGAP42 rs633185 is associated with blood pressure ARHGAP42 variants suggestively associated with either AM or ANM in this study. A recent study by Lu et al., found SNPs in both TNFSF11 and TNFRSF11A significant for AM and ANM PHACTR1 and ARHGAP42 play in atherosclerosis, osteoporosis and the development of lactation glands in pregnancy, further investigation on the influence of these genes in AM and ANM is warranted Different pathways appear to be involved in the initiation and cessation of menses. Prior GWAS and linkage studies performed in European descent or Asian populations for AM and ANM show little concordance with specific genes  which may have impacted our results. Additionally, prior studies that identified SNPs associated with AM and ANM were performed primarily in European-descent cohorts. Though our study included over 4,000 African American women, we had limited power to identify significant associations in most previously identified loci, which may explain why we failed to detect the same associations identified in European-descent GWAS. For specific tests of association, our power was impacted by sample size and by minor allele frequencies. For example, the allele frequency for rs7861820 in this African American cohort was 0.11 compared to a higher frequency observed in HapMap CEU  of SNPs targeted by the Metabochip passed rigorous quality control with high call rates et al. demonstrated that Metabochip data can be used to replicate known GWAS-identified SNP-trait relationships. Furthermore, the pilot study demonstrated that Metabochip data can be used to fine-map GWAS-identified regions to uncover potential novel index SNPs specific to African Americans in an established locus for that trait. Fine-mapping data for AM/ANM was not included in the Metabochip content. While we were able to use the Metabochip to identify potentially novel SNP-trait relationships for AM/ANM, additional fine-mapping efforts of other loci already implicated for these traits are needed. Furthermore, additional studies in general are warranted for diverse (non-European descent) populations using Metabochip or other arrays designed for fine-mapping. Admixture in the African American population and its associated decreased LD compared to European Americans challenge identification of trait-associated SNPs. Targeted fine mapping, such as use of the Metabochip, may be more appropriate in some circumstances than GWAS to evaluate specific SNPs and regions associated with particular traits.Metabochip performance in non-European populations was recently evaluated in a pilot study in African American PAGE participants Although the Metabochip was designed for genotyping of cardiovascular and metabolic SNPs, this study demonstrates the feasibility of utilizing such a targeted chip to identify SNP associations with age at menarche and age at natural menopause. We identified potentially novel associations with AM/ANM at loci implicated in cardiovascular traits, obesity and cancer. This may result from pleiotropic loci or may suggest that the AM/ANM timing mechanisms influence underlying disease process. With numerous genes implicated in both metabolic and cardiovascular phenotypes and both AM and ANM, further studies will allow us to consider how specific genes may influence the reproductive lifespan in women.Table S1Comparison of SNPS in Elks et al. meta-analysis for AM to African American women in PAGE Study.(DOCX)Click here for additional data file.Table S2Comparison of SNPs in Stolk et al. meta-analysis for ANM to African American women in PAGE Study.(DOCX)Click here for additional data file.Table S3AM Discovery—SNPs associated (p<1e−4) with AM in African American women from the PAGE Study.(DOCX)Click here for additional data file.Table S4Minor allele frequency comparisons of African American women in PAGE Study to HapMap CEU Panel.(DOCX)Click here for additional data file.


Lymphoma is the most common malignancy of the spleen. The imaging features of splenic lymphoma are nonspecific and mostly lymphomas present as a diffusely enlarged spleen. Focal lesions are described but remain of low density or intensity on CT or MRI, respectively. We describe a histologically proven case of splenic lymphoma that showed an atypical hyperdense/hyperenhancing appearance on imaging suspicious for a vascular pathology. To the best of our knowledge and based on review of English literature, such an appearance of splenic lymphoma is extremely unusual and rare. Lymphoma is the most common tumor of the spleen and it may be primary or secondary to a diffuse lymphomatous process . Splenic3/mm3, and platelet count of 70 × 103/μL. Peripheral blood smear did not show any abnormal lineage cells.A 60-year-old Caucasian male presented to the ER with one-month history of weakness, fatigue, and left upper quadrant pain. Past medical history was significant for diabetes Mellitus, hypertension, and hyperlipidemia. Patient worked in a fertilizing chemical business with history of exposure to chemicals and pesticides over the last 2 decades. Physical examination was remarkable for pallor and splenomegaly extending 7-8 cm below costal margin. Laboratory analysis showed hemoglobin of 9 g/dL, hematocrit 28%, white count 4.0 × 10Noncontrast CT abdomen showed massive splenomegaly with multiple hyperdense foci within the spleen  revealed Bone marrow biopsy, however, revealed a B-cell lymphoma and an initial staging PET scan showed massively enlarged spleen with increased FDG uptake  with an CT is the most widely used and currently the imaging modality of choice for assessment of lymphoma. The overall accuracy of CT in depicting splenic lymphoma is approximately 22–65% , differeConventional T1- and T2-weighted MR sequences have poor sensitivity for splenic involvement in lymphoma, which is due to the similar relaxation times of lymphoma and normal splenic tissue using these sequences . IdentifThe differential diagnoses based on CT and MRI imaging characteristics involved pathologies with a high vascular content, namely, splenic peliosis, cavernous atypical hemangioma, or an angiosarcoma. The bone marrow biopsy obtained given the history of chemical exposure and the finding of pancytopenia, however, showed B cell lymphoma. This was confirmed by PET scan SUV 6.23) and splenic pathology when patient presented with a splenic rupture. In a study of 68 patients with known malignant disease, an SUV threshold of 2.3 correctly differentiated benign from malignant lesions with the sensitivity, specificity, PPV, and NPV of 100%, 100%, 100%, and 100%, respectively [.23 and sIn conclusion, splenic lymphoma could present with imaging features simulating a vascular malformation or tumor and should be considered in differential of a multifocal hypervascular lesion, in an appropriate clinical setting.


GRN) in a German population of patients with schizophrenia (n = 271) or BPD (n = 237) as compared with 574 age-, gender- and ethnicity-matched controls. Furthermore, we measured plasma progranulin levels in 26 German BPD patients as well as in 61 Italian BPD patients and 29 matched controls.Basing on the assumption that frontotemporal lobar degeneration (FTLD), schizophrenia and bipolar disorder (BPD) might share common aetiological mechanisms, we analyzed genetic variation in the FTLD risk gene progranulin , rs4792938  and rs5848 . Mean±SEM progranulin plasma levels were significantly decreased in BPD patients, either Germans or Italians, as compared with controls  and were not correlated with age.A significantly decreased allelic frequency of the minor versus the wild-type allele was observed for rs2879096 (23.2 versus 34.2%, GRN variability decreases the risk to develop BPD and schizophrenia, and progranulin plasma levels are significantly lower in BPD patients than in controls. Nevertheless, a larger replication analysis would be needed to confirm these preliminary results.In conclusion,  GRN) are the most frequent cause of autosomic dominant frontotemporal lobar degeneration (FTLD). Age at disease onset, as well as clinical phenotypes associated with such mutations are extremely wide, even in the same family GRN mutation. Additional evidence of a clinical overlap between psychiatric disorders and genetically determined FTLD comes from the recent description of a patient with heterosexual pedophilia GRN mutation and developed bvFTD over time, and from a second article reporting two clinically different, apparently sporadic FTLD cases sharing the previously described Thr272fs GRN mutation, who had had a premorbid BPD history GRN was found GRN mutation carriers Mutations in the progranulin gene  from patients with AD. GRN is localized in a region of chromosome 17q21 previously shown to be associated with BPD Besides autosomic dominantly inherited GRN association study in patients with BPD and schizophrenia compared with controls. In addition, we measured progranulin plasma levels and correlated them with genetic data.Given these premises, we carried out a GRN was analyzed in a German population of 508 patients with schizophrenia and BPD as compared with 574 matched controls  or controls . Stratification according to each of the SNPs studied, no significant differences in progranulin plasma levels were shown (data not shown).Progranulin plasma levels were measured in 26 German BPD patients. As one of them had levels below the reference range GRN variability decreases the risk to develop BPD and schizophrenia. In addition, progranulin plasma levels are significantly decreased in patients as compared with controls. Despite both the SNPs and progranulin levels were associated with the target phenotypes, no association between such SNPs and progranulin levels were observed. This could be due to a number of reasons, including the possible regulation of translation by microRNA, the interaction of additional variants not included in this study, or the effect of medications taken at time of plasma sampling. Unfortunately, at time of DNA sampling, no matched plasma samples were taken from German controls. Therefore, these preliminary results need a further confirmation in a larger and ethnically matched population.To our best knowledge, this is the first evidence that Progranulin and the various granulin peptides derived by elastase cleavage are implicated in a range of biological functions, including development, wound repair, inflammation and tumorigenesis GRN, has been shown in Latino populations with schizophrenia GRN mutation was found, even in family members diagnosed with schizophrenia To date, a number of Genome Wide Association Study (GWAS) have been performed in patients with either schizophrenia or BPD In our study, the association observed in the whole population was maintained after stratifying in patients with BPD and schizophrenia, suggesting common pathogenic pathways. In line with this hypothesis, two large GWAS in BPD and schizophrenia patients, respectively, lead to the identification of the same susceptibility genes Regarding progranulin levels, we acknowledge that the majority of patients were treated at time of sampling, thus we can't exclude an influence of therapies on progranulin levels.GRN variability contributes to schizophrenia and BPD development and that progranulin plasma levels are lower in patients with BPD than in controls, although this findings need a replication in a larger cohort.In conclusion, we demonstrated that Five hundreds and eight patients with psychiatric disorders, including 229 males, 273 females, and 6 gender unknown, mean age±SEM at sampling: 48.99±0.65 years (range 22–85), mean age±SEM at disease onset: 28.30±047 years (range: 9–72) were recruited at the Department of Psychiatry, Psychosomatics and Psychotherapy, University of Würzburg, Germany. Two hundreds seventy one patients, including 146 males, 120 females, and 5 gender unknown, mean age±SEM at sampling: 47.00±0.84 years (range: 22–85), mean age±SEM at disease onset: 27.13±0.59years (range: 9–72) were diagnosed with schizophrenia P>0.05). The control sample was composed of blood donors, staff members, and volunteers all originating from the Lower Franconia region. The sample was not systematically screened for psychiatric disorders; however, all subjects were free of medication, and the study was explained to them, so that the likelihood of severe psychiatric disorders in the control sample was low. Only subjects who gave written informed consent were enrolled in the study, which complied with the Declaration of Helsinki and was approved by the Ethics Committees of the University of Würzburg.The control group consisted of 574 German volunteers matched for ethnic background and age  and was recruited at the Department of Psychiatry, Psychosomatics and Psychotherapy, University of Würzburg, Germany. The age of controls did not significantly differ from that of patients' age at disease onset . Additional plasma samples were collected from 61 Italian patients with BPD, including 25 males and 36 females, mean age±SEM at sampling: 52.35±1.63 years (range: 20–64) and 29 controls , mean age±SEM at sampling: 67.89±1.83 years (range: 50–83) at the University of Milan, Policlinico Hospital, Milan, Italy. Psychiatric diagnoses were performed through the administration of a semi-structured clinical interview for a DSM-IV-TR Axis 1 disorders (SCID-I/P) This study has been approved by the Institutional Review board of the Fondazione Cà Granda, IRCCS Ospedale Maggiore Policlinico. Details of patients and controls are shown in GRN sequence were analyzed . Chi2 was used for differences in SNP distribution between cases and controls. Bonferroni's correction was applied. The Odds Ratio (OR) was calculated along with its 95% Confidence Interval (CI). Haploview 3.2 software was used to test for LD and for differences in haplotype distribution between cases and controls. Statistical significance was estimated empirically using the bootstrap function in Haploview. Bootstrap P-values are calculated using 10000 bootstrap samples. Calculation of D' is based on block definition by Gabriel et al. Allelic and genotypic frequencies were obtained by direct counting. Chi square test was used to test for Hardy Weinberg Equilibrium (HWE,


Dietary fibers have been proposed to play a role in cardiovascular risk as well as body weight management. Flaxseeds are a good source of dietary fibers, and a large proportion of these are water-soluble viscous fibers.Here, we examine the effect of flaxseed dietary fibers in different food matrices on blood lipids and fecal excretion of fat and energy in a double-blind randomized crossover study with 17 subjects. Three different 7-d diets were tested: a low-fiber control diet (Control), a diet with flaxseed fiber drink (3/day) (Flax drink), and a diet with flaxseed fiber bread (3/day) (Flax bread). Total fat and energy excretion was measured in feces, blood samples were collected before and after each period, and appetite sensation registered 3 times daily before main meals.Compared to control, Flax drink lowered fasting total-cholesterol and LDL-cholesterol by 12 and 15%, respectively, (p < 0.01), whereas Flax bread only produced a reduction of 7 and 9%, respectively (p < 0.05). Fecal fat and energy excretion increased by 50 and 23% with Flax drink consumption compared to control (p < 0.05), but only fecal fat excretion was increased with Flax bread compared to control (p < 0.05).Both Flax drink and Flax bread resulted in decreased plasma total and LDL-cholesterol and increased fat excretion, but the food matrix and/or processing may be of importance. Viscous flaxseed dietary fibers may be a useful tool for lowering blood cholesterol and potentially play a role in energy balance.NCT00953004ClinicalTrials.gov:  Prospective cohort studies suggest that consumption of dietary fibers protects against coronary heart disease , althougDietary fibers may also play a role in body weight regulation, through both hunger suppression and diminished nutrient absorption . AlzuetaIn the present study we test the hypothesis that addition of flaxseed fibers to a controlled diet will increase excretion of fat and energy, lower blood cholesterol and suppress hunger. Furthermore, a potential difference due to food types will be explored as the flax dietary fiber will be given both as a viscous drink and baked into bread.http://clinicaltrials.gov database (NCT00953004).Seventeen young subjects (10 women and 7 men) were recruited through advertising at university campuses in the Copenhagen area. The exclusion criteria were as follows: known chronic illnesses , smoking, exceesive physical activity (> 10 h/week), regular use of medication , use of dietary supplements and food intolerances or dislikes of relevance to the composition of the meals. All study subjects gave written consent after having received verbal and written information about the study. The study was carried out at the Department of Human Nutrition, Faculty of Life Sciences, University of Copenhagen, Denmark, and was approved by the Municipal Ethical Committee of The Capital Region of Denmark in accordance with the Helsinki declaration (KF 01-309595) and registered in the The study had a double-blind randomized crossover design, where three different iso-caloric diets were examined during three 7-d dietary intervention periods separated by ≥1 week washout. All food was provided by the Department of Human Nutrition, in daily portions matching the individual subject's energy requirement (ER). Most of the food was pre-portioned. The meals were ready-prepared and only had to be heated up. The subjects were instructed to follow the diet plan very strictly, report any deviation from the diet plan and to maintain their habitual activity level throughout the study. On days 1 and 8 in each intervention period, the subjects came to the Department after an overnight fast (> 10 hours) and abstention from alcohol and physical exercise for 24 hours. Their body weight in underwear was measured to the nearest 0.1 kg  and height was assessed at baseline to the nearest 0.5 cm using a wall-mounted stadiometer . Blood pressure measurements were performed on the right arm in the supine position after 10 min of rest with an automatically inflated cuff . Two measurements with at least one minute in between were performed and a mean value was calculated at each visit. Hereafter blood samples were taken. All feces were collected in pre-weighed plastic containers during the last 5 days of each diet period. Transit time was measured by using non-absorbable radio-opaque transit markers. With breakfast on days 1-5 of each diet period, the subjects ingested capsules containing transit markers (20 markers per day), differing in size and shape for each day. Transit time was determined as described elsewhere [ad libitum, and a maximum of two cops of coffee or tea daily. Three daily menus were prepared to vary the diet, but all subjects consumed the same standardized diet on each day. The breakfast meals were served at the Department of Human Nutrition, while the lunch and evening meals were provided for home consumption. On Fridays, the subjects were provided all foods for consumption at home during the weekend. Besides the standardized diet, the subjects were provided with drinks or breads containing a non-fiber thickener (modified corn starch (MCS))  or flaxseed fiber  three times daily as follows: Control period: MCS drink and plain breads; Flax drink period: Flaxseed fiber drink and plain breads; Flax bread period: MCS drink and Flaxseed fiber breads.A standardized diet was provided for the subjects consisting of breakfast, lunch and evening meals, as well as fruits and snacks. The subjects were allowed to consume water MCS and Flaxseed fibers were provided as powders to be mixed with water and blackberry syrup for immediate consumption 30 minutes prior to all three main meals. The Flaxseed fibers were baked into breads which were consumed as part of the three main meals. No MCS breads were made, as the plain breads were similar in appearance to the Flax breads, whereas the viscosity of Flax drinks needed to be disguised. The dose of MCS was 12 g/10 MJ and the dose of Flaxseed extract was 7.5 g/10 MJ (providing 5.2 g dietary fibers/10 MJ), and were adjusted according to ER.The energy distribution of the standardized diet including Flax dietary fiber/MCS was as follows: ~15E% from protein, ~29E% from fat, and ~56E% from carbohydrates (Table -1 and temperatures of 10 (temperature at ingestion) and 37°C . Flax dietary fiber and MCS was mixed with cold water and blackberry syrup until fully suspended and measurements were preformed immediately after preparation of the solutions. Viscosities at 30 s-1 was compared along with the overall viscosity behavior approximated with a flow behavior index n from the Power Law model as SS = c SRn where SS (Pa) is shear stress and SR (s-1) is shear rate.Measurement of viscosity was performed with a StressTech rheometer  using a cup (26.0 mm) and bob (25.0 mm) geometry over a shear rate range of 1-100 sTo assess appetite sensation, visual analogue scales (VAS) were used. They are 100 mm in length with words anchored at each end, expressing the most positive and the most negative rating, and were used to assess hunger, satiety, fullness, thirst, plus well-being. The subjects were instructed to fill in these questionnaires three times daily before consumption of the drinks . The use of VAS to assess subjective appetite sensation has however only been validated for use in postprandial single meal studies .At the end of each period, the subjects evaluated gastrointestinal discomfort. They were asked to report whether they had experienced any of the following during the last week: heart burn, reflux, bloating, nausea, stomach pain, stomach rumbling, stomach gas, diarrhea or constipation as well as the severity of these .XT15 Extraction System, ANKOM Technology, NY, USA).Before analysis, the fecal samples were freeze-dried and homogenized. For each subject, all samples from the same diet period were pooled. Fecal energy was measured by bomb calorimetry . Total fecal fat was determined using the ANKOM filter bag technique, where fecal samples are acid hydrolyzed with 3 N HCl at 90°C for 1 h, and fat was extracted using petroleum ether , whereas the dietary fiber content of the flax fiber was measured using AOAC method No. 985.29. Energy and fat content of the three test diets were measured in the same way as described for the fecal samples.nd gene-ration, Roche Diagnostics GmbH, Mannheim, Germany); intra-assay precisions were 1.8%. All analyses were performed on a COBAS MIRA Plus . LDL-cholesterol was calculated using the Friedewald equation  [Plasma glucose was measured with the use of an enzymatic endpoint method (Hexokinase)  using an ABX Pentra 400 chemistry analyzer ; intra-assay coefficient of variation (CV) was 1.4%. Concentrations of triacylglycerols and total cholesterol were assessed using colorimetric test kits ; intra-assay variations were 0.6% and 0.9%, respectively. HDL-cholesterol was measured using a homogeneous enzymatic colorimetric test kits (Roche HDL-C plus 2lycerol) . InsulinAll statistical analyses and calculations were performed using the Statistical Analysis System software package, version 9.1 . All dependent variables were controlled for homogeneity of variance and normal distribution by investigation of residual plots, normal probability plots and histograms. Analysis of co-variance (ANCOVA) was performed using the PROC MIXED procedure to compare baseline values between the three dietary intervention periods, in which period and diet were modeled as fixed variables; sex and energy intake as covariates and subject was included as a random variable. An ANCOVA analysis was used to investigate the effect of diet, where subject was modeled as a random variable and sex, energy intake, corresponding baseline values and body weight changes were modeled as covariates, and period as well as period × diet interaction were included as fixed variables. A similar model was applied to investigate the effect of diet on body weight, fecal parameters and mean appetite scores, however, without a corresponding baseline value  and using baseline body weight as a covariate. For all analyses, period × diet interaction was only omitted from the model when p > 0.1. Posthoc pairwise comparisons between diets were made when effect of diet was significant. Results are presented as means ± SD and the statistical significance level defined as p < 0.05.2. The subjects' estimated ER ranged from 9 to 17 MJ/d (Table Sixteen subjects (seven men and nine women) completed all three diet periods, and one subject (woman) completed only two diet periods and dropped out due to dislike of the diet. The mean age of the subjects was 24.8 ± 3.3 years and mean BMI was 23.8 ± 1.7 kg/mn≈1 the MSC drink was Newtonian whereas the flax drink was shear thinning (n = 3). At physiological temperature (37°C) the viscosity of the flax drink dropped to 361 mPas, whereas the MSC drink remained at same level. Both solutions exhibited same flow behavior as for 10°C.The viscosity of the reference MCS and Flax drink was 6 mPas and 528 mPas, respectively, at the moment of ingestion (10°C). With a flow behavior index of Flax drink lowered fasting total-cholesterol and LDL-cholesterol by 12 and 15%, respectively, compared to Control (p < 0.01), whereas smaller declines were observed for Flax bread in comparison to Control  Figure .Daily fecal volume and excretion of DM did not differ between diets. However, fecal excretion of energy was affected by diet (p < 0.05). Post-hoc pairwise comparison showed that a 23% (~129 kJ/d) greater fecal excretion of energy was seen with consumption of Flax drink compared to the Control (p = 0.05), and the proportion of energy excreted of intake increased correspondingly (p < 0.05). Also, fecal fat excretion was affected by diet (p < 0.01) as 4.96 ± 0.31 g fat/d was excreted when consuming the Flax drink diet as compared to only 3.20 ± 0.33 g fat/d with the Control diet, corresponding to a 55% increase. The percentage of fat intake excreted increased correspondingly (p < 0.01). No significant effect of Flax bread was observed on any of the fecal parameters compared to the Control diet. Transit time was not significantly affected by diet.There was no difference in the appetite ratings between days, thus data from day 2-7 were pooled  and are presented as mean ratings Figure .The results of the present study demonstrate that consumption of 5 g of dietary fibers from flaxseeds daily for one week significantly increased fecal excretion of fat and reduced total and LDL-cholesterol markedly. Despite similar doses of dietary fiber, the effect of the dietary fiber was less pronounced when incorporated into bread than when administered as a drink, which emphasizes the importance of food matrix.We observed a lowering of both total-cholesterol and LDL-cholesterol by 12 and 15%, respectively, within just seven days in young healthy adults with normal blood cholesterol concentrations. It can be speculated that the effect may be overestimated as the short intervention period does not allow for equilibrium to occur, but the short duration was chosen due to the high level of controlled diet and the fact that fecal parameters were the main end point. However, in support of our findings, effects of similar magnitude are reported in a recently published study, in which 5 g of flaxseed gum per day for three months reduced total and LDL cholesterol by 10 and 16%, respectively in type 2 diabetics , althougFecal energy excretion increased by 129 kJ/d with Flax drink consumption compared to Control. The amount of fat and energy which escapes digestion may appear small in numbers, but corresponds to ~47 MJ excreted per year and thus of relevance in the prevention of weight gain as a decrease in energy uptake of this magnitude equals a difference in body weight of ~1.6 kg. The increase in fecal energy and fat excretion is in accordance with our previous studies on flaxseed fibers showing a reduction in energy digestibility and weight gain in growing Wistar rats fed a diet with 10% of flaxseed dietary fibers . Studies using other viscous dietary fibers have shown similar results -27, and Appetite sensation was assessed three times daily throughout the present study, and there was an overall trend towards increased fullness during the Control period compared to both Flax drink and bread. This was unexpected as we have previously observed that flax fibers significantly increases both satiety and fullness  and no sOne of the objectives of the present study was to explore the effect of food matrix on physiological responses. We found that the effect of Flax bread was less pronounced than with a similar dose of dietary fiber provided as a drink, both with regard to cholesterol-lowering properties and reduction of apparent fat digestibility, although only the latter was significantly different between Flax drink and Flax bread. An effect of food matrix was also seen in a study, in which β-glucan enriched juices produced a more pronounced effect on total and LDL-cholesterol compared to β-glucan enriched cookies , but in In conclusion, we studied the effect of flax fiber-enriched drinks and breads on fecal energy and fat excretion, blood lipids as well as subjective appetite sensation. We found that addition of a flax dietary fiber extract rich in viscous dietary fibers significantly increased fat excretion and lowered total and LDL-cholesterol although no effect on appetite was observed. Viscous flaxseed fibers appear useful for lowering blood cholesterol and may play a role in energy balance; however, food type and/or processing may be of importance.The University of Copenhagen has a patent pending concerning the present research and Arne Astrup is a consultant for Basic Research, Salt Lake City, Utah, USA. All other authors have no competing interests.MK participated in design of the study, performed the statistical analyses and drafted the manuscript. MGJ participated in design of the study. JA, KENP and LS performed the experimental work, MSM performed the viscosity measurements. AA participated in design of the study All read and approved the final manuscript.


Vibrio diabolicus and Alteromonas infernus was previously demonstrated using a bioproduct manufacturing process. The objective of this study was to determine which sterilization method, other than heat sterilization, was the most appropriate for these marine exopolysaccharides and was in accordance with bioprocess engineering requirements. Chemical sterilization using low-temperature ethylene oxide and a mixture of ionized gases (plasmas) was compared to the sterilization methods using gamma and beta radiations. The changes to both the physical and chemical properties of the sterilized exopolysaccharides were analyzed. The use of ethylene oxide can be recommended for the sterilization of polysaccharides as a weak effect on both rheological and structural properties was observed. This low-temperature gas sterilizing process is very efficient, giving a good Sterility Assurance Level (SAL), and is also well suited to large-scale compound manufacturing in the pharmaceutical industry.Polysaccharides are highly heat-sensitive macromolecules, so high temperature treatments are greatly destructive and cause considerable damage, such as a great decrease in both viscosity and molecular weight of the polymer. The technical feasibility of the production of exopolysaccharides by deep-sea bacteria  Carbohydrates, particularly those found on cell surfaces, play critical biological roles, such as glycoconjugates or proteoglycans and especially their polysaccharidic chains called glycosaminoglycans. The discovery of the biological importance of these carbohydrates marked the beginning of glycobiology, glycomics, carbohydrate-based drug discovery and glycomimetic drug development ,2. Bacteetc.) is possible naphthothiazolium (Sigma)) prepared as follows: 5 mL of 0.001% (w/v) Stains All solution in dimethylformamide; 5 mL of 300 mM Tris-HCl pH 8.8; 5 mL of dimethylformamide; 25 mL of isopropanol, and 60 mL of H2O .−1. All the spectra were corrected from H2O and CO2 absorptions, using OMNIC software (Thermofischer Scientific).Pellets were obtained by careful grinding of a mixture of 2 mg of polysaccharide with 200 mg of dry KBr. Infrared spectra were recorded with a Magna IR 550 Fourier Transform Infrared Spectrophotometer (Nicolet) equipped with a DTGS detector with a resolution of 4 cmSterilization efficiency was evaluated using a pour plate method. Every polysaccharide (0.2-0.4 g) was aseptically weighted, dissolved with 20 mL of sterile water and left at room temperature for 24 h for resuscitation and solubilization of polymers. The solutions were then serially diluted and 0.1 mL of each appropriate dilution was placed in an empty sterile Petri dish followed by the addition of molten agar tempered to 45 °C. For total flora (marine and non marine flora) 15 mL of PCA (Plate Count Agar) medium  was used. For marine flora, 15 mL of ZoBell medium (2216E medium) composed of sea-salts , yeast extract and tryptone  were added. After swirling and solidification of the agar plates, the latter were incubated aerobically for 48 h at 37 °C to develop colonies. Colonies were then counted and the data recorded. −3 µg in 100 µg of EPS or <30 ppm). As positive and negative controls, RAC were also cultured in absence of polysaccharides or in the presence of actinomycin-D (5 µg/mL), an inhibitor of RNA polymerase, which was used as a potent inducer of cell death. Samples were incubated at 37 °C, 5% CO2. After 24 h, 48 h or 72 h of culture, culture media were removed. A Tetrazolium Salt (MTS) test  to evaluate their mitochondrial activity was carried out. For the latter, a MTS solution was added in each well for 1 to 3 h according to the manufacturer’s instructions. The optical density of formazan dye was measured on a spectrophotometer at 490 nm. Each condition was tested in quadruplicate and the results were expressed as relative MTS activities as compared to the positive control.To evaluate whether the level of ethylene oxide residue in treated polysaccharides was toxic, cellular viability of primary articular cartilage cells from rabbits (RAC) was measured using an MTS assay. RAC were isolated from five-week-old New Zealand white rabbits (Grimaud) as previously described by . RAC werP < 0.05.Results are expressed as mean ± of quadruplicate determinations. Comparative studies of means were performed by using one-way ANOVA followed by post-hoc test (Fisher’s projected least significant difference) with a statistical significance at In our study, the results show that gamma or beta irradiation is completely unsuitable for the sterilization of the polysaccharides examined. For both EPSs, radiation induces great alterations in their physical structure with a loss of their rheological polymer properties and morphological structure, as demonstrated by a considerable decrease in their viscosity and molecular weight respectively. Surprisingly, radiation did not alter the chemical properties of the EPSs studied. The results obtained with cold plasma treatment were disappointing. This method is often described as a novel alternative for the sterilization of heat-sensitive substances. Our results showed that cold plasma treatment induces changes in both rheological and structural properties of the EPSs, with a decrease in their viscosity and molecular weight respectively. Our results also showed that the most adequate sterilization method for the sterilization of polysaccharides is EO treatment. This method provides both a good SAL and the lowest physical and chemical damage to the EPSs. The results obtained with EO are acceptable and compatible with subsequent use of these EPSs to investigate their therapeutic potential in animal models. Currently this treatment enables: (i) preservation of the non-Newtonian pseudo plastic behavior characteristic of both treated EPSs; and also (ii) retention of a high molecular weight close to the untreated EPSs. This study highlights the importance of the choice of the sterilization method, as an essential part of any product development.


After six infusions of AIET, the carcinoembryonic antigen (CEA) level was decreased from 901 to 437 U/ml, regression of lesions was noted and there were no adverse reactions during the course of this therapy. Thus, AIET may be a promising anticancer approach to eradicate tumor cells with other conventional therapies.Current modalities of cancer treatment, including surgery, chemotherapy and radiotherapy, show marginal therapeutic responses in cancer patients. In adoptive immunotherapy, interleukin-2 (IL-2) activated immune cells demonstrated notable results in patients with advanced malignant disease. The present study reports the efficacy and safety of repetitive infusions of autologous immune enhancement therapy (AIET) in a stage IV colonic cancer patient who had already received first-line chemotherapeutic drugs. Peripheral blood was aspirated from the patient. Specifically, natural killer (NK) cells and T-lymphocytes were isolated from the peripheral blood mononuclear cells (PBMCs). These cells were activated and expanded  Colorectal cancer is statistically recorded as the second leading cause of mortality in Western countries and ∼50% of mortalities which occur as a result of this type of cancer are associated with progression . In coloImmunotherapy is based on the principle that the host immune system is capable of generating immune responses against cancer cells. There are plethoras of natural killer (NK) cells and antigen-induced cytotoxic T-lymphocytes (CTLs) in the human immune system. Initial efforts to generate anticancer immune responses have focused on adaptive immunity, particularly on the induction of tumor-specific CTLs. While it is appreciated that tumor-specific CTLs are critical for successful immunotherapy, it is becoming increasingly apparent that cellular components of the innate and adaptive arms of the immune system are able to control tumor growth .in vivo.NK cells are a component of the innate immune system, which perform immunosurveillance via the recognition of altered or missing ‘self’ surface markers in damaged, infected or transformed malignant cells ,11. NK cThe main functions of NK cells are to suppress tumor cell initiation, growth and metastasis through mechanisms mediated by perforin and the granzyme-containing granule-mediated death receptor and interferon-γ-mediated pathways . These vin vitro derived NK cells and CTLs, which may be adoptively transferred to patients via a painless procedure for cancer treatment  in 1,205 patients with metastatic cancer who had undergone different types of active specific immunotherapy (ASI).Autologous immune enhancement therapy (AIET) has had a successful clinical history in Japan, Europe and the USA over the past two decades. Immune cell therapy using autologous activated lymphocytes ,19 was fIn the present study, we report the results from a colorectal cancer patient who underwent 6 infusions of immune cell therapy.KRAS mutation. CT scans revealed a malignancy in the liver and the carcinoembryonic antigen (CEA) level was 500 U/ml. The patient was administered FOLFOX-6 between June 2008 and September 2008. The disease progressed and selective internal radiation therapy (SIRT) was administered. After the therapy, the patient remained healthy for 2 months and the CEA level began to increase. Thus, Bevacizumab and FOLFIRI was restarted between November 2008 and April 2009. The patient was unable to tolerate the chemotherapeutic side effects and opted to receive AIET.A 50-year-old female diagnosed with stage IV colonic cancer in May 2008 presented with a 6 and 19.5×106 for NK cells and TLs, respectively. After 2 weeks of expansion, the average number of expanded lymphocytes for six injections was 14.4×108 for NK cells and 32.9×108 for TLs. The average frequency of NK (CD3−/CD56+) cells was 6.3% initially and 40.5% at final culture. For TLs (CD3+), the average frequency was 88.9% initially and 62.1% at final culture, as evaluated by flow cytometry. The total infused cell numbers are shown in After informed consent was obtained from the patient, 60 ml peripheral blood was withdrawn. The activated NK cells and TLs were isolated from the peripheral blood mononuclear cells (PBMCs) of the patient, and later were activated and expanded according to previously described methods , for 14 After 6 infusions of NK cells and TLs, the CEA level decreased considerably from 901 to 437 U/ml . The CT ex vivo adoptively transferred, activated and expanded NK cell s and TLs derived from the peripheral blood of a colorectal cancer patient. The patient was followed up for 8 months from the first infusion of AIET. CEA levels were recorded before and after injection of the AIET  was not administered. Following standard procedures, the effector cells were cultured with IL-2, which ranged from 350 to 700 IU/ml over the duration of 0–14 days. For all six infusions, the fold expansion of lymphocytes from PBMCs was ∼44 and 168 for NK cells and TLs, respectively. With regard to this fold expansion, it was concluded that the total number of activated and expanded cells are highly dependent on the initial lymphocyte population of PBMCs of the whole blood. Notably, inverted phase contrast microscopy (data not shown) revealed that cell viability and the frequency of the healthy population was clear in each sample processing during the culture of PBMCs.The expanded cells were administered intravenously. We determined that the infusion of heterogeneous TL and NK cell populations induced a decrease in tumor mass in this case, as shown by the CT scan results . We concin vitro studies (Several case reports demonstrate that CTLs and NK cells improve the cytotoxic effects in cellular immune responses to similar levels as those identified in  studies –23. NK c studies ,24.+ T cells are correlated with an effective antitumor response (CTLs also play a critical role in the adaptive immune system. CTLs and NK cells secrete granzyme and perforin, which are packaged in cytoplasmic granules  to lyse response , patientresponse  and the response .in vitro and in vivo. Todaro et al(ex vivo derived CTLs and γδT cells. CTLs and γδT cells kill their targets via the secretion of perforin and granzyme B, which supports the clinical observations in the present case.A number of studies support the sensitivity of CTLs against colorectal cancer aro et al showed tin vivo, whether γδT cells are able to effectively recognize cancer cell populations is questionable. This has been clarified by a large cohort study. Ogino et al(However, the cancer stem cell population and its dysfunction in the presence of CTLs and γδT cells requires further investigation in clinical studies. Due to the minor population of γδT cells ino et al showed tNumerous studies which explored NK cells and TLs have demonstrated the impact of immune contexture in human tumors and its clinical outcome. In a study involving colon cancer patients with a history of surgery, 20–25% of patients had recurrence of their disease, suggesting that occult metastases are already present at the time of surgery . Adjuvanex vivo clonal expansion of CD8+ T cells and CD4+ T cells to activate and endure CD8+ killer cells (ex vivo derived therapies are effective with a reasonable dose of IL-2 or other combined therapies (These results encourage most attempts at cancer immunization, which directly involve er cells . A numbeherapies ,35. Althherapies .In conclusion, considering the immune contexture, CTLs and NK cells may be useful tools for the benefit of patients by enhancing their internal tumor infiltrating lymphocytes. Adoptive therapy demonstrated long-term disease-free survival, a notable improvement in quality of life and also an improved overall survival and therefore, may be recommended to patients with no other treatment options. Although AIET has been shown to benefit patients, longer follow-up is required before a final conclusion may be determined.