The p53 homologs, p63 and p73, share ∼85% amino acid identity in their DNA-binding domains, but they have distinct biological functions.in vivo, suggesting that p63 and p73 bind primarily as heterotetrameric complexes in ME180 cells.Using chromatin immunoprecipitation and high-resolution tiling arrays covering the human genome, we identify p73 DNA binding sites on a genome-wide level in ME180 human cervical carcinoma cells. Strikingly, the p73 binding profile is indistinguishable from the previously described binding profile for p63 in the same cells. Moreover, the p73∶p63 binding ratio is similar at all genomic loci tested, suggesting that there are few, if any, targets that are specific for one of these factors. As assayed by sequential chromatin immunoprecipitation, p63 and p73 co-occupy DNA target sites The observation that p63 and p73 associate with the same genomic targets suggest that their distinct biological functions are due to cell-type specific expression and/or protein domains that involve functions other than DNA binding. Eukaryotic organisms contain families of DNA-binding transcription factors comprised of structurally related proteins that are encoded by different genes. Individual members of the family are often co-expressed in the same cell, and in many cases they can associate with each other to generate heteromeric transcription factors. In addition, transcription factor families can exhibit cross-regulation, in which one family member affects the expression and/or function of another family member. In general, individual transcription factors within the family have both distinct and overlapping biological functions.An important transcription factor family in mammalian cells includes the p53 tumor suppressor and two other proteins, p63 and p73, that are strikingly similarity to each other and less similar to p53 Despite the very high degree of similarity between p63 and p73, mouse knockout models reveal distinct and non-redundant physiological roles. p63-deficiency is associated with severe defects in epithelial development in vivo, either due to subtle differences in their DNA-binding domains and/or to differences in cooperative interactions with other DNA-binding proteins. Third, the two proteins can have functionally distinct domains that differentially mediate transcriptional activation or repression, interactions with co-activators or co-repressors, or interactions with other regulatory proteins. In cases where the proteins themselves are functionally distinct, the differences could be intrinsic to the protein sequence and/or reflect differences in phosphorylation or other post-translational modifications.There are multiple explanations, not mutually exclusive, for how two highly related members of the same protein family can have distinct biological functions. First, differences in tissue- and cell type-specific expression patterns can underlie distinct biological functions, even if the proteins are functionally equivalent. Differences in expression patterns might involve some or all of the structurally distinct isoforms that arise via alternative splicing, promoter usage, or 3′ end formation. Second, the two proteins can have distinct target specificities in vivo binding behavior of highly related transcription factors in the same cells has rarely been examined in a global, unbiased manner. In the case of the ETS family of transcription factors, analysis of in vivo binding using genome-wide promoter microarrays revealed redundant and specific occupancy by individual members of the family in vivo, albeit with different kinetics that may underlie differences in Stat5 biology The in vivo target specificity to that of p63. We show that the p73 and p63 binding profiles are indistinguishable, with a similar p73∶p63 binding ratio at essentially all genomic loci. Furthermore, we show that p63 and p73 co-occupy DNA target sites in vivo, suggesting that p63 and p73 bind primarily as heteromeric complexes. The observation that p63 and p73 directly associate with the same set of genomic targets suggests that their distinct biological functions are due to cell-type specific expression and/or protein domains that involve functions other than intrinsic or cooperative DNA binding to target sites.In previous work, we used tiled microarrays covering the human genome to identify ∼5800 target sites for p63 in ME180, a cervical carcinoma cell line ME180 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum. Chromatin immunoprecipitation was performed with a mouse monoclonal antibody to p73 (11C12), essentially as described previously 8 cells was immunoprecipitated with the 4A4 anti-p63 or 11C12 anti-p73 antibodies as described above. 10% of the eluted material was removed, de-crosslinked, and designated “1st IP.” The remaining eluate was incubated with antibody-coupled protein A/G sepharose beads , BSA (5 mg/ml), phage lambda DNA (25 µg/ml), and E. coli tRNA (50 µg/ml) in a total volume of 2 ml IP dilution buffer (approximately 10-fold dilution of eluate). Washes and elution were performed as described above, and eluted samples designated “2nd IP.” Precleared chromatin from the 1st IP was used as “input” DNA for both 1st and 2nd IP samples.These were performed essentially as described in GTTTCCCAGTCACGGTCNNNNNNNNN), using the following cycling conditions: 95°C, 4 min; 10°C, 5 min; +27°C at 1°C per 20 sec; 37°C, 8 min. Round A material was purified using column purification (Qiagen PCR Purification kit) and PCR amplified with primer B(GTTTCCCAGTCACGGTC). PCR program used was: 95°C, 3 min; followed by 30 cycles of 95°C, 30 sec; 40°C, 45 sec; 50°C, 45 sec, 72°C, 1 min; and a final extension at 72°C for 10 min. The samples were then purified using column purification (Qiagen PCR Purification kit) and ready for array hybridization protocols.Input and ChIP DNA was amplified by four rounds of primer extension (Round A) with random primers  one oligonucleotide pair every 35 bp. There are 7 chips in a full genome set and approximately 3,200,000 probe sets per chip (PM probes only). Array data from three biological replicates were scaled to target intensity of 500 and quantile normalized using Affymetrix Tiling Analysis Software (Version 1.1.02). A binding p-value was then determined for each genomic position by Wilcoxon rank sum test and binding sites were generated from those more significant than specified thresholds with a maximum gap of 500 and minimum run of 350. For every binding site, a binding enrichment score was computed from a smoothed “peak” estimator using the five genomic positions with the highest binding p-values (in the form of -10logP) within the region and one-step Tukey's biweight alogorithm. Data for p73 binding is stored at GEO (GSE18650).−(ΔCTexpt-ΔCTref) where ΔCT is the cycle threshold (Ct) difference between ChIP DNA and input material, calculated for experimental and reference regions, and 1.9 is the mean primer slope. For each site, we calculated the occupancy units defined as the fold enrichment value minus background . Based on our previous observations of p63 occupancy for various control negative regions qPCR was performed essentially as described previously de novo motif discovery algorithm MEME For every binding site, we retrieved repeat-masked sequence and used Based on the multiz-8-way alignments for human, chimp, mouse, rat, dog, chicken, fugu and zebrafish Immunoblotting was performed with the 4A4 anti-p63 and 11C12 anti-p73 antibodies using standard procedures. Briefly, proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, blocked in 5% milk , and incubated with primary antibody followed by a horseradish peroxidase conjugated anti-mouse secondary (Jackson ImmunoResearch Laboratories). Chemiluminescent detection was performed with the SuperSignal Pico Chemiluminescent Substrate (Pierce) according to manufacturer's instructions.−5, the same cut-off used for the p63 analysis previously described Chromatin from ME180 cells was immunoprecipitated with an anti-p73 monoclonal antibody (11C12) that shows no detectable cross-reactivity with p63 . The immP≤10−5 were verified as “true positives,” defined as showing at least an average 2.5-fold enrichment in three biological replicates, relative to a negative control region. We also tested regions with lower binding scores and verified four of five sites for 10−5≤P≤10−4, and eight of nine targets for 10−4≤P≤10−3  to verify p73 enrichment at several targets identified from the ChIP-Chip experiment. Five of five sites with 4≤P≤10−3 . These r−5) located within 1 kb and 5 kb upstream of the transcriptional start, respectively. p73 binding sites included previously reported p73 targets, such as the PUMA, mdm2, and p63 genes, and they show strong evolutionary conservation , supporting the similarity between p63 and p73 binding, and a further indication that sites in this range are bona fide p73 targets. A comparison of p63 and p73 binding enrichment scores shows a strong correlation (Pearson correlation = 0.414), with p63 generally showing higher scores than p73 (Comparison of the p73 binding profile with the previously described p63 binding profile ous work . The perthan p73 . These o−5 cutoff used previously to define p63 targets −5 cutoff) that had very low levels of p63 occupancy (P<10−2). In all such cases tested, these putative p73-only sites showed no detectable levels of either p63 or p73, indicating that these were false positives from the p73 array results. Most importantly, for all sites tested, the relative occupancy of p63 and p73 appeared similar, with p63 enrichment being approximately 2–4 fold higher than that of p73  genomic targets. Such co-occupancy indicates that p63 and p73 can bind to their targets as heteromeric complexes. As p63 and p73 form stable heterotetramers in solution The ability of p63 and p73 to bind as heterotetramers provides a simple explanation for why they have indistinguishable binding specificites in ME180 cells. However, our experiments do not indicate that the similar target profiles of p63 and p73 are due exclusively to binding by heterotetrameric complexes. It is possible that p63 and p73 homotetramers have similar target specificity to each other and likely  to the heterotetrameric complexes.Numerous genome-wide ChIP experiments reveal that fold-enrichments for association of a given protein to target sites vary over a wide range. This indicates that most target sites are not fully occupied by the transcription factor, and hence that the concentration of the protein is limiting for binding, except perhaps for the strongest sites. As a consequence, and as observed in our previous work on p63 As p63 and p73 can bind as a heteromeric complex, the difference in protein levels affects the stoichiometry of the complexes. As these proteins, like p53, bind as tetramers, p73 will be typically under-represented in hetero-tetramers, and there should be a significant number of p63 homo-tetramers in ME180 cells. In accord with theoretical considerations of sequential ChIP experiments Mutational analysis in mice indicates that p63 and p73 control very different physiological processes Other cell types express either p73 or p63, but not both. In such cell types, phenotypic effects can only be observed when the gene encoding the expressed protein is mutated. Similarly, if one protein is much more prevalent than the other, as is the case in ME180 cells, it is likely that mutation of the more abundant protein will cause stronger phenotypic effects. Thus, many, and perhaps most, of the distinct biological functions of p63 and p73 are likely to reflect differences in cell-type-specific expression.The DNA-binding profiles of p73 and p63 in cell types that express only one of these proteins are unknown. In such cell types, it is likely, that p63 and p73 will associate with some sites in common, but interact differentially with other genomic regions. However, individual DNA-binding proteins typically have cell-type specific binding profiles, because binding to chromatin templates in mammalian cells generally requires cooperative interactions with other proteins Our findings address reports of p63 and p73 antagonism in head and neck squamous cell carcinomas Table S1(1.02 MB PDF)Click here for additional data file.Table S2(0.06 MB PDF)Click here for additional data file.Figure S1Figure S1(0.27 MB PDF)Click here for additional data file.


The ability to select an action by considering both delays and amount of reward outcome is critical for maximizing long-term benefits. Although previous animal experiments on impulsivity have suggested a role of serotonin in behaviors requiring prediction of delayed rewards, the underlying neural mechanism is unclear.To elucidate the role of serotonin in the evaluation of delayed rewards, we performed a functional brain imaging experiment in which subjects chose small-immediate or large-delayed liquid rewards under dietary regulation of tryptophan, a precursor of serotonin. A model-based analysis revealed that the activity of the ventral part of the striatum was correlated with reward prediction at shorter time scales, and this correlated activity was stronger at low serotonin levels. By contrast, the activity of the dorsal part of the striatum was correlated with reward prediction at longer time scales, and this correlated activity was stronger at high serotonin levels.Our results suggest that serotonin controls the time scale of reward prediction by differentially regulating activities within the striatum. R after delay D is given byG(D) is a discounting function that decreases with delay D. A steep rate of discounting results in impulsive choice, defined by an abnormally frequent choice of the more immediate reward When hungry and looking for a restaurant, do you choose a well-reputed restaurant with many people waiting in line or a fast food restaurant where you can have a quick but perhaps less palatable meal? In our daily life, we constantly make such choices between actions leading to rewards of various sizes after different delays. “Delay discounting” is a theoretical concept in which the “value” of reward P<0.0001; multiple comparison) than those under the control condition, while the levels of those under the trp+ condition were significantly higher (P<0.0001)  see . Based o001) see .Under each tryptophan condition, all subjects performed a multi-step delayed reward choice task in an fMRI scanner : the subject predominantly chose large rewards when the delay to the large reward was relatively short (bottom right part of the s-DlD space), but preferred small rewards when the delay to the large reward was relatively long (top left part). No subject adopted an extreme strategy of choosing only small or large rewards in any session under any tryptophan condition (Subjects performed eight sessions lasting three minutes each. ondition . There wondition .V(t) represents a discounted future reward R  acquired at time step T and evaluated at time step t. Because V(t) decreases exponentially with delay D = T-t, it grows exponentially as time t approaches T  with six different settings of γ  and used each of them as explanatory variables in a regression analysis.The levels of tryptophan significantly affected subjects' brain activity. We performed model-based fMRI data analyses based on an exponential discounting model see :which waes T see . DiscounV(t)  of BOLD signals with V(t) at all γ values (0.6≤γ≤0.99) in the striatum , with a ventral to dorsal gradient (−4≤z≤28) from small to large γ. In the tryptophan depletion condition  of BOLD signals with V(t) only at smaller γ values  in the ventral parts of the striatum (−12≤z≤−4). Conversely, in the tryptophan loading condition  with V(t) only at larger γ values  in the dorsal parts of the striatum (16≤z≤28). These ventro-dorsal gradients could not reflect slice-timing effects within single scans as we sought a correlation with regressors varying across multiple scans, and also due to the fact that we acquired images by interleaved scanning (see V(t) with steeper discount rate only in the ventral putamen, and significant correlation of V(t) with slower discount rate only in the dorsal putamen and caudate nucleus.We found that blood oxygen level-dependent (BOLD) signals in the striatum and the cortex correlated significantly with estimated V(t) see . Under tning see . We alsoning see . We founV(t) computed with small and large values of γ (0.6 and 0.99). While the activity of the ventral ROI  correlated with V(t) with γ = 0.6 was strongest under the tryptophan depletion condition (V(t) with γ = 0.99 was strongest under tryptophan loading (P = 0.0215 in a one-tailed one-sample t-test), while the betas at large γ (γ = 0.99) showed no significance (P = 0.184). In the dorsal ROI, the betas at large γ showed a significantly increasing relationship with tryptophan levels (P = 0.010), while those at small γ showed no significance (P = 0.118).To quantify the differential modulation of the ventral and dorsal parts of the striatum by tryptophan levels, we set regions of interest (ROI) in the ventral and dorsal parts of the striatum see  and compondition , the act loading . These rP = 0.29, with anatomically defined ROI of V1 To check for any effects of tryptophan levels on reward-unrelated brain activities or vascular responses, we investigated the event-related responses of the visual cortex at the beginning of each trial. We found no significant differences in the responses under different tryptophan conditions  and Hiroshima University. On the screening day, trained psychiatrists interviewed each volunteer and screened for previous psychiatric problems by using the Structured Clinical Interview for DSM-IV (SCID). We excluded volunteers with any previous psychiatric disorders, including mood or psychotic disorders, or substance abuse. Each volunteer had a physical that included blood and urine tests, a chest X-ray and an electrocardiogram. We excluded participants with health problems or those who disliked the isotonic drink used as the reward in the experiment.Each subject participated for four days: one day for screening and task practice and three days for fMRI experiments under three different tryptophan conditions . Three days of experiments were scheduled over a minimal interval of one week to completely remove the effects of tryptophan dietary control induced in the preceding experiment. The experiment was of a counter-balanced, placebo-controlled, double-blind, within-subject design, in which a controller who was not an experimenter prepared three types of amino acid mixtures for each subject. To maximize dietary effect, subjects were instructed to consume the provided low-protein diet  24 hours before the experiment and to fast overnight before each experimental day F = 205.64, P<0.0001). After the second venipuncture, all subjects entered an fMRI scanner and performed the multi-step delayed reward choice task.On each experimental day, subjects consumed one of three amino acid mixtures  and underwent two venipunctures to determine their total plasma tryptophan concentration, which is known to be correlated with CSF serotonin levels We prepared amino acid mixtures consisting of the following quantities of 14 amino acids partially dissolved in 350 ml of water: L-tryptophan: 0 g (trp−), 10.3 g (trp+), 2.3 g (control), 5.5 g L-alanine, 4.9 g L-arginine, 3.2 g glycine, 3.2 g L-histidine, 8.0 g L-isoleucine, 13.5 g L-leucine, 11.0 g L-lysine monohydrochloride, 5.7 g L-phenylalanine, 12.2 g L-proline, 6.9 g L-serine, 6.5 g L-threonine, 6.9 g L-tyrosine, and 8.9 g L-valine. This aqueous suspension was flavored with 10 ml of chocolate syrup. In addition, 2.7 g of L-cysteine and 3.0 g of L-methionine were administered in a little water with each of the trp−, trp+ and control drinks, due to their unpalatability.SM and LM respectively, were SM = 18±9 and LM = 72±24 in all sessions. The numbers of patches added to white and yellow squares, SS and LS respectively, were varied between sessions so that subjects had to remain alert to adapt to the changes in settings:  =  in four sessions,  in two sessions, and  and  in one session each.Each experiment consisted of eight sessions lasting three minutes each. The initial numbers of black patches on white and yellow squares, SD and LD, respectively, are given by:τ is the time step (2.5 sec). Thus, the ranges of expected delays for the average values of SS and LS were:  =  in four sessions,  in two sessions, and  and  in one session each. However, because the experimental step was 2.5 sec, the actual delays were the above delays rounded to the next 2.5 sec increment.The expected delays to small and large rewards As a liquid reward, an isotonic drink  was delivered through a plastic tube from a computer-controlled pump  outside the MRI room. All subjects were told that they would receive a small drink (0.8 ml) after they completely filled the white square by selecting white squares; they would receive a larger drink (3.2 ml) when they filled the yellow square by choosing yellow squares.At the beginning of each session, “baseline” blocks (25 sec) only showed a fixation cross. Each session lasted three minutes; the entire task took about 28 minutes. Although subjects could choose either square at any step, they did not usually reverse their choice; reversals accounted for about 8% of all choices, and trials with a reversed choice were excluded from analysis.A 1.5 Tesla scanner  was used to acquire both structural T1-weighted images and T2*-weighted echo planar images  with BOLD contrast. Images were acquired with axial orientation in an interleave sequence in which even-numbered slices were first acquired from the top to the bottom of the brain, followed by the odd numbered slices. We used SPM2  for preprocessing and statistical analyses. The first five volumes of images were discarded to avoid T1 equilibrium effects. The images were realigned to the first image as a reference, spatially normalized with respect to the Montreal Neurological Institute (MNI) EPI template, and spatially smoothed with a Gaussian kernel . We entered event-related regressors at the reward delivery event to capture head motion effects caused by swallowing the liquid reward.V(t) at each step t as follows. By assuming an exponential discounting model, we estimated the discount factor γ for each subject in each condition , we set γ for the value estimation as 0.6, 0.7, 0.8, 0.9, 0.95, and 0.99. We assumed that the subjects knew the mean number of patches filled at each step s and its range of variation Δs. The range of possible steps n until the reward from the current number M(t) of black patches isV(t) as the sum of n-step discounted value Rγn weighed by probability nP(M(t)) for all possible steps n until the reward:n steps is given byWe estimated subjects' value tion see . Based oV(t) at each γ as the explanatory variable in a general linear model (GLM) by multiplying the simple event regressor (δ-function) at the timing of the stimulus presentation of each step. To remove any effects of factors other than V(t), we concurrently used other variables in the regression, namely, reward R  at reward delivery timing and a boxcar function representing baseline blocks (25 sec) for eight sessions. All explanatory variables were convolved with a canonical hemodynamic response function (HRF). For each tryptophan condition, images of parameter estimates were created for each subject and entered into a second-level group analysis using a one sample t-test at a threshold of P<0.001, uncorrected for multiple comparisons . We repeated the same process for each γ and tryptophan condition.We used estimated http://www.cns.atr.jp/multi_color/) that can overlay multiple activation maps in different colors on a single brain structure image. When a voxel is significantly activated in multiple values of γ, it is shown by a mosaic of multiple colors with apparent subdivision of the voxel.To compare the results of regression analyses with six different values of γ, we used display software (multi_color: P<0.001 (uncorrected) for the entire brain) correlations with V(t) at γ = 0.6 in trp-  =  and ; see 3, union of two clusters peak at  =  and ) within an anatomical ROI of the striatum, determined from normalized T1 images. We again used a GLM with averaged BOLD signal of all voxels within ROIs at the single-subject level and computed the value of the regression coefficient (beta) of V(t) averaged across subjects for each tryptophan condition. We used the MarsBaR toolbox for ROI analyses (http://marsbar.sourceforge.net/).We defined ROIs as clustered voxels in which we found significant (with a peak level threshold of P>0.05 in Kolmogorov-Smirnov test), and performed a one-sample t-test.To check for an increased or decreased tendency between betas and tryptophan levels, we performed nonparametric analysis (Spearman's rank correlation coefficients) for each subject. In the group (second level) analysis, we found a significant normality of distribution of data for four groups, 1) γ = 0.6 in the ventral ROI, 2) γ = 0.99 in the ventral ROI, 3) γ = 0.6 in the dorsal ROI, and 4) γ = 0.99 in the dorsal ROI Click here for additional data file.Figure S1s-Dl space  and the indifference line, where the probabilities of the two choices are equal. Each asterisk (*) indicates where the subject chose a large reward with corresponding Dl against a small reward with corresponding Ds, and each circle (o) indicates a choice of small reward against large reward.A subject's choice . Small and large reward choice on the D(0.28 MB PDF)Click here for additional data file.Figure S2Choice rate of large reward choices against small reward choices. The line plot shows the individual choice rate at each tryptophan level. A repeated measures ANOVA shows no effect of tryptophan levels on choice ratio of large rewards  = 0.053, P = 0.948). All bar plots show mean across subjects and error bars indicate standard error.(0.23 MB PDF)Click here for additional data file.Figure S3Time course of estimated V(t) . Each color corresponds to a value of γ used for calculating V(t) (corresponding to color code used in (0.38 MB PDF)Click here for additional data file.Figure S4Regression analysis of BOLD signal by expected future reward using a hyperbolic model with different discount rates. We checked for a correlation between BOLD signal and hyperbolic discounted values (equation S2 in (0.65 MB PDF)Click here for additional data file.Table S1Total plasma tryptophan level before consumption and six hours after consumption.(0.04 MB PDF)Click here for additional data file.Table S2Voxels significantly correlated with estimated V (t) at each γ.(0.07 MB PDF)Click here for additional data file.


For robust performance, walking movements must adapt at each step, not just on average. Here, we propose an analytical framework that reconciles issues of optimality, redundancy, and stochasticity. For human treadmill walking, we defined a goal function to formulate a precise mathematical definition of one possible control strategy: maintain constant speed at each stride. We recorded stride times and stride lengths from healthy subjects walking at five speeds. The specified goal function yielded a decomposition of stride-to-stride variations into new gait variables explicitly related to achieving the hypothesized strategy. Subjects exhibited greatly decreased variability for goal-relevant gait fluctuations directly related to achieving this strategy, but far greater variability for goal-irrelevant fluctuations. More importantly, humans immediately corrected goal-relevant deviations at each successive stride, while allowing goal-irrelevant deviations to persist across multiple strides. To demonstrate that this was not the only strategy people could have used to successfully accomplish the task, we created three surrogate data sets. Each tested a specific alternative hypothesis that subjects used a different strategy that made no reference to the hypothesized goal function. Humans did not adopt any of these viable alternative strategies. Finally, we developed a sequence of stochastic control models of stride-to-stride variability for walking, based on the Minimum Intervention Principle. We demonstrate that healthy humans are not precisely “optimal,” but instead consistently slightly over-correct small deviations in walking speed at each stride. Our results reveal a new governing principle for regulating stride-to-stride fluctuations in human walking that acts independently of, but in parallel with, minimizing energetic cost. Thus, humans exploit task redundancies to achieve robust control while minimizing effort and allowing potentially beneficial motor variability.It is widely accepted that humans and animals minimize energetic cost while walking. While such principles predict average behavior, they do not explain the  Existing principles used to explain how locomotion is controlled predict average, long-term behavior. However, neuromuscular noise continuously disrupts these movements, presenting a significant challenge for the nervous system. One possibility is that the nervous system must overcome all neuromuscular variability as a constraint limiting performance. Conversely, we show that humans walking on a treadmill exploit redundancy to adjust stepping movements at each stride and maintain performance. This strategy is not required by the task itself, but is predicted by appropriate stochastic control models. Thus, the nervous system simplifies control by strongly regulating goal-relevant fluctuations, while largely ignoring non-essential variations. Properly determining how stochasticity affects control is critical to developing biological models, since neuro-motor fluctuations are intrinsic to these systems. Our work unifies the perspectives of time series analysis researchers, motor coordination researchers, and motor control theorists by providing a single dynamical framework for studying variability in the context of goal-directedness. Walking is an essential task most people take for granted every day. However, the neural systems that regulate walking perform many complex functions, especially when we walk in unpredictable environments. These systems continuously integrate multiple sensory inputs variability ubiquitously observed in movements like walking overcome all variability as a limiting constraint regulate variability in ways that help maximize task performance The principal idea used to explain how humans and animals regulate walking has been energy cost Others have sought to determine how muscles are organized into functional synergies to resolve the inherent redundancy of complex movements every step (not just on average) to be able to respond to externally and/or internally generated perturbations from stride to stride remain unknown. Small stride-to-stride fluctuations in gait dynamics are typically assumed to reflect random noise. Indeed, there is ample evidence supporting multiple sensory and motor sources of physiological noise During walking, humans need to adapt at regulate these variations?Here, we formulate goal functions at each stride. This yields a decomposition of stride-to-stride variations into new gait variables explicitly related to achieving this strategy. Time series analyses confirm that humans do indeed adopt this hypothesized strategy. We similarly analyze three alternative strategies that equally achieve the task requirements, but make no reference to the hypothesized goal function. Humans do not adopt any of these alternatives. Finally, we develop a sequence of stochastic optimal control models of stride-to-stride dynamics to determine if they replicate our observations. These models confirm that healthy humans do carefully regulate their movements explicitly to maintain constant speed at each stride. However, humans do not use strategies that are precisely “optimal” with respect to the employed cost functions, but instead slightly but consistently over-correct small deviations in walking speed from each stride to the next.We present a mathematical definition of a specific hypothesized task strategy v is to not walk off the treadmill. The net change in displacement, relative to the laboratory reference frame, for stride n is determined by the stride length, nL, and stride time, nT, as N strides and TML is the length of the treadmill belt. A key observation is that any sequence of nL and nT that satisfies this inequality will successfully accomplish the treadmill walking task. Many possible strategies for generating such a sequence of nL and nT exist. The simplest strategy can be formulated using the goal functionit is not required by Eq. (1). It is instead only one possible movement strategy. The solid line in nT, nL] pairs that equally satisfy Eq. (2). We hypothesized that humans minimize errors relative to this GEM. Thus, for the present analyses, the relevant stride-to-stride walking dynamics are entirely captured by the impact Poincaré section nT, nL] plane  and perpendicular (Pδ) to it and converted  coordinates into GEM-specific  coordinates , stride lengths (nL), and stride speeds (nS = nL/nT) for all strides within each trial were obtained and analyzed.To test GEMs of different location/orientation, subjects walked on a motorized treadmill at each of 5 constant speeds, from 80% to 120% of their preferred walking speed (PWS). Time series of stride times  α>½ indicates statistical persistence: deviations in one direction are more likely to be followed by deviations in the same direction. α<½ implies anti-persistence: deviations in one direction are more likely to be followed by deviations in the opposite direction. α = ½ indicates uncorrelated noise: all deviations are equally likely to be followed by deviations in either direction. In the context of control, statistical persistence (α>½) is interpreted as indicating variables that are not tightly regulated. Conversely, variables that are tightly regulated are expected to exhibit either uncorrelated or anti-persistent fluctuations (α≤∼½).Therefore, to quantify temporal correlations across consecutive strides, we computed scaling exponents, nT and nL time series (α>½). Conversely, nS time series (anti-persistence (∼0.4<α<0.5). Thus, at all walking speeds, deviations in both nT and nL were allowed to persist, while deviations in nS were rapidly reversed on subsequent strides. This provides indirect evidence that subjects did not regulate nT or nL independently, but instead adjusted both nT and nL in a coordinated manner to maintain walking speed.Consistent with previous results e series  both exhe series  exhibiterequired by the inequality constraint of Eq. (1). However, these movements also exhibited a high degree of statistical persistence (∼1.25<α<∼1.55) at all walking speeds  = 139.93; p = 2.53×10−9; nT, nL] points were solely a reflection of average metabolic costs, given the nearly circular energy contours seen in Tδ time series all exhibited standard deviations >>1, while the Pδ time series all exhibited standard deviations <<1  nT or nL time series.Plots of Tn e.g.,  exhibiteions <<1 . Thus, sTδ and Pδ time series exhibited temporal correlation structures qualitatively very different from each other  = 368.21; p = 1.81×10−12; anti-persistence  for the goal-relevant Pδ deviations at all five walking speeds. Thus, subjects rapidly corrected Pδ deviations from each stride to the next, while allowing Tδ deviations to persist across multiple strides, independent of the magnitudes of these fluctuations.The ch other . As hypoonly viable strategy subjects could have used. Rejecting this possibility requires only that we identify at least one alternative strategy that still satisfied the fundamental task requirements (Eq. 1), but was completely “ignorant” of the proposed GEM defined by Eq. 2. Here, we present three such alternatives using “surrogate” data data-based model of the observed stride-to-stride dynamics. Each surrogate model directly tested a specific null hypothesis that subjects could have successfully completed the treadmill walking task  using a strategy that made absolutely no reference to the GEM.One obvious question is whether these observed dynamics represented the T*, L*]  = 2.614; p = 0.125; α exponents for Pδ and Tδ were both≈½ and also not significantly different  = 0.413; p = 0.529; These surrogates exhibited approximately isotropic distributions  about [n] plane . Likewisch other . StandarnT and nL independently of the GEM, but in a way that retained the statistical persistence observed in humans  nT. Subjects did not adopt either of these two viable alternative control strategies. Full details and results of these analyses are presented in Supplementary n humans  [22], , was assumed to be equal to the mean stride time and stride length  for both nL and nT that, while still greater, were much closer to those of humans (α<½) exhibited by humans for nS  = 2,916.30; p = 1.55×10−37; α exponents for Tδ fluctuations than for Pδ fluctuations  = 597.27; p = 7.61×10−25; α exponents for Tδ were greatly reduced compared to the MIP model. However, this model still failed to replicate the anti-persistent (α<½) Pδ fluctuations exhibited by humans . Conversely, humans consistently exhibited statistical anti-persistence (α<½) in their Pδ fluctuations  exhibited by humans for nS   . Human s and δT) . Moreove the GEM , and two the GEM –8. InsteP errors  [25], , in the  plane OptT, OptL], induced for example by neuromuscular noise OptT, OptL] should, on average, approximate the shape of the contours shown in OptT, OptL]. We did not observe that here. Instead, all  data were strongly oriented along the GEM , we computed a POP of  = . Mechanical walking models of Minetti OptT, Optl] =  and  = , respectively, for this speed. Simplifications in both models account for their slightly under-estimating the preferred  of actual humans Our findings, however, remain compatible with the idea that humans also try to minimize energy cost while walking. The failure of the MIP model  to captuOP model , is precover-corrected Pδ deviations  can very easily lead to incorrect conclusions about control ) and “unhealthy” (according to α(Pδ)) ) . Insteado α(δP)) –9. ThisnT and nL similar to nSnL and nS remained strongly persistent nT became anti-persistent speed (timing (nT), but not nL or nSbeyond minimizing energy cost help determine how stride-to-stride movements are regulated. The critical first step is to identify the appropriate goal function for each task One question is whether the theoretical framework developed here will generalize to other contexts. During unconstrained overground walking nt speed . Remove All participants provided written informed consent, as approved by the University of Texas Institutional Review Board.Seventeen young healthy adults , participated. Subjects were screened to exclude anyone who reported any history of orthopedic problems, recent lower extremity injuries, any visible gait anomalies, or were taking medications that may have influenced their gait.Subjects walked on a level motor-driven treadmill  while wearing comfortable walking shoes and a safety harness  that allowed natural arm swing nd phalanx and 5th metatarsal, dorsum of the foot, inferior to the fibula, and calcaneous). The movements of these markers were recorded using an 8-camera Vicon 612 motion capture system . All data were processed using MATLAB 7.04 . Brief gaps in the raw kinematic recordings were filled using rigid-body assumptions. Marker trajectories were low-pass filtered with a zero-lag Butterworth filter at a cutoff frequency of 10 Hz. A heel strike was defined as the point where the heel marker of the forward foot was at its most forward point during each gait cycle.Five 14-mm retro-reflective markers were mounted to each shoe  for each stride, n, was calculated as the time from one heel contact to the next ipsilateral heel contact. Step length was defined as the anterior-posterior distance between the heel and the contralateral heel at each heel contact, when both feet were in contact with the treadmill belt. Stride length (nL) was calculated as the sum of the 2 consecutive step lengths composing each stride. Individual stride speeds (nS) were then calculated as n strides. Means, standard deviations, and DFA scaling exponents  were computed across all strides for each nT, nL, and nS time series obtained from each walking trial Standng trial .randomly shuffled surrogates . This was easily verified by computing the net cumulative distance (netd) each surrogate time series would have walked relative to the treadmill at each stride, n:d = 0 represents the center of the treadmill belt. We then extracted the maximum forward [max(netd)], and backward [min(netd)] distances each surrogate walked during the entire trial ≥−0.864 m and max(netd)≤+0.864 m in all cases). We generated 20 total such surrogates for each original trial, or 3,320 of each type of surrogate. Thus, all surrogates analyzed  represented hypothetical walking trials that would have successfully completed the entire trial without walking off of the treadmill.All surrogates were constrained so they did not “walk off” the treadmill  time series by dividing the surrogate nL by the surrogate nT time series. These surrogates were then subjected to the same GEM decomposition and analyses as the original time series. For each trial, the average value of each dependent measure computed across all 20 surrogates for that trial was computed and extracted for statistical analyses.For each surrogate, we then computed a new stride speed  noise matrix with nonzero diagonal elements. η was a 2×1 vector of additive  noise. Non-zero elements of N and η were taken to be independent, Gaussian random variables with mean zero and standard deviation σk  in a finite amount of time (nT). Overall, we assume walking dynamics are governed by central pattern generator (CPG) processes xn+1 = xn), reflecting the fundamentally cyclical nature of walking. Many suitable differential equation models of such continuous-time walking dynamics exist, ranging from relatively simpler mechanical models any such reasonable model could be used to generate  time series. Thus, our results have broad potential impact both for experimental studies of human walking and also for anyone developing computational simulations of walking or actual  walking robots, regardless of their complexity.The state update equation (Eq. 5) is intended to model only the discrete-time αe2, depended on the definition of the goal-level error for the task nL/nT = v . Thus, the error the controller sought to minimize was n+1. This cost function directly reflects the strategy (Eq. 2) we hypothesize subjects adopted to regulate stride variability while satisfying the fundamental task requirement defined in Eq. 1. While the underlying task requirement (Eq. 1) does not change, different hypothesized control strategies could be obtained by defining different GEMs (possibly including more and/or different state variables) and would thus change the definition of the error term, e, used in the above cost function. The second term in Eq. (6), βp2, penalized the distance, n+1p, of the state at stride n+1 from the preferred operating point, . The last two terms in Eq. (6) were effort penalty terms where u = T was the control input used to drive the state from stride n to stride n+1 (Eq. 5). Here, α, β, γ, and δ were positive constants that weighted the different components in C.The controller was modeled as an unbiased stochastic optimal single-step controller with direct error feedback. This controller design was based on the Minimum Intervention Principle (MIP) C in a probabilistic sense across each trial. That is, we did not minimize the cost itself function directly, but rather its expected value, u1 and u2 were then determined by solving a classic quadratic optimal control problem with an equality constraint. This process yielded optimal control inputs obtained analytically as a function of the current state, xn , depended only on the goal-level error e. This strict MIP controller only corrected Pδ deviations off of the GEM  was already minimized. Since this was true at all points along the GEM, the strict MIP controller was neutrally stable along the GEM. Because of the stochastic nature of the trial-to-trial dynamics (Eq. 5), we expected consecutive strides to exhibit random walk behavior  along the GEM. Indeed, this was what we obtained in our simulations  or noise amplitudes The optimal POP controller  was impl100% PWS , and L* e-series  could noPδ time-series , as well as the same weightings for the remaining cost function terms , and the same noise amplitudes Tδ and Pδ directions for the OVC model to the average variability observed in the human (HUM) data  data .It is important to note that for each model, no explicit or rigorous attempts were made to find “best fits” to our experimental data. For example, we could adjust model parameters to fit different values for the means and SD's of different stride variables to try to more closely replicate the data of any of our individual subjects. However, our overall results were insensitive to the precise parameter values: i.e., the contrasts in the fundamental qualitative features of each of these models will remain the same.nT) and stride length (nL) time series. Time series of stride speeds were then calculated as nS = nL/nT, as before. As with our surrogate analyses, we computed the net cumulative distances walked (Eq. 4) by each simulation to ensure no simulation “walked off” the treadmill. Means, standard deviations, and DFA α exponents were computed for all primary stride variables , as we did for the experimental trials. The same GEM decomposition (Eq. 3) was applied to compute Tδ and Pδ deviations along and perpendicular to the GEM. Standard deviations and DFA α exponents were then computed for each Tδ and Pδ time series obtained from each simulated walking trial  and Speed (80%–120% of PWS) were taken as fixed factors. Subjects (n = 17) was taken as a random factor. There were 2 repeated trials obtained for nearly all subjects and walking speeds . These models tested for main effects for each factor and also for any interaction effects. For the three computational models, the standard deviations and DFA α exponents computed from each model (Tδ vs. Pδ) repeated measures, balanced ANOVA, with 20 repeated observations. For all statistical tests, standard graphical analyses of the model residuals were performed to ensure each test met the linearity and normality assumptions of each ANOVA model.All statistical tests were performed in Minitab 15 . For all dependent measures, we computed between-subject means and ±95% confidence intervals at each walking speed. Where appropriate , linear rimental  and surrrimental  and 7F–Gch model –9, F–G Text S1Extended description of the construction of (0.30 MB PDF)Click here for additional data file.Text S2Additional surrogate data analyses and results.(0.44 MB PDF)Click here for additional data file.Text S3Derivation of the GEM-based inter-stride optimal controller for treadmill walking.(0.26 MB PDF)Click here for additional data file.Text S4Extended description of the detrended fluctuation analysis algorithm.(0.27 MB PDF)Click here for additional data file.


Although p73L and p51 are suspected to play oncogenic and tumour suppressive roles in mammalian cells, respectively, no evidence of linkage between the expression of these isoforms and human cancers has been reported so far. In this study, we first investigated the expression profile of p51 and p73L in various human tumour cell lines and found that a novel isoform, termed ΔNp73L, was predominantly expressed in squamous cell carcinomas. The expression profile of ΔNp73L/p73L/p51 in primary human skin cancer specimens showed that the expression of p51 was frequently lost (62%) but was detected in all normal skin samples. In p51-expressing skin cancers, ΔNp73L expression was associated at a high frequency (75%) though it was not detected in normal skin tissues. Transient co-transfection data indicate the possibility that ΔNp73L can inhibit p53-, and more preferentially, p51-mediated transactivation. These data suggest that the loss of expression of p51 and/or the expression of ΔNp73L might contribute to the pathogenesis of human squamous cell carcinomas. © 2001 Cancer Research Campaign http://www.bjcancer.comWe have recently identified a second


Saccharomyces cerevisiae genome are demonstrated by statistical analyses of sequence data. This studyincludes: (a) The random frame rule—that the six reading frames W1, W2, W3,C1, C2 and C3 in the double-stranded genome are randomly occupied by ORFs. (b) The inhomogeneityrule—coding and non-coding ORFs differ in inhomogeneity of base composition inthe three codon positions. By use of the inhomogeneity index (IHI), one can makea distinction between coding (IHI > 14) and non-coding (IHI ≤ 14) ORFs at 95%accuracy. We find that ‘spurious’ ORFs (with IHI ≤ 14) are distributed mainly inthree classes of ORFs, namely, those with ‘similarity to unknown proteins’, those with‘no similarity’, or ‘questionable ORFs’. The total number of spurious ORFs (whichare unlikely to be regarded as coding ORFs) is estimated to be 470. (c) The evaluationof ORF length distribution shows that below 200 amino acids the occurrence of ATGinitiator ORFs is close to random.Some rules on gene recognition and ORF organization in the


Correction to:British Journal of Cancer (2006) 95, 401–405, doi:10.1038/sj.bjc.6603246Owing to an author error, there was an erroneous statement presented in the Abstract of the above paper.Where it was stated, ‘Assuming average lead times of 5 to 10 years, 40–64% of the PSA-detected cases were estimated to be overdiagnosed’, the figure 64% corresponds to average lead time of 7 years whereas, the corresponding figure for 10 years would be 98%.The correct statement should read ‘Assuming average lead times of 5–10 years, 40–98% of the PSA-detected cases were estimated to be overdiagnosed’.


Many men suffering from stress, including post-traumatic stress disorder (PTSD), report sexual dysfunction, which is traditionally treated via psychological counseling. Recently, we identified a gastrin-releasing peptide (GRP) system in the lumbar spinal cord that is a primary mediator for male reproductive functions.in vivo. Remarkably, pharmacological stimulation of GRP receptors restores penile reflexes in SPS-exposed males, and induces spontaneous ejaculation in a dose-dependent manner. Furthermore, although the level of plasma testosterone is normal 7 days after SPS exposure, we found a significant decrease in the expression of androgen receptor protein in this spinal center.To ask whether an acute severe stress could alter the male specific GRP system, we used a single-prolonged stress (SPS), a putative rat model for PTSD in the present study. Exposure of SPS to male rats decreases both the local content and axonal distribution of GRP in the lower lumbar spinal cord and results in an attenuation of penile reflexes We conclude that the spinal GRP system appears to be a stress-vulnerable center for male reproductive functions, which may provide new insight into a clinical target for the treatment of erectile dysfunction triggered by stress and psychiatric disorders. Post-traumatic stress disorder (PTSD) is a psychiatric disorder involving long-lasting symptoms that may occur after exposure to a life-threatening traumatic event, and is characterized by intrusive memories (flashbacks), a hyperarousal state and avoidance of stimuli associated with the trauma Bombina bombinavia intrathecal catheters to this spinal region significantly attenuates penile reflexes and ejaculation rate in normal male rats Gastrin-releasing peptide (GRP), a member of the bombesin-like peptide family first discovered in the skin of the frog in vivo more than a week later. Remarkably, administration of GRP agonist restores penile reflexes in SPS-exposed males, and also induces spontaneous ejaculation in a dose-dependent manner. In the SPS model, the stress-induced effects on the rat GRP system are not due to any detectable differences in circulating levels of androgens or corticosteroids. Our data suggest that pharmacological targeting of the spinal GRP system might relieve ED in men suffering PTSD.In the present study, we examined whether an acute severe stress could alter the male specific GRP system. Here we show that exposure to a single-prolonged stress (SPS), a putative rat model for PTSD 2,14F = 7.71, *P = 0.015; L5–6, 2,14F = 7.42, *P = 0.002) (P = 0.002) and lower (*P = 0.005) lumbar spinal cord  (Using a competitive enzyme-linked immunosorbent assay (ELISA) specific for GRP, we quantified the local contents of GRP in two separate regions of the lumbar spinal cord by dividing the lumbar spinal cord into upper  . Seven dnal cord . In fact = 0.57) .2,12F = 7.28, P = 0.008) (P = 0.83)  and DGC   (We next used immunocytochemistry (ICC) to examine the sexually dimorphic expression and stress response of GRP in the lumbar spinal cord. As expected, the number of GRP neurons in the upper lumbar spinal cord (L3–4) was much higher in control males than in females (= 0.008) . In cont = 0.83) , althoug = 0.83) . In the  = 0.83) . Further = 0.83) . Additio = 0.83) , a markefemales) . GRP-con = 0.81) .in vivo to evaluate male reproductive function, as assessed by penile reflexes, after SPS exposure. Seven days after SPS exposure males showed significantly reduced spinal reflexes of the penis, including simple erections and cup-like flaring erections of the distal glans , and tended to reduce dorsal flips of the penis  (20–29) restored penile erections in a dose-dependent manner (†P<0.003), without affecting flips and cups   . We hypoand cups . Cups anol males . On the  = 1.65) .P = 0.023) (P = 0.57) (P = 0.31) (data not shown). In addition, we found no significant differences in plasma T and corticosterone (CORT) levels of the SPS-exposed and control rats , but not estrogen receptor alpha (ERα) = 0.023) . On the  = 0.57) , nor wasrol rats .per se and the expression of ARs that regulate penile reflexes. Furthermore, pharmacological stimulation of GRP receptors restores male reproductive function in male rats exposed to SPS. These data suggest that the effects of SPS to inhibit male reproductive function are mediated, at least in part, by effects on the spinal GRP system. To the best of our knowledge, this is the first demonstration of a center for male reproductive function in the spinal cord that can be altered in the expression and/or distribution of protein by exposure to stress. Since a significant reduction in the frequency of penile reflexes was observed in SPS-exposed males, this may also serve as a viable paradigm to study the pathophysiology of psychogenic ED in PTSD or other disorders.PTSD has been found to affect emotional and social functioning Considerable evidence suggests that GRP has complex molecular interactions in various tissues. GRP stimulates neuroblastoma growth and the expression of angiogenic markers, platelet endothelial cell adhesion molecule-1 and vascular endothelial growth factor expressions Castration of adult male rats significantly reduced the expression of GRP in the lumbar spinal cord, and this reduction was averted by androgen replacement Stress responses are typically mediated through glucocorticoid receptors in the central nervous system In the present study, we did not use the GRP antagonist RC-3095 in SPS-exposed rats because they already display few reflexes. Furthermore, installing intrathecal catheters to the lower lumbar spinal cord to deliver the antagonist, would itself act as a stressor, making it difficult to see an effect of SPS. However, we previously inserted intrathecal catheters in otherwise unstressed rats and found that RC-3095 suppresses sexual reflexes in a dose-dependent manner In summary, we found that stress significantly attenuates the spinal GRP system regulating male reproductive function, reducing expression of both GRP and the ARs mediating T effects on the system, interfering with male reproductive function. These findings suggest that the use of selective GRP-R agents may provide new avenues for the treatment of stress-related ED in men.Adult male Sprague-Dawley rats were maintained in air-conditioned rooms (22±1°C) on a 12 h light/dark cycle with free access to food and drinking water. All experimental procedures were authorized by the Committee for Animal Research, Kyoto Prefectural University of Medicine, Japan and/or Michigan State University, USA.Rats were randomly assigned to one of two experimental groups: control group and SPS group, and were housed in pairs. After an acclimation period, male rats were subjected to SPS as previously described We performed the ICC analysis according to established methods To determine the projection site of GRP-immunoreactive axons, double-immunofluorescence staining of GRP  and nNOS  , a marker protein for neurons in the SPN, was performed as described previously 20–29, a specific agonist for GRP-R, diluted in saline .All behavior tests were conducted between 08:30 and 13:30 h and included at least 9 animals per group. Penile reflex tests consisted of holding a rat in a supine position in a Plexiglas cylinder for a period of 25 min as described previously Peptides were extracted according to our previous methods Western blot analyses were conducted as previously described g at 4°C, and blood plasma was stored at −80°C until assay. Plasma concentrations of T and CORT were measured using specific enzyme immunoassay kits  as described previously Rats were intraperitoneally injected with sodium pentobarbital between 10:00 and 11:00 h before cage movement, and they were then decapitated and blood samples were collected at 30 min after the injection. The samples were centrifuged immediately at 4,000×P values for post hoc Bonferroni tests were performed. Significance in t-test. Differences were considered significant if P<0.05.Data are expressed as the standard error of the mean (s.e.m.). We derived


Several herbal plants improve medical conditions. Such plants contain many bioactive phytochemicals. Terpenoids  constitute one of the largest families of natural products accounting for more than 40,000 individual compounds of both primary and secondary metabolisms. In particular, terpenoids are contained in many herbal plants, and several terpenoids have been shown to be available for pharmaceutical applications, for example, artemisinin and taxol as malaria and cancer medicines, respectively. Various terpenoids are contained in many plants for not only herbal use but also dietary use. In this paper, we describe several bioactive terpenoids contained in herbal or dietary plants, which can modulate the activities of ligand-dependent transcription factors, namely, peroxisome proliferator-activated receptors (PPARs). Because PPARs are dietary lipid sensors that control energy homeostasis, daily eating of these terpenoids might be useful for the management for obesity-induced metabolic disorders, such as type 2 diabetes, hyperlipidemia, insulin resistance, and cardiovascular diseases. Nature relies on an intricate network of biosynthetic pathways to produce a lot of small organic molecules required to support life. Terpenoids  constitute one of the largest families of natural products accounting for more than 40,000 individual compounds of both primary and secondary metabolisms. Most of them are of plant origin, and hundreds of new structures are reported every year –3. All oMethanocaldococcus jannaschii [Isopentyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) are the universal five-carbon precursors of all terpenoids. After the discovery of the mevalonate (MVA) pathway in yeast and animals, it was assumed that IPP was synthesized from acetyl-CoA via MVA and then isomerized to DMAPP in all eukaryotes and some Gram-positive prokaryotes , 3 Figu. The orinnaschii . Before nnaschii , it has nnaschii . This alnnaschii , 3. Plannnaschii .Plants have an enormous capacity to synthesize huge amounts of diverse terpenoids, particularly via the combination of the terpenoid biosynthetic route and other secondary metabolic pathways. For instance, tocopherol biosynthesis occurs as a result of combination of the shikimate and isoprenoid pathways, which lead to homogentisic acid phytyl diphosphate, which in combination ultimately lead to the formation of tocopherols (formed by a chromanol head group and a phytyl tail) .In addition to universal physiological, metabolic, and structural functions, many specific terpenoids function in various situations, including communication and defense. Members of the isoprenoid group also include industrially useful polymers  and agrochemicals .It is known that several herbal plants improve medical conditions. Such plants contain many bioactive phytochemicals. In particular, terpenoids are contained in many herbal plants, and several terpenoids have been shown to be available for pharmaceutical applications, for example, artemisinin and taxol as malaria and cancer medicines, respectively. Various terpenoids are contained in many plants for not only herbal medicine use but also dietary use .In this paper, we describe several bioactive terpenoids  containeMembers of the nuclear receptor superfamily of ligand-dependent transcription factors play a multitude of essential roles in development, homeostasis, reproduction, and immune functions –14. Liga+-dependent protein deacetylase enzyme implicated in the biology of aging [Nuclear receptors are frequent biological targets of active compounds contained in herbal and dietary plants. This is perhaps not surprising, since nuclear receptors evolved to be regulated by lipophilic molecules derived from diet and the environment , 16. At of aging . Other pα, δ, and γ, were found [PPARs are members of the nuclear receptor superfamily, which are activated by fatty acids and their derivatives. PPARs are dietary lipid sensors that regulate lipid and carbohydrate metabolism . In mammre found . PPARs fre found –24. Ligaα is highly expressed in the liver, cardiac muscle, and digestive tract, and regulate the expression of target genes involved in lipid catabolism. Activators of PPARα, such as fibrates, decrease circulating lipid levels and are commonly used to treat hypertriglyceridemia and other dyslipidemic states [δ is expressed in many tissues including the skeletal muscle and brown adipose tissue. Recently, it has been suggested that PPARδ activation attenuates obesity and type-2 diabetes [γ is abundant in adipose tissues functioning as the key transcription factor for adipogenesis. Synthetic ligands for PPARγ, such as thiazolidinediones, are increasingly used to treat type-2 diabetes [PPARc states . PPARδ idiabetes , 27. PPAdiabetes .α regulates inflammatory processes, mainly by inhibiting inflammatory gene expression. In recent years, several molecular mechanisms responsible for the immunosuppressive effects of PPARα have been uncovered [δ during inflammation is limited. So far, an anti-inflammatory effect has been observed in macrophages suggesting a possible role for PPARδ in the process of atherogenic inflammation. [α, PPARγ is involved in governing inflammatory response, particularly in macrophages. Currently, two different molecular mechanisms have been proposed by which anti-inflammatory actions of PPARγ are effectuated: (1) via interference with proinflammatory transcription factors [PPARs are involved in not only energy homeostasis but also inflammation. PPARncovered . These incovered . The nummmation. . Similar factors  and (2)  factors .α (TNF-α) [α. Macrophage-derived TNF-α establishes a vicious cycle that augments inflammatory changes and insulin resistance in obese adipose tissues [α.Recently, it has been indicated that obesity is associated with a low-grade chronic inflammation state . The inf (TNF-α) . MCP-1,  (TNF-α) . Macroph tissues . TherefoSeveral herbal and dietary plants improve medical conditions including diabetes mellitus, hyperlipidemia, and cardiovascular disease associated with an abnormality of lipid metabolism , 39. To We carried out screening for novel PPAR ligands in natural compounds contained in medicinal plants. We used several terpenoids including carotenoids and polyisoprenoid alcohols (isoprenols) for the screening, because these compounds are contained in many medicinal and dietary plants . These tγ at a concentration of 50 or 100 μM [γ as novel ligands. At 100 μM, the activations by geraniol, farnesol, and geranylgeraniol were 2.2-, 4.1-, and 3.7-fold that by the vehicle control, respectively. On the other hand, squalene had no effect on PPARγ transactivation. Although geraniol had no effect on PPARα, farnesol and geranylgeraniol also dose-dependently activated PPARα  in the PPARα ligand assay system [μM fenofibrates, one of the fibrates (antihyperlipidemia drugs) used as a positive control for PPARα. In this regard, these farnesol and geranylgeraniol isoprenols have the effects of dual activation of PPARγ and PPARα.As shown in r 100 μM . In thisy system . These aγ activation in adipose tissues results in the improvement of insulin resistance [α activation in the liver induces the lowering of circulating lipid levels [γ and PPARα by isoprenols is very important for thinking of the mechanisms understanding the effects of medicinal plants and valuable for the management of diabetic and hyperlipidemic conditions in herbal medicine. Indeed, in our preliminary study, farnesol ameliorated hyperglycemia in high fat diet-fed wild-type mice but not in PPARα deficient mice . These findings indicate that improvement of obesity-associated metabolic disorders by farnesol is mainly dependent on PPARα activation. These results provide not only a significant molecular basis on how herbal plants containing phytochemicals such as isoprenols induce the improvement of diabetes or hyperlipidemia, but also possibilities that phytochemicals might have therapeutic applications in lipid abnormalities, such as obesity, diabetes mellitus, and hyperlipidemia.PPARsistance  and PPARd levels . These ed levels . TherefoPhytol, a diterpene alcohol, which is a carbon side chain of a chlorophyll molecule , is invoγ and the retinoid-X-receptor (RXR) [α to regulate lipid metabolism in some types of cell [α target genes involved in peroxisomal and mitochondrial β-oxidation and fatty acid metabolism [Phytanic acid is a branched-chain, terpenoid-derived fatty acid constituent of diet . In survor (RXR) , 53 so tor (RXR) . In addi of cell . Therefotabolism .However, since the conversion of phytol into phytanic acid is not very rapid, a phytol-enriched diet also induced accumulation of phytol in the liver . Moreoveα was activated the most markedly following the addition of phytol [α activation were larger than those of phytanic acid under our experimental conditions. Phytol induced the mRNA and protein expression of PPAR target genes in a manner dependent on the level of PPARα expression in HepG2 hepatocytes. In our in vitro coactivator recruiting assay, it was revealed that phytol can activate PPARα directly [α as its ligand.Therefore, we evaluated the effects of phytol on PPAR activity using our advanced highly sensitive luciferase assays . Among tf phytol . The effdirectly . These fα is one of the most important factors in lipid metabolism in peripheral tissues including the liver and muscles, the ability of phytanic acid and phytol to activate PPARα is very important in the management of lipid metabolism using food factors. Induction of PPARα target gene expression in mice fed a phytol-enriched diet [α activation in the liver.Because the activation of PPARhed diet  is likelAbies grandis) and lodgepole pine (Pinus contorta) [The amount of variety of hydrocarbons and their derivatives used in industrial and commercial activities has been increasing over the years. Abietic acid is a tricyclic-diterpene carboxylic acid , and is ontorta) . Abieticontorta) . Moreoveontorta) .α and PPARγ but not PPARδ [γ ligand, abietic acid suppressed mRNA expressions of TNF-α and cyclooxygenase 2 (COX2), which are induced in inflammatory reactions, in LPS-stimulated macrophages [α and PPARγ more potently than abietic acid [α, and NO in LPS-stimulated macrophages and in the coculture of macrophages and adipocytes [α levels and their mRNA expressions in white adipose tissues. Moreover, dehydroabietic acid improved carbohydrate and lipid metabolism [To investigate whether the activation of PPARs is related to the anti-inflammatory effects of abietic acid and its derivatives, we evaluated the effects of abietic acid and its derivatives on PPAR activity . Abieticot PPARδ , 62. Simrophages . Dehydrotabolism . These fα and PPARγ ligand [γ antagonist [γ. In addition, mRNA expressions of several PPARα target genes involved in FA catabolism, were also induced in PPARα-expressing HepG2 hepatocytes by auraptene treatment [γ and PPARα target genes as a dual agonist, and these activities might contribute to the anti-cardiovascular disease effect of citrus fruits.Citrus-fruit-derived compounds have many beneficial bioactivities  , 65. Thrγ ligand , 67. Aurtagonist , suggestBixa orellana is a natural pigment extensively used in many processed foods [γ ligand assay, both bixin and norbixin activated PPARγ (γ target genes in 3T3-L1 adipocytes, resulting in the promotion of adipocyte differentiation and insulin-stimulated glucose uptake [γ.Annatto obtained from the pericarp of seeds from ed foods , 69. Bixed foods –72. Furted foods , 74. In ed PPARγ , and bixe uptake . TherefoIn this paper, we mentioned the diversity of terpenoids, functions of PPARs, and several terpenoids activating PPARs. The prevalence of obesity worldwide has progressively increased over the past decades. This unabated rise has spawned proportionate increases in obesity-associated metabolic disorders. Currently, synthetic PPAR agonists are widely used for the treatment of metabolic disorders. Daily intake of dietary terpenoids, which activate PPARs as we described above, may be valuable for the control of carbohydrate and lipid disorders. Dietary patterns rich in vegetables and fruit are associated with a lower prevalence of metabolic syndrome , 76. Bec


Modern hard corals  are widely studied because of their fundamental role in reef building and their superb fossil record extending back to the Triassic. Nevertheless, interpretations of their evolutionary relationships have been in flux for over a decade. Recent analyses undermine the legitimacy of traditional suborders, families and genera, and suggest that a non-skeletal sister clade  might be imbedded within the stony corals. However, these studies either sampled a relatively limited array of taxa or assembled trees from heterogeneous data sets. Here we provide a more comprehensive analysis of Scleractinia  and various outgroups, based on two mitochondrial genes , with analyses of nuclear genes  of a subset of taxa to test unexpected relationships. Eleven of 16 families were found to be polyphyletic. Strikingly, over one third of all families as conventionally defined contain representatives from the highly divergent “robust” and “complex” clades. However, the recent suggestion that corallimorpharians are true corals that have lost their skeletons was not upheld. Relationships were supported not only by mitochondrial and nuclear genes, but also often by morphological characters which had been ignored or never noted previously. The concordance of molecular characters and more carefully examined morphological characters suggests a future of greater taxonomic stability, as well as the potential to trace the evolutionary history of this ecologically important group using fossils. Molecular analyses have been used to study higher-level relationships among the Scleractinia only comparatively recently, in part because of technical difficulties. Rates of molecular evolution in mitochondrial genes are extremely slow in anthozoans cox1) and cytochrome b (cob). A subset of taxa was analyzed using two nuclear gene regions: ß-tubulin and/or parts of the nuclear ribosomal genes . The nuclear analyses were targeted to test mitochondrial results that strongly contradicted traditional morphological classification. Unless noted otherwise, all conclusions are supported by both the mitochondrial dataset  is in a clade that contains the type for the family Mussidae Ortmann, 1890 (Mussa angulosa)  (group XIII Leptastrea)  (group XIX in Micromussa and members the genus Acanthastrea (and even the monophyly of Acanthastrea) are unstable  has no other close relative in our analyses  that appears to be close to Stephanocoenia in morphology.The family Astrocoeniidae Koby, 1890 also contains two highly divergent clades  – one represented by the Atlantic Cladocora and Solenastrea, as noted above, but loses Galaxea to the Euphylliidae Veron, 2000 . The Fungiidae Dana, 1846, as noted above, gains Leptastrea, Psammocora, Coscinaraea and Oulastrea; although our tests did not reject monophyly for this family based on the mitochondrial data , Physogyra (1 spp), and Plesiastrea (2 spp)]. Examples of the latter are several genera that appear to have members which are highly divergent even within ocean basins, for example Acanthastrea ; Carrie Bow Cay, Belize; Togian Island, Indonesia; Aka Island, Okinawa and Shirahama, Wakayama, Japan; Yeiliu, Suao, Kenting National Park and Penghu Island, Taiwan (see Supporting Information cox1 gene was amplified by polymerase chain reaction (PCR) with previously reported primers MCOIF and MCOIR 5′-GAC ATG GCT ATT TTT AGC CT-3′), SeaMCOIF (5′-CTA CTA ATC ATA AAG ATA TCG G-3′), AcMCOIR (5′-AAG CAT AGG AGT GTC GTC TAA TC-3′), SeaMCOIR (5′-CAA AGT CAG AGT ATC GTC TTG G-3′).The mitochondrial cob was amplified by PCR with previously reported primers MCytbF and MCytbR for “robust” corals 5′-GCC GTC TCC TTC AAA TAT AAG-3′) or MCytbF, and AcCytbR (5′-AAA AGG CTC TTC TAC AAC-3′) for “complex” corals and other hexacorallians except for the three sea anemone species. For the sea anemones, SeaCytbF (5′-GTG GAA CTT CGG TTC TTT ATT-3′) and SeaCytbR (5′-ATA CAG AGG CTA ATT GTC C-3′) were used for PCR.Mitochondrial The basic protocol for mitochondrial gene amplifications was 94°C for 120 s, followed by 30 cycles at 94°C for 45 s, 55°C for 45 s and 72°C for 90 s, ending with a final phase of 72°C for 5 min. In the case of amplification failure, the following protocol was used: 94°C for 120 s, followed by first 10 cycles at 94°C for 45 s, 45°C for 45 s and 72°C for 90 s, and next 20 cycles at 94°C for 45 s, 45°C for 45 s and 72°C for 90 s, ending with a final phase of 72°C for 5 min. PCR products were directly sequenced as described previously The ß-tubulin gene (intron and exon regions) was amplified, cloned, and sequenced following the methods described previously The ribosomal DNA (rDNA) segment containing the 3′-end of 18S, internal transcribed spacers, 5.8S, and the 5′-end of 28S was amplified, cloned, and sequenced following the methods described elsewhere DNA sequence accession numbers of all genes analyzed here  are listed in the cox1 (607 bp) and cob (776 bp) without gaps; 666 parsimony-informative sites] by searching for optimal topologies, as measured by the criteria maximum likelihood (ML) and parsimony (MP) methods, as well as by taking a Bayesian approach. For the ML and Bayesian analyses, we assumed a model of nucleotide evolution obtained by using the Akaike Information Criterion (AIC) as implemented in ModelTest For mitochondrial DNA, we conducted phylogenetic analyses on the dataset  and evidence supporting those changes. We list provisional placement based on mitochondrial data (from cox1 and cob from (0.10 MB DOC)Click here for additional data file.


M. paratuberculosis (MAP) in feces, internal condition and lesions, as well as humoral and cell-mediated immune responses for 210 days. Study showed marked overall improvement in physical condition of vaccinated goats and average body weight gain was significantly higher (P < .05) in “Bison” group as compared to “Sham-immunized” goats. Mortality due to JD was significantly (P < .05) lower in vaccinated groups than in “sham-immunized”. Morbidity rates (due to diarrhea and weakness) were lower in “Bison” group as compared to other groups. Died goats from vaccinated groups showed regression of gross JD lesions and regeneration of fat layer around visceral organs while “Sham-immunized” goats exhibited frank lesions. Vaccinated goats had higher protective CMI response and also higher antibody titer for the trial period as compared to “Sham immunized”. Both vaccines also reduced shedding of MAP in feces significantly (P < .05). Though the two vaccines effectively restricted the severity of clinical symptoms of JD, however “Indigenous vaccine” was superior in many respects.Therapeutic efficacy of an “Indigenous vaccine” has been evaluated with respect to a commercial vaccine , for the control of clinical Johne's disease (JD) in naturally infected goatherds. Seventy-one goats (JD positive) were randomly divided into 3 groups . After vaccination, goats were monitored for physical condition, morbidity, mortality, body weights, shedding of  Mycobacterium avium subspecies paratuberculosis (MAP) is the cause of chronic incurable infection of animals called Johne's disease (JD) and is also associated with Crohn's disease (CD) in human beings  dye (5 mg/mL) was added to each well of the tissue culture plate. Plate was incubated at 37°C for 4 hours. After incubation 150 μl of DMSO (dimethyl sulfoxide) was added to each well and incubated overnight at 37°C and absorbance was read at 570 nm. Proliferation of lymphocytes was indicated by optical density value of the well with test samples and that of the unpulsed wells. For analysis of data, a signal-to-noise ratio, that is, Stimulative Index (SI) was calculated for individual goats using the following formula: Average OD at 570 nm in stimulated wells/Average OD at 570 nm in nonstimulated control wells. Average SI value for each group of goats was calculated and compared to assess cellular immune response in vaccinated and sham-immunized groups at 60 DPV.LTT was performed as per Uma et al. , with moPre- and post-vaccination antibody response was measured by ELISA. Antibody titers were monitored in all the goats from zero to 210 DPV at 30-day interval as per Singh et al. . OD valuNitric Oxide was estimated at 0, 15, 45, 90 and each 30 DPV after 90 days up to 210 DPV in the serum samples as per Sastry et al. . OpticalFecal culture was performed on zero and 210 DPV from all the goats to check the efficiency of two vaccines in decreasing number of MAP shedders. Fecal samples were grounded, centrifuged, as well as decontaminated (0.9% HPC), and sediment was inoculated on Herrold's egg yolk medium with mycobactin J as per Merkal .There was marked improvement in the overall body conditions of vaccinated goats as compared to sham immunized. Vaccinated and Sham-immunized goats could be easily differentiated from distance on the basis of differences in the body conditions. Vaccinated goats were alert, active, healthy, regained skin luster, shining, pliability, regeneration of hairs and had shining bright eyes.P > .05) and “sham-immunized” (P < .05) groups. Few goats from each group lost weight (in comparison to weight at zero DPV) during the trial period. Percentage of goats loosing body weight was higher in sham-immunized (38.9%) followed by Gudair (33.3%) and Bison (17.2%) groups. Lowered number of vaccinated goats that lost weight after vaccination was indicative of the maximum “individual effect” of the “Indigenous vaccine”.Average body weights, at the time of vaccination in 3 groups, were almost similar . At the X2 = 6.28, df = 1, P < .02) deaths in the vaccinated groups than in sham-immunized goats.Overall mortality rate was lower in vaccinated groups than in “Sham-immunized” group . PercentThere was minor outbreak of contagious ecthyma (Orf) in 12 vaccinated goats just after vaccination and lasted for 7 days. In “Bison” group, one goat of Marwari breed suffered from maggotic wound and was under treatment for 22 days. There was significant decrease in the body weight of this goat. In “sham-immunized” group besides diarrhea, pneumonia was frequent cause of sickness. Number of goats sick (due to diarrhea and weakness) and those that received treatment during trial period were higher in sham-immunized group as compared to vaccinated groups.Four goats, out of 7 died in “Sham-immunized group”, had marked gross lesions of JD, and typical acid fast bacilli indistinguishable from MAP were demonstrated in impression smears. Whereas, 2 goats that died at 60 DPV in “Gudair” group showed mildre inflammatory lesions in mesenteric lymph nodes  and intestines (lowered thickening and no corrugations). Substantial fat layer was generation around all the visceral organs as compared to goats that died in sham-immunized group. Similarly, in “Bison” group also gross lesions of JD were mild in goats that died within few months of vaccination. Marked improvements were seen in the internal condition of one goat of “Bison” group that died at 165 DPV. Wherein gross lesions for JD were very mild, carcass yield was average and there was extensive regeneration of fat layer  around all the visceral organs in comparison to sham-immunized goats . This goAbility of the PBMCs to recognize and respond to MAP antigen was investigated. PBMCs of vaccinated groups had greater, “Stimulative Index” (SI), when pulsed with protoplasmic antigen from native MAP strain than “Sham-immunized” group at 60 DPV . HoweverP < .05) concentration of NO as compared to “sham-immunized” group. In “Bison” and “Gudair” groups, peak NO concentration was seen at 45 DPV and subsequently concentration declined slightly at 90 DPV. After 90 DPV at each sampling interval, goats in vaccinated groups maintained NO concentration with slight up and down and remained higher than sham-immunized goats at all the postvaccination intervals.Results of nitric oxide (NO) production were expressed as geometric means at each sampling interval and are graphically presented in After vaccination high seroconversion rates were seen in vaccinated goats as compared to “Sham-immunized” . Number X2 = 4.293, P = .038; Gudair: X2 = 3.938, P = .047) in feces of goats in “Bison” and “Gudair” groups. Two vaccines reduced shedding of MAP in feces dramatically and only 17.2 and 29.1% goats were excreting MAP in “Bison” and “Gudair” groups, respectively, at 210 DPV. In “sham-immunized” group, all the goats (18 = 100%) were positive for excretion of MAP at 210 DPV.Fecal culture of goats in 3 groups was performed at zero and 210 DPV. Percent of goats positive for MAP at 0 day were 88.8, 79.3, and 79.1 in “sham-immunized”, “Bison”, and “Gudair” groups, respectively. At 210 DPV there was significant reduction in shedding of MAP (Bison: Mycobacterium avium subspecies paratuberculosis (MAP) genotyped as “Indian Bison Type”. This “indigenous vaccine” has been shown to provide good protection (prophylactic) in experimentally vaccinated and twice-challenged goats [Vaccination has been known to offer good protection and recovery against MAP infection and shown to reduce the prevalence of clinical JD by 90% and prevalence of JD in herds by about 50% . First Ied goats . There aSigurdsson and Gunnarson  achievedResults of the present study also indicated that vaccination could be practiced in any stage of disease , in any age group (kids above 3 months old to adults) and in any physiological stage . Initial priming of the goats with MAP infection at the start of trial could have elicited higher immune response against vaccine (second inoculation of the antigen). JD was endemic in CIRG herds; therefore, continuous and slow reinfection of vaccinated animals from environment might have booster effect, thereby complete turnaround in the condition of ready to cull goats when trial was started. Incomplete degree of the response in 15-day-old kids as observed by Corpa et al.  might beLymphoproliferative response to antigen stimulation has been widely used as in vitro correlates of cell-mediated immunity . PBMCs fIt is generally believed that reactive nitrogens, such as NO, are most effective in direct killing of mycobacteria . Signifiγ can play stimulatory effect on B-lymphocytes and antibody production [Significant rise in the peripheral blood antibodies was seen in vaccinated groups after vaccination and “peak titers” were attained at 60 DPV that gradually declined, such pattern was absent in “sham-immunized” group. Comparative antibody titers, in the vaccinated groups, were higher in “Bison” group. In vaccination of sheep by a killed vaccine maximum ELISA reactors were observed around 30 DPV . In a stoduction . Antibodγ, IL-12, IL-2, TNFα, etc.). Though NO concentration also showed decreasing trend in vaccinated goats, they still maintained significantly higher concentration of NO as compared to “sham-immunized” goats. These results were further supported by antibody results, in “sham-immunized” goats, where titers increased slightly towards the end of the experiment. As “‘sham-immunized” goats approached terminal stage of JD, inhibitory Th2 responses might be increased with suppressed activity of antiinflammatory cytokines to limit the host tissue damage.There was decreasing trend of NO concentration in “sham-immunized” goats. Probably protective Th1 response decreased due to decrease in the concentration of pro-inflammatory cytokines , and degeneration of visceral fat was marked in these goats. Goats from vaccinated groups did not show severe lesion of JD grossly due to remission of lesions after vaccination. In a study of vaccination in infected animals, Corpa et al.  also repVaccination reduced fecal shedding in significant number of vaccinated goats. Initially, 16 (88.8%) goats were positive in “sham-immunized” group, but at 210 DPV all the goats were positive in fecal culture. At zero and 210 DPV, the number of goats excreting MAP decreased from 23 (79.3%) and 19 (79.1%) to 5 (17.2%) and 7 (29.1%) in “Bison” and “Gudair” groups, respectively. Number of goats negative for MAP infection were more in “Bison” group. Colony-forming units (cfu) counts also decreased in vaccinated goats as compared to 0 DPV. However, in “sham-immunized” group, cfu increased in 55.0% of goats.Vaccination of lambs against ovine JD reduced deaths up to 90% and also reduced amount of bacilli passed in feces by up to 90% . Rapid dPresent trial indicated that “Indigenous vaccine” used in this study exhibited “Therapeutic effect” by reversing the clinical signs. There was reduction in shedding of number of MAP bacilli per goat, number of goats with clinical, JD and number of goats positive bacteriologically , and complete turnaround was visible in the physical condition of the vaccinated goats as compared to “sham immunized”. Internally the lesions of JD reduced and there was regeneration of visceral fat layer and fat layer around all the visceral organs. Goat became alert active, and udders were filled with milk despite continuous suckling by kids. “Indigenous vaccine” using aluminum hydrooxide gel as adjuvant was superior to “Gudair” using mineral oil, in controlling JD in the naturally infected goats. However, these minor differences in vaccine-induced immune response may have resulted due to use of two different adjuvants, differences in the genetic makeup of individual animals and level of MAP infection.Single dose of “Indigenous vaccine” developed from highly pathogenic locally isolated “Indian Bison Type” genotype of MAP significantly reduced morbidity and mortality, reversed clinical signs as well as reduced shedding of MAP, and there was marked improvement in physical and internal body condition (Therapeutic effect). Therefore, “Indigenous vaccine” worked as “Therapeutic Vaccine” in goats suffering from clinical to advance clinical symptoms of JD.


To analyse associations between the clinical status of patients with rheumatoid arthritis (RA) and the gross domestic product (GDP) of their resident country.The Quantitative Standard Monitoring of Patients with Rheumatoid Arthritis (QUEST–RA) cohort includes clinical and questionnaire data from 6004 patients who were seen in usual care at 70 rheumatology clinics in 25 countries as of April 2008, including 18 European countries. Demographic variables, clinical characteristics, RA disease activity measures, including the disease activity score in 28 joints (DAS28), and treatment-related variables were analysed according to GDP per capita, including 14 “high GDP” countries with GDP per capita greater than US$24 000 and 11 “low GDP” countries with GDP per capita less than US$11 000.2  =  61%). Disease activity levels differed substantially between “high GDP” and “low GDP” countries at much greater levels than according to whether patients were currently taking or not taking methotrexate, prednisone and/or biological agents.Disease activity DAS28 ranged between 3.1 and 6.0 among the 25 countries and was significantly associated with GDP (r  =  −0.78, 95% CI −0.56 to −0.90, rThe clinical status of patients with RA was correlated significantly with GDP among 25 mostly European countries according to all disease measures, associated only modestly with the current use of antirheumatic medications. The burden of arthritis appears substantially greater in “low GDP” than in “high GDP” countries. These findings may alert healthcare professionals and designers of health policy towards improving the clinical status of patients with RA in all countries. Health disparities, including high mortality rates, are associated with low socioeconomic status in many specific diseases in many countries.A multinational database Quantitative Standard Monitoring of Patients with Rheumatoid Arthritis (QUEST–RA)QUEST–RA was established in 2005 with the objectives of promoting quantitative assessment in usual rheumatology care, and to develop a baseline cross-sectional database of consecutive RA patients seen outside of clinical trials in regular care in many countries. Three or more rheumatologists were asked to enroll 100 consecutive unselected patients in each country.11The study was carried out in compliance with the Helsinki Declaration. Ethics Committees or Internal Review Boards of participating institutes approved the study, and a written informed consent was obtained from the patients.All patients were assessed according to a standard protocol to evaluate RA.Patients completed a 4-page self-report questionnaire, which included demographic data, physical function, pain, global estimate and psychological distress. Demographic data included date of birth, gender, years of formal education, race and smoking status, as well as weight and height, to calculate body mass index (BMI). A standard health assessment questionnaire (HAQ)15The DAS28The GDP of each country in 2005 was obtained from a database of the International Monetary Fund2 test for categorical variables. Linear regression models were applied to analyse possible associations between DAS28 and GDP.Descriptive results for each country are presented as mean, median and percentages. Comparisons of demographic variables, clinical characteristics, RA disease activity measures and treatment-related variables within and/or between “high GDP” and “low GDP” countries were performed using parametric and non-parametric tests for continuous variables and the χThe 6004 patients in the QUEST–RA database, from 71 clinics in 25 countries, represent a typical RA cohort in demographic features, with a mean age of 56 years, 79% women, and a mean disease duration of 11 years . PatientPatients in “low GDP” countries had statistically significantly higher disease activity levels in all individual disease activity measures, including physician-derived measures, patient self-report scores and laboratory measures, as well as the composite index DAS28 . The perDMARD were taken by 88–100% of all patients in the 25 countries , with noDifferences in the DAS28 scores of patients who were taking or not taking methotrexate were small within “high GDP” countries (3.6 vs 3.8) and “low GDP” countries (5.1 vs 5.2) but large between “high GDP” versus “low GDP” countries p<0.001; . Similar2  =  61%); a stronger association  was seen when only 18 European countries were included , process  and outcomes , which may contribute to differences in quality of care for patients with RA in different countries. Recognition of major differences in outcomes can lead to more informed efforts to improve the structure and process of care. The ultimate goal of these efforts is to improve outcomes for patients with RA in all countries.


A “registry” of all known cases of nasopharyngeal carcinoma in Malaysia, 1968-72, was established. Attention was focused on the State of Selangor where conditions are best for case finding. Age-adjusted incidence rates among Chinese males and females were 17·3 and 7·3 per 100,000; among Malay males and females, the rates were 2·5 and 0·3 and among Indian males, 1·1. The detailed ethnicity of 192 cases in Selangor was established. Estimated incidence rates for the Chinese sub-groups agreed with the pattern observed elsewhere: highest among the Cantonese, lowest among the Hokkien/Teochiu, with the Khek in between. There was no correlation between histological type and sub-ethnic group among the Chinese cases.


Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors controlling many important physiological processes, including energy homeostasis, lipid, and glucose metabolism, inflammation, as well as cell proliferation and differentiation. PPARs and their heterodimeric partner  have been found in early embryo, developing fetus, and in various compartments of the reproductive system  of many species  . The revβ-oxidation of fatty acids are important for testicular functions . In addition, some testicular toxicants such as phthalates bind to PPARα and PPARγ and modify their activities. In female, invalidation of PPARγ in the mouse ovary [β/δ, PPARγ, or RXRα [α and γ expression drops at the start of parturition. After birth, PPARs continue to play a role in the relation mother/neonates through the mammary gland functions. Indeed, in transgenic mice, a constitutive activation of PPARα in mammary gland alters its development and the lactation leading to mortality of neonates [γ in mammary gland produce a “toxic milk” containing elevated levels of inflammatory lipids. Despite these strong phenotypes, the mechanisms of action of these receptors remain unclear in the control of fertility and further investigations are needed to better use them in medical treatments.Over the last 10 years, a number of studies in vivo and in vitro have strongly suggested that these nuclear receptors might play an important role from gametogenesis to parturition, including gestation and the links mother/fetus . Thus, Pse ovary  leads to or RXRα –6 displaneonates . Furtherα, or glitazar molecules, which bind to PPARα/PPARγ), currently being tested in clinical studies, may prove particularly useful for the treatment of certain types of infertility associated with metabolism disorders such as insulinoresistance in polycystic ovary syndrome (PCOS) [β/δ ligands. Recent studies have shown that the development and implantation of IVF embryos may be augmented by supplementing culture media with PGI2 analogs, a synthetic PPARβ/δ ligand, or retinoic acid [In the future, these drugs might also be used in a large spectrum of treatments targeting reproduction such as improvement of follicular development, in vitro fertilization, certain complications of pregnancy, and sex hormone-sensitive cancers affecting the reproductive tissues, including breast, prostate, ovary, or cancers affecting pituitary cells (pituitary adenomas). For example, new generation of pharmacological drugs targeting these receptors are already in clinical use or are undergoing testing for use as therapeutic agents. Synthetic molecules  . The theoic acid . Of noteFinally, in this issue, we report the putative functions of PPARs and RXRs in gonads, placenta, and embryo and we will discuss the possible role of PPARs as mediators of environmental toxicity for reproductive function.P. FromentP. Froment