Normalization is the process of removing non-biological sources of variation between array experiments. Recent investigations of data in gene expression databases for varying organisms and tissues have shown that the majority of expressed genes exhibit a power-law distribution with an exponent close to -1 (i.e. obey Zipf's law). Based on the observation that our single channel and two channel microarray data sets also followed a power-law distribution, we were motivated to develop a normalization method based on this law, and examine how it compares with existing published techniques. A computationally simple and intuitively appealing technique based on this observation is presented.Using pairwise comparisons using MA plots (log ratio vs. log intensity), we compared this novel method to previously published normalization techniques, namely global normalization to the mean, the quantile method, and a variation on the loess normalization method designed specifically for boutique microarrays. Results indicated that, for single channel microarrays, the quantile method was superior with regard to eliminating intensity-dependent effects (banana curves), but Zipf's law normalization does minimize this effect by rotating the data distribution such that the maximal number of data points lie on the zero of the log ratio axis. For two channel boutique microarrays, the Zipf's law normalizations performed as well as, or better than existing techniques.Zipf's law normalization is a useful tool where the Quantile method cannot be applied, as is the case with microarrays containing functionally specific gene sets (boutique arrays). DNA microarrays have become a widely used biotechnology for assessing expression levels of tens of thousands of genes simultaneously in a single experiment ,2. WhethIt is now clear that simple inter-array normalization techniques, such as simple scaling to housekeeping genes or normalizing to a global mean, are not adequate for microarray data . Housekey) is inversely proportional to the rank (r) of that data point in the data set,Analysis of publicly available large-scale SAGE gene expression data sets ,12 and ay∝rc     (1)c is a coefficient close to -1. Our microarray data can be classified as a generalized form of Zipf's law because the coefficient (c) is not always close to -1 and, in fact, varies between individual microarrays, making simple linear normalization procedures, such as global normalization to the same mean, inappropriate. However, the normalization procedure proposed here demonstrates that by taking Zipf's law into account, it is possible to apply a simple intra-array normalization procedure such that all filters have the same coefficient c and proportionality.where a priori selection of 'housekeeping' genes as a proxy for normalization.We demonstrate the Zipf's law based normalization technique on microarray data sets representing both single channel and two channel technologies. In the single channel category, we produced two radio-labeled, nylon membrane based cDNA data sets, one commercial and one generated "in-house". Both systems contain a selection of genes chosen without regard to functional or pathway considerations, which make them especially appropriate for normalization using Zipf's law. These data sets were also normalized to a global mean  , and theet. al. [The finding that our microarray data distributions conform to a power law distribution agrees with predictions based on genome wide gene expression studies -13, howeet. al.  observedet. al. .a priori selected subset 'housekeeping' genes, however it should not be confused with the a posteriori set of genes typically envisioned when the term is used. Another possible proxy that could meet the 'well-behaved' criteria are control spots which are included on the microarray during it's manufacture. We tested our normalization method on data from a two channel boutique microarray experiment using two types of control spots as proxies for normalization . The Zipf's law normalization methods were then compared with the variant of the loess method developed by Wilson et. al. [The two assumptions upon which the normalization method are based are the same as those used in other normalization methods ,6, namel et. al.  using hoe-loge transformed data set. Our regression showed a good fit, with a correlation coefficient of -0.98 and a slope of -0.56 for microarrays representing human colon  to Zipf's law was verified. The most common method of verifying conformity to Zipf's law is a linear regression on the logn Figure  set A, an Figure  set B, an Figure  set H. Ic of the Zipf's power function (formula 1) than that observed after normalization by the Zipf's law method  and to the quantile method was conducted on the single channel microarray data sets. Five human Unigene microarrays from the panel of thirty-two microarrays used in the sigmoidal colon experiments were selected to represent the greatest variability in pre-normalized data observed in the experiment Figure . Normalid Figure  or the qd Figure . The Ziphods see . HoweverIn order to access the effectiveness of the normalization method, pairwise comparisons using MA-plots   were carThe Kolmogorov-Smirnov test is often used to determine whether data distributions differ significantly and provides a test statistic that measures the proportion of overlap between distributions which ranges from 0  to 1 (for non-overlapping distributions) . Mean Koe intensity versus loge rank fitted with linear regressions show that the Zipf's law normalization based on internal controls  as evidenced by the similarity in slopes of the regression lines. Loess normalization using selected housekeeping genes  on one microarray which had low median intensity and high variance due to low labelling efficiency  performed approximately equally well. Here again, raw (d), quantile normalized (e), and loess normalized (f) plots are provided for reference only.It should be noted that much of the variation in We generated MA plots for each of the normalization methods we compared Figure . TypicalFinally, to quantify the differences between distributions after normalization, pairwise Kolmogorov-Smirnov values were computed for both the complete boutique array data set Table  and afteet. al. [2) of the log mean intensity vs. log rank are a measure of conformation to a power-law distribution and ranged from 0.9968 to 0.7773 for microarray data sets, 0.9982 and 0.9978 for the SAGE experiments . The quantile normalization method would effectively remove this difference by replacing the data distribution of each microarray with the mean distribution of all arrays. In contrast, the principle of normalization according to Zipf's law can also apply to arrays of this type if a group of control spots are included on the microarray. These control spots could be an external reference probe which hybridises to a concentration gradient of matching spots on the array, or internal positive (highly expressed genes) and negative (spotting buffer) control spots on the microarray, or an  et. al.  or Schad et. al. . A lineaThe results of our comparison between methods which are designed to normalize boutique microarray data show that Zipf's law normalization using internal control spots results in a relatively well normalized data set when compared to Zipf's law normalization using selected housekeeping genes and the modified loess method using selected housekeeping genes. In addition, the Zipf's law method produced data distributions which are more similar between arrays allowing for between-array comparisons which are advantageous in terms of both cost, because of the reduced number of microarrays that need to be run, and, statistical power, by allowing for greater numbers (n), experimental design permitting.In summary, we examined the applicability of using Zipf's law as the basis for a novel normalization technique, which is applicable to both one channel microarray data and two channel microarrays. This method is shown to out-perform such methods as global normalization to the mean but would appear to be inferior to quantile normalization. The quantile method was superior to Zipf's law in removing intensity dependent effects commonly seen in microarray data. While the latter method cannot be applied to boutique arrays, we show that the Zipf's normalization method used with internal positive and negative controls or with selected housekeeping genes normalizes boutique arrays as well as currently existing methods. Additionally, data normalized with the Zipf's method using internal control spots seems more amenable to between-array gene intensity comparisons when compared to other methods.33P-labeled cDNA derived from total RNA extracted from biopsy material from the sigmoidal colon of normal , and patients with Crohn's disease  and ulcerative colitis . To emphasize that our normalization technique can be used to normalize other array systems, the second array set used was a smaller, but widely used, commercially available microarray system. Data set B  are identical for all microarrays. This is easily achieved using a regression model on the loge intensity versus loge rank transformed data, which has the general form,Normalization was accomplished by transforming the data such that the coefficient y) = a + bln (r) + e     (2)ln , b is the regression coefficient (corresponding to the coefficient c in Zipf's power function), and e is an error coefficient, which is assumed to be normally distributed.where ymed) of each gene, over all microarrays on which it was measured, and sorting the resulting list of medians to obtain their median ranks (rmed). The regression model (2) is applied to the loge median intensities and their ranks to estimate amed and bmed using the least squares method,The first step in this three step procedure was to compute the median intensity of each gene over all microarrays to establish ranks, which were used as the 'reference' to which all microarrays were normalized. This was done by taking the median intensity  genes, which do not (or only slightly) vary, act to stabilize the regression line and allow normalization to be performed.In the second step of the normalization procedure, the regression model was applied individually to each microarray using the same ranking as the reference curve,ak and bk which are estimated individually for each array using the least squares method, where k is equal to the number of microarrays in one channel systems, and equal to 2 time the number of microarrays (one for each channel) in two channel systems. Data from two channel arrays were treated in the same way as one channel systems, i.e. each channel was treated independently.This results in a set of coefficients kth array and that of the reference curve was applied as the normalization factor,In the third step, the difference between the expected gene intensity value on the yk,  and  were always greater than one to avoid negative values after log transformation. After normalization, the same scaling factor was applied to the data to back transform to their original magnitude. For example, if the smallest raw value in the data set was 0.1, the unlogged raw data was multiplied by a scaling factor of 10 before normalization, and the unlogged normalized data was divided by the same scaling after normalization.A scaling factor was applied to the raw data before normalization such that the values med and bmed, and only the 64 internal control spots from each array were used to estimate ak and bk. In both cases, the ranks generated from the entire data set, were used. The normalization factor was then applied over the entire data set as described above.In the special case of our third microarray data set (see Methods: Data Acquisition) which was a boutique array, the same procedure as described above was applied with the following modifications. Each microarray contained 32 spots each of internal positive controls  and internal negative controls (spotting buffer). The medians of all gene intensities were computed , and median ranks were assigned as described. However, only the medians of the 64 internal control spots were used to estimate aa priori from the data set by virtue of their low variance in intensity and such that the entire range of intensities observed on the microarrays is uniformly represented. We also applied the Zipf's law normalization technique to our boutique microarrays using the set of housekeeping genes selected using the method of Wilson, et. al.An alternative to the used of internal control spots for the normalization of boutique microarrays was also explored. Wilson, et. al.  describeIn addition to the normalization method based on Zipf's law, all data sets were normalized to a global mean  and the quantile method. The quantile method is applied by ranking the genes in each array by intensity, taking the median intensity at each rank, and replacing each gene intensity with the median intensity corresponding to the same rank. All normalization methods were compared to each other and to the raw data distribution using box plots and MA plots (pairwise array comparisons of the log-intensity ratio (M) to the mean log-intensity (A)). The two channel boutique microarray data set allowed further normalization methods not possible on one channel array systems to be applied. We normalized this data set using the popular loess method , and a mThe Zipf's normalization procedure was initially implemented as an SQL stored procedure in a relational database. However, because this is not easily transferable to other systems, we provide two further implementations, a Perl script and an Excel macro [see Additional files e intensity versus loge rank fitted with linear regressions are a way to visually evaluate the normalization procedure according to the criteria of the Zipf's Law normalization. Specifically, all arrays have identical coefficients c and proportionality for the Zipf's power function when the slops and y-intercepts of the regression lines are identical. Finally, to quantify the similarity between microarray distributions after normalization, the mean Kolmogorov-Smirnov value was calculated over all possible pairwise combinations of microarrays within an experiment. In the case of two channel arrays, the mean of within-array Kolmogorov-Smirnov values was also computed (n = the number of arrays). It should be emphasized that even though the Kolmogorov-Smirnov values are technically a test statistic, no statistical test is performed. The values are here used only as a measure of similarity between microarray distributions.To compare and evaluate the effectiveness of the various normalization methods applied in this paper, several well established methods were used along with some less common techniques. MA plots  are a co2 are the mean and variance, was made for each data set to visualize the conformity to log normal distribution.The underlying premise of the Zipf's normalization method is that microarray data distributions follow a power law distribution such that the relationship between the log intensities and the log ranks is clearly linear. While this assumption holds true for the three data sets we present in this paper, to evaluate the general applicability of the method we also examined eight publicly available data sets (Table EST – Expressed Sequence TagMA – log ratio (M) vs. mean log intensity (A)NCBI – National Center for Biotechnology InformationRZPD – Deutsches Ressourcenzentrum für Genomforschung GmbHSAGE – Serial analysis of gene expressionSQL – Structured Query LanguageTL conducted the data analysis and implementation of algorithms, participated in the development of the normalization method and is principle author of this manuscript. CMC generated the Unigene and Clontech microarray data set, participated in the development of the normalization method and participated in manuscript preparation. PJPC conceived of and participated in the development of the normalization method. RH participated in the generation of microarray data sets and participated in the development of the normalization method. GD conceived of and coordinated neurology related aspects of this study. SS conceived of and coordinated gastrointestinal related aspects of this study.Clontech microarray log plots Five rat Clontech microarrays from the panel of thirty-nine microarrays probed with rat-brain tissue. Upper left to lower right: a. Loge median gene intensity vs. loge rank – conformity to Zipf's law is demonstrated by the linear regression line (in red) b. Five microarrays chosen to maximize pre-normalization variability, each plotted according to the gene ranks determined by their median gene intensity levels. c. The same five microarrays, normalized to a global median, with regression lines. d. The same five microarrays, normalized with the quantile method, with regression lines. e. The same five microarrays normalized taking Zipf's law into account, with regression lines. For plots b-d, a sub-sample of 50% of the data points are plotted for readability.Click here for fileMean of squared log ratios from MA plots in Figure Click here for fileBoutique microarray log plots Four mouse apoptosis boutique microarrays used in the mouse cell line experiments. This is the same data set as shown in Figure e median gene intensity vs. loge rank – a. Normalized according to Zipf's law, using internal positive and negative controls as proxies for the whole data set. b. Normalized with a loess curve fit using a selected set of housekeeping genes as proxies (see Methods). c. Normalized according to Zipf's law, using the same selected set of housekeeping genes as in b. as proxies d. The raw data. e. For comparison purposes only, normalized using the quantile method. f. For comparison purposes only, normalized using the standard loess method.Click here for fileRequires: Microsoft Excel Click here for fileRequires: Perl (which runs on many platforms), the PDL perl module Click here for fileMicroarray type: Filter based cDNA from the RZPD Number of genes: 33,792 Number of microarrays: 31 Probed with: Total RNA from human sigmoidal colon. Within microarray normalization: NoneClick here for fileMicroarray type: Clonetech Atlas Rat cDNA 7738-1 Number of genes: 558 Number of microarrays: 33 Probed with: Total RND from rat cerebellum and olive. Within microarray normalization: NoneClick here for fileMicroarray type: custom made glass slide Number of genes: 1024 Number of microarrays: 5 Probed with: Total RND from mouse cell lines. Within microarray normalization: NoneClick here for file


Although less clinical intervention studies have been performed with fibrates than with statins, there are evidences indicating that fibrates may reduce risk of cardiovascular events. The potential clinical benefit of the fenofibrate will be specified by the ongoing Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study, which rationale, methods and aims have been just published.Controlled clinical trials show similar or even greater cardiovascular benefits from statins-based therapy in patient subgroups with diabetes compared with overall study populations. Therefore, statins are the drug of first choice for aggressive lipid lowering actions and reducing risk of coronary artery disease in these patients. However, current therapeutic use of statins as monotherapy is still leaving many patients with mixed atherogenic dyslipidemia at high risk for coronary events. A combination statin/fibrate therapy may be often necessary to control all lipid abnormalities in patients with metabolic syndrome and diabetes adequately, since fibrates provide additional important benefits, particularly on triglyceride and HDL-cholesterol levels. Thus, this combined therapy concentrates on all the components of the mixed dyslipidemia that often occurs in persons with diabetes or metabolic syndrome, and may be expected to reduce cardiovascular morbidity and mortality.Safety concerns about some fibrates such as gemfibrozil may lead to exaggerate precautions regarding fibrate administration and therefore diminish the use of the seagents. However, other fibrates, such as bezafibrate and fenofibrate appear to be safer and better tolerated. We believe that a proper co-administration of statins and fibrates, selected on basis of their safety, could be more effective in achieving a comprehensive lipid control as compared with monotherapy. Due to their beneficial effects on glucose and lipid metabolism, peroxisome proliferator activated receptors (PPAR's) alpha agonists (fibrates) are good potential candidates for reducing the risk of myocardial infarction (MI) in subjects with metabolic syndrome -3. AlthoThere have been no direct head-to-head comparisons of a statin with a fibrate in any clinical endpoint trial. However, compared with statins, fibrates appear to more selectively target the therapeutic goals in obese individuals with features of insulin resistance and metabolic syndrome -cholesterol and inappropriate high-density lipoprotein (HDL)-cholesterol and triglyceride levels).The primary-prevention trial Helsinki Heart Study showed that treatment with gemfibrozil led to a significant reduction in major cardiovascular events . RegardiIn two previous small studies bezafibrate decreased the rate of progression of coronary atherosclerosis and decreased coronary event rate ,7. In anRecently, reduced incidence of type 2 diabetes in patients with impaired fasting glucose level on bezafibrate has been demonstrated . The potDespite increasing use of statins, a significant number of coronary events still occur and many of such events take place in patients presenting with the metabolic syndrome. Whereas statins remain the drug of choice for patients who need to achieve the LDL-cholesterol goal, fibrate therapy may represent the alternative intervention for subjects with atherogenic dyslipidemia typical for metabolic syndrome and an LDL-cholesterol already close to goal values. In addition, the concomitant use of fibrates seems to be attractive in patients whose LDL-cholesterol is controlled by statin therapy but whose HDL-cholesterol and/or triglyceride levels are still inappropriate -19. ThisThe factor that dominates in overweight-related metabolic syndrome is the permanent elevation of plasma free fatty acids (FFA) and the predominant utilization of lipids by the muscle inducing a diminution of glucose uptake and insulin resistance. Currently, an insulin-resistant state – as the key phase of metabolic syndrome – constitutes the major risk factor for development of macrovascular complications -23. On tIt is important to note that on a whole-body level, lipid and glucose metabolisms interact intimately. Briefly, PPAR alpha is activated by fibric acids (e.g. bezafibrate) and form heterodimers with the 9-cis retinoic acid receptor (RXR). These heterodimers bind to peroxisome proliferator response elements, which are located in numerous gene promoters and increase the level of the expression of mRNAs encoded by PPAR alpha target genes. Bezafibrate reduces triglyceride plasma levels through increases in the expression of genes involved in fatty acid-beta oxidation and by decrease in apolipoprotein C-III gene expression. Fibric acids increase HDL-cholesterol partly by increasing apolipoprotein A-I and apolipoprotein A-II gene expression. Their triglyceride-lowering and HDL-cholesterol raising effects lead to decreased systemic availability of fatty acid, diminished fatty acid uptake by muscle with improvement of insulin sensitization and reduced plasma glucose level -28.Evidence also suggests that there is a 'fibrate effect' that mediates the reduction in CHD risk beyond the favorable impact of these agents on HDL-cholesterol levels. This last notion is consistent with the pleiotropic effects of fibrates which are known to be related to their mechanisms of action . Being PControlled clinical trials show similar or even greater cardiovascular benefits from statins-based therapy in patient subgroups with diabetes, impaired fasting glucose, and metabolic syndrome, compared with overall study populations. Therefore, statins are the drug of first choice for aggressive lipid lowering actions and reducing risk of coronary artery disease in these patients. However, current therapeutic use of statins as monotherapy is still leaving many patients with mixed atherogenic dyslipidemia at high risk for coronary events.A combination statin/fibrate therapy may be often necessary to control all lipid abnormalities in patients with metabolic syndrome and diabetes adequately, since fibrates provide additional important benefits, particularly on triglyceride and HDL-C levels. Thus, this combined therapy concentrates on all the components of the mixed dyslipidemia that often occurs in persons with diabetes or metabolic syndrome, and may be expected to reduce cardiovascular morbidity and mortality.Safety concerns about some fibrates such as gemfibrozil may lead to exaggerate precautions regarding fibrate administration and therefore diminish the use of the seagents. However, other fibrates such as bezafibrate and fenofibrate appear to be safer and better tolerated -36. We bThe author(s) declare that they have no competing interests.


PLoS Biology, volume 2, issue 6:In Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA ClonesTadashi Imanishi, Takeshi Itoh, Yutaka Suzuki, Claire O'Donovan, Satoshi Fukuchi, et al.DOI: 10.1371/journal.pbio.00201621. The abbreviation FLJ was expanded incorrectly (as full-length long Japan) in the abbreviations list and in the first paragraph of the Results/Discussion section. FLJ stands for full-length cDNA Japan.2. In Table 1, the number of cDNAs and the number of library origins for two of the cDNA sources were incorrect. The correct numbers are as follows:


Apolipoprotein A-V (Apo A-V) gene has recently been identified as a new apolipoprotein involved in triglyceride metabolism. A single nucleotide polymorphism (SNP3) located in the gene promoter (-1131) was associated with triglyceride variation in healthy subjects. In type 2 diabetes the triglyceride level increased compared to healthy subjects. Hypertriglyceridemia is a risk factor for coronary artery disease. We aimed to examine the interaction between SNP3 and lipid profile and coronary artery disease (CAD) in Tunisian type 2 diabetic patients.The genotype frequencies of T/T, T/C and C/C were 0.74, 0.23 and 0.03 respectively in non diabetic subjects, 0.71, 0.25 and 0.04 respectively in type 2 diabetic patients. Triglyceride level was higher in heterozygous genotype (-1131 T/C) of apo A-V (p = 0.024). Heterozygous genotype is more frequent in high triglyceride group (40.9%) than in low triglyceride group (18.8%) ; p = 0.011. Despite the relation between CAD and hypertriglyceridemia the SNP 3 was not associated with CAD.In type 2 diabetic patients SNP3 is associated with triglyceride level, however there was no association between SNP3 and coronary artery disease. Dyslipidemia in type 2 diabetes are most frequently characterized by elevation of total serum triglycerides, of very low density lipoprotein-triglyceride (VLDL-TG) and low level of high density lipoprotein-cholesterol (HDL-C) . HypertrWe have addressed the issue to examine the interaction between SNP3 and lipid profile and coronary artery disease (CAD) in type 2 diabetic patients compared to controls in Tunisian population.Type 2 diabetic patients have a BMI values more important than non diabetic subjects. Whereas the WHR in lower in controls. Males and smokers are more frequent in patients with CAD Table . To compAccording to SNP3 of the apo A-V gene, variation of lipid parameters in diabetic or non diabetic patients are shown in Table To investigate the relation of SNP3 polymorphism with coronary artery disease we studied the association in all subjects (those who are taking lipid lowering drugs are included). There was no association between coronary artery disease and SNP3 either in non diabetic subjects or in type 2 diabetic patients Table .Elevated serum lipid levels are an important risk factor for atherosclerosis. Both environmental and genetic factors contribute to variability in serum lipid levels . In thisIn type 2 diabetes the triglyceride level is increased, which is due to multiple factors related to insulin and carbohydrate metabolism, LPL activity, CETP activity... The absence of an unusual allele frequency of SNP3 in type 2 diabetic patients compared to non diabetic subjects, in spite of triglyceride level variation, shows that there is no association between apo A-V SNP3 polymorphism and the presence or absence of diabetes. Diabetic homozygous for the major allele are more frequent in low triglyceride group, showing that SNP3 is associated with triglyceride variation in type 2 diabetic patients. Contrary to Esteve et al., who have reported no significant difference in triglyceride concentration with the apo A-V polymorphism in type 2 diabetic patients , our res target gene [The relationship between SNP3 and coronary artery disease is under discussion. The LOCAT study showed that there is no association between SNP3 and progression of coronary heart disease . Our resget gene . While SIn summary, the apo A-V SNP3 is associated with triglyceride level in Tunisian type 2 diabetic patients. However, this SNP is unlikely to be associated with the presence of diabetes. Although SNP3 is associated with hypertrigyceridemia, there was no relationship between this polymorphism and coronary artery disease. Further investigations were needed to determine the effect of SNP3 on lipid lowering drug response.Three hundred and eight subjects, aged 45–70 years, participated in this study. They belong to four groups. The first group contained 74 type 2 diabetic patients with CAD, among whom 12 are taking lipid lowering drugs. The second group contained 78 type 2 diabetic patients without CAD, all of them did not take lipid lowering drugs. The third group contained 57 patients with CAD and without diabetes, 70% of the patients are taking lipid lowering drugs. The last group contained 99 controls without diabetes nor CAD, 11 subjects are taking lipid lowering drugs. The clinical and biological characteristics of each group are summarized in Table 2 and glycosylated hemoglobin (Hb A1c) more than 12%. Post menopausal women had no hormone replacement therapy. BMI was calculated using the formula : weight (Kg)/height2 (m2). Obesity was defined as BMI>30 kg/m2. Waist-to-hip ratio (WHR) was calculated from measurements of the waist circumference taken at the mid point between umbilicus and xiphoid and hip circumference, at the widest point around the hips, respectively. Blood samples were drawn after subjects had fasted overnight (12 hours) into tubes containing EDTA. Plasma was immediately separated by centrifugation.All participants were recruited in the departments of internal medicine and cardiology in Monastir university hospital. Written or verbal informed consent were obtained from all patients and controls before the study. All written informed consent were not possible because the majority of eligible subjects in our study were illiterate. The diagnosis of diabetes was based on a previous history of diabetes according to American Diabetes Association criteria . The patAfter DNA extraction, the single nucleotide polymorphism SNP3 within the apo A-V gene (-1311 T/C) was determined by PCR-RFLP analysis using MseI restriction endonuclease as described previously .2) test was used to examine distribution of categorical variables. A value of p < 0.05 was considered significant.Data management and statistical analysis were performed using SPSS 10.0 software. Results are summarized as mean ± SD. Since triglyceride levels were not normally distributed, logarithmic transformation of triglyceride concentration was performed before the statistical analysis. Student's test was used to compare continuous variables and Chi square (χApo : apolipoproteinBMI : Body Mass IndexCAD : Coronary Artery DiseaseHDL : High Density LipoproteinsHDL-C : HDL cholesterolPCR-RFLP : Polymerase Chain Reaction-Restriction Fragment Length PolymorphismSNP : Single Nucleotide PolymorphismTG : TriglycerideVLDL : Very Low Density LipoproteinsWHR : Waist-to-Hip RatioR.Ch and N.A: carried out the molecular studies, participated in the design of the study and drafted the manuscript ; M.S : carried out the biochemical essay ; S.H and Sy.M : interested in the clinical aspect ; M.H : conceived of the study, and participated in its design and coordination and helped to draft the manuscript and revised it critically for important intellectual content and have given final approval of the version to be published; MS.M : revised the article critically for important intellectual content and have given final approval of the version to be published.


Percutaneous coronary intervention can be associated with distal embolization of thrombotic material causing myocardial necrosis and infarction.We discuss the role of intravascular imaging to guide the use of a distal protection device by describing the outcome of a young woman presenting with non-ST elevation myocardial infarction. Coronary angiography demonstrated an isolated minor stenosis in the proximal left anterior descending coronary artery with slight haziness beyond the lesion. Intravascular ultrasound confirmed an extensive thrombus overlying a bulky atherosclerotic plaque. A distal filter wire was therefore successfully used to reduce the risk of distal embolization.The use of intravascular ultrasound in patients presenting with acute coronary syndrome may reveal large thrombi that are difficult to image using conventional angiographic techniques. Intravascular ultrasound can therefore be used as a tool to select lesions requiring distal protection. Patients with acute coronary syndromes (ACS) are increasingly treated with an early invasive strategy. During percutaneous coronary interventions (PCI) interventionalists have often to deal with thrombus-laden lesions in native coronary vessels. This poses the serious problem of preventing macroscopic and microscopic distal embolization . There hIn native vessels cost and efficacy  considerAngiography alone is known to underestimate the risk of distal embolization: for example, only overt signs of massive thrombus burden are predictive of no-reflow in myocardial infarction patients treated with primary PCI . IntravaWe present a case of successful prevention of macroscopic distal embolism in a young patient with ACS obtained combining IVUS and distal filter protection.A 41-year-old lady presented with a short history of cardiac sounding chest pain. She was a smoker, had hypertension and had a strong family history of premature ischaemic heart disease. The ECG showed widespread anterior T wave inversion. Conventional treatment was started immediately for ACS including intravenous nitrates, low molecular weight heparin, aspirin and clopidogrel. The patient remained pain-free following admission and serial biochemistry demonstrated a rise in cardiac troponin I to 24 mmol/l at 24 hours. There was no evidence of Q waves on the ECG and left ventricular function was normal on echocardiography.Coronary angiography was undertaken four days after admission. This demonstrated mural thickening of the proximal left anterior descending (LAD) coronary artery with a possible filling defect at the distal end of the lesion but no evidence of coronary atherosclerosis elsewhere Fig. . InterroPercutaneous coronary intervention was performed using distal filter protection with the EZ Filterwire . The GpIIb/IIIa antagonist Abciximab was administered. A 4.0 × 15 mm stent was placed directly with good final angiographic result Fig.  and no eNo further Troponin I elevation occurred; the patient was discharged home the next day, and remains asymptomatic at two months.Acute coronary syndromes with extensive thrombosis in the absence of widespread coronary atheroma are detected most frequently in young smokers ,6.The necessity of preventing distal embolic during PCI is becoming increasing recognised, as traditional angioplasty may be associated with distal myocardial necrosis .In our case, angiographic appearance was not suggestive of either obstructive coronary disease or a high embolic risk: none of the criteria set up by Yip et al  (cutoff However, we suspected the presence of a significant thrombotic mass, mainly on the clinical grounds of widespread ECG change and Troponin I elevation, and performed IVUS examination. The importance of the IVUS results is clear: the visualization of a lobulated thrombus, loosely attached to an eroded, non-ruptured plaque, prompted us to use distal protection, which in turn may have prevented significant embolism.IVUS proved also useful in ensuring full coverage of the lesion, resolving the relation of the lesion with the LAD ostium, correctly sizing the stent, and ruling out thrombus or plaque prolapse through stent struts.We present a case of successful management of an intracoronary thrombus, which was accomplished combining IVUS data and a distal protection device.IVUS proved invaluable in the diagnosis and treatment of this challenging case. We believe IVUS guidance should be considered for assessing thrombotic burden and embolism potential particularly in young patients with ACS when angiographic data are inconclusive.The authors declare that they have no competing interests.IP and AM have written the first draft of the manuscript. AB and IP have performed the coronary intervention. IP, AM, VA and AB participated in the design and coordination of the final manuscript. All authors have read and approved the final manuscript.


Insulin-like growth factor binding protein 2 (IGFBP2) is overexpressed in ovarian malignant tissues and in the serum and cystic fluid of ovarian cancer patients, suggesting an important role of IGFBP2 in the biology of ovarian cancer. The purpose of this study was to assess the role of increased IGFBP2 in ovarian cancer cells.Using western blotting and tissue microarray analyses, we showed that IGFBP2 was frequently overexpressed in ovarian carcinomas compared with normal ovarian tissues. Furthermore, IGFBP2 was significantly overexpressed in invasive serous ovarian carcinomas compared with borderline serous ovarian tumors. To test whether increased IGFBP2 contributes to the highly invasive nature of ovarian cancer cells, we generated IGFBP2-overexpressing cells from an SKOV3 ovarian cancer cell line, which has a very low level of endogenous IGFBP2. A Matrigel invasion assay showed that these IGFBP2-overexpressing cells were more invasive than the control cells. We then designed small interference RNA (siRNA) molecules that attenuated IGFBP2 expression in PA-1 ovarian cancer cells, which have a high level of endogenous IGFBP2. The Matrigel invasion assay showed that the attenuation of IGFBP2 expression indeed decreased the invasiveness of PA-1 cells.We therefore showed that IGFBP2 enhances the invasion capacity of ovarian cancer cells. Blockage of IGFBP2 may thus constitute a viable strategy for targeted cancer therapy. Ovarian cancer is the most lethal gynecological malignancy. Indeed, epithelial ovarian cancer is detected at a late clinical stage in as much as 75% of patients, in whom the overall survival rate is a dismal 14–30% -4. A recIGFBP2 is also overexpressed in a wide spectrum of other cancers, including glioma, prostate cancer, synovial sarcoma, neuroblastoma, colon cancer, adrenocortical cancer, lung cancer, Wilms' tumor, and hepatoblastoma -17. The In this study, we found that overexpression of IGFBP2 enhanced the invasiveness of ovarian cancer cells. Further, attenuation of IGFBP2 expression by siRNA reduced the invasiveness of ovarian cancer cells.p = 0.03) and between borderline tumors and invasive carcinomas (p = 0.03)  Figure , Table 1Thus far, the biological or pathophysiological role of IGFBP2 in ovarian cancer is unknown. To elucidate the role of increased IGFBP2 in ovarian cancer, we therefore generate IGFBP2-overexpressing cells. To obtain suitable cells for this study, we first examined the endogenous expression of IGFBP2 in six ovarian cancer cell lines: NIH:OVCAR3, SKOV3, PA-1, OV-90, TOV-112D, and TOV-21G. IGFBP2 was expressed at different levels in all six cell lines Figure . Both SKp < 0.05; Figure We studied the cellular phenotype of IGFBP2-overexpressing cell lines. Similar to observations in gliomas , we did p < 0.05; Figure The observation that IGFBP2 increased the invasive capacity of ovarian cancer cells and those of previous studies prompted us to determine whether IGFBP2 could serve as a target of therapy for ovarian cancer. We used PA-1 ovarian cancer cells for this experiment because they express a high level of endogenous IGFBP2 and show a relatively high invasive capacity compared with other ovarian cancer cell lines. We designed siRNAs for four different target sites of IGFBP2 mRNA because not all siRNAs were expected to effectively attenuate IGFBP2 expression. Western blotting analysis showed that the IGFBP2 level after siRNA-3 transfection was comparable to the levels after the transfection of Lamin A/C and negative control siRNA in OVCAR3 and PA-1 ovarian cancer cell lines, suggesting that the siRNA-3 was not effective Figure . ConsideIn this study, we showed that IGFBP2 was significantly overexpressed in invasive ovarian carcinomas compared with borderline ovarian tumors as well as normal ovarian tissues and that IGFBP2 increases invasion capability of ovarian cancer cells. These results provide evidence that IGFBP2 could promote ovarian cancer progression by augmenting the invasion potential. Although further investigations of molecular mechanisms are required, our findings using siRNA study support IGFBP2 as a novel target for the treatment of ovarian cancer.2. Media were routinely changed every 3 days.Ovarian cancer and normal control tissues were obtained from The University of Texas M. D. Anderson Cancer Center tumor tissue bank with the approval of the Institutional Review Board. The ovarian cancer cell lines NIH:OVCAR3, SKOV3, OV-90, TOV-112D, TOV-21G, and PA-1 were purchased from the American Type Culture Collection . NIH:OVCAR3 cells were maintained in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS); SKOV3 and PA-1 cell were maintained in McCoy's 5a medium and Dulbecco's modified Eagle medium (DMEM)/F12, respectively, supplemented with 10% FBS; OV-90, TOV-112D, and TOV-21G cells were maintained in a 1:1 mixture of MCDB 105 medium and medium 199, supplemented with 15% FBS. All cells were kept at 37°C in a humidified atmosphere with 5% COFormalin-fixed, paraffin-embedded archival tissue blocks from ovarian cancer patients who had undergone surgery at The University of Texas M. D. Anderson Cancer Center between 1990 and 2001 were used to construct progression tissue microarrays according to previously described methods . The proPolyclonal antibody against IGFBP2  was used in the immunohistochemistry studies. This antibody is specific and does not cross-react with other isoforms of IGFBP. A standard indirect immunoperoxidase procedure  was used for all stains. In brief, antigen retrieval was performed by first placing unstained slides in a steamer for 25 min. The antibody against IGFBP2 (in a 1:1000 dilution) was overlaid on the tissue sections of tissue arrays, and incubation was performed at 4°C overnight. Secondary antibody incubation was performed at room temperature for 60 min. Mayer's hematoxylin nuclear stain was used as a counterstain. Staining intensity was graded on a 0–3 scale, where 0 = no staining as assessed by staining with anti-goat secondary antibody alone, 1 = weak (<10%), 2 = moderate (10–50%), and 3 = strong (50–100%). The results of the immunohistochemistry studies were statistically analyzed using a Mann-Whitney nonparametric U test.To establish stable cell lines that overexpressed IGFBP2, we transfected SKOV3 ovarian cancer cell lines with a pcDNA3.1 expression vector encoding IGFBP2 cDNA using FuGENE6 reagent . Transfected cells were subsequently selected in the presence of G418 (300 μg/ml) for 5 weeks. The expression of IGFBP2 clones was determined from western blots of cell extracts with anti-IGFBP2 antibody (C-18). Two vector-transfected cell lines and three IGFBP2 stable cell lines were used in this study. Established stable cells were maintained without antibiotics.Equal amounts of proteins from the total cell lysates was separated by 10% SDS-PAGE and transferred electrophoretically to a Hybond ECL nitrocellulose membrane . The membrane was blocked in 5% skim milk in 1× PBS and probed with a 1:1000 dilution of a goat polyclonal anti human IGFBP2 (C-18) overnight at 4°C. An enhanced chemiluminescence kit  was used to visualize the proteins.4 to 2 × 105 cells/well were placed in triplicate wells and incubated for 22 h at 37°C in a humidified incubator with 5% CO2. After incubation, the cells that had passed through the filter into the lower wells were stained with Giemsa  and counted by analyzing images under a microscope using software program as described previously [p value of <0.05 was considered statistically significant.We used 24-well BioCoat Matrigel invasion chambers  with an 8-μm pore polycarbonate filter coated with Matrigel to measure chemoinvasion. The lower compartment contained 0.75 ml of medium with 0.5% FBS as a chemoattractant. In the upper compartment, 5 × 10eviously . All assFour different siRNA molecules designed for IGFBP2 mRNA and siRNA molecules for Lamin A/C and negative control were synthesized and purified . siRNA that targets Lamin A/C and siRNA that bears no homology with relevant human genes was used as a negative control.5) were plated to a 6-well plate and allowed to adhere for 24 h; the confluency of the cell monolayer at the time of transfection was 40–60%. 5 μg of siRNA and 15 μl of RNAiFect Transfection Reagent  was used. The cells were incubated under normal cell culture conditions. All assays were performed 72 h after treatment.AATGGCGATGACCACTCAGAA was the target sequence for siRNA1; AAGGGTGGCAAGCATCACCTT was the target sequence for siRNA2; AAGCGCCGGGACGCCGAGTAT was the target sequence for siRNA3; AACCTCAAACAGTGCAAGATG was the target sequence for siRNA4; AACTGGACTTCCAGAAGAACA was the target sequence for Lamin A/C; and AATTCTCCGAACGTGTCACGT was the target sequence for the negative control. siRNAs were dissolved in siRNA suspension buffer to a final concentration of 20 μM, and the mixture was heated to 90°C for 1 min and incubated at 37°C for 60 min. PA-1 ovarian cancer cells (2 × 10EL carried out Western blotting analysis, establishing stable cells, invasion assay and siRNA transfection, participated in the analysis of all data and drafted the manuscript. CM and IS contributed to the statistical analysis. HW and JL provided frozen tissues and carried out tissue microarray and its analysis. AN performed the analysis of invasive assay. JK participated in its design of the study. JL and WZ participated in its design and coordination and helped draft the manuscript. All authors read and approved of the final manuscript.


Some cells—such as the cells of the female germline (oocytes)—do not contain centrosomes, and the chromosomes themselves seem to arrange and stabilize the microtubules into spindles. These spindles are referred to as anastral.As cells can only arise from cells that already exist, continuity of life depends on the highly regulated sequence of events that control cell division. This process is mediated by a complex macromolecular structure called the mitotic spindle. The most conspicuous components of the spindle are microtubules, which are made of tubulin and other associated proteins. In most animal cells—body cells and male germline cells (spermatocytes)—spindle assembly is orchestrated by organelles called centrosomes, which actively polymerize  and stabilize microtubules. The spindles found in these cells are known as Drosophila melanogaster) spermatocytes, Cayetano Gonzalez and his colleagues at the European Molecular Biology Laboratory in Germany  have been able to observe the assembly and sorting of microtubules of noncentrosomal origin in cells that contain centrosomes. The task of flagging such microtubules is complicated by the fact that centrosomes become quite active microtubule organizers once cell division begins. Thus, as soon as the membrane around the nucleus breaks down, microtubules from the centrosome invade the nuclear region, making it hard to identify any noncentrosomal microtubules that might appear. To get around this problem, Elena Rebollo in the Gonzalez lab set up two experimental conditions under which centrosomes remain functional but are kept affixed to the cell membrane—and, therefore, away from the nucleus—in Drosophila spermatocytes. One takes advantage of a genetic mutation ; the other uses a transient treatment with a drug  that depolymerizes microtubules.To gain insight into the mechanisms of spindle assembly, scientists are increasingly relying on techniques that allow them to directly observe dynamic, complex processes in the living cell. Using time-lapse microscopy of fluorescently labeled fruitfly (In these modified cells, microtubules can be seen growing not only over the membrane-bound centrosomes, as expected, but also over the nuclear region, away from the centrosomes. Nucleation, or formation, of such noncentrosomal microtubules has a relatively late onset, starting only once chromosomes are condensed, and takes place on the inner side of the remnants of the nuclear envelope. In a fraction of cells, these microtubules are sorted into bipolar spindle-shaped structures, highly reminiscent of the anastral spindles found in oocytes. Chromosome segregation—a critical stage of cell division—and cell division itself tend to be aberrant in these cells.Drosophila spermatocytes. It is unlikely, the researchers also conclude, that the anastral spindles they have observed can fill in as a backup to ensure successful cell division. More likely, they argue, both centrosomal and noncentrosomal microtubules are required for proper spindle assembly and robust cell division in cells with centrosomes. As the authors point out, Drosophila is a rich model system that should help scientists further investigate the intricacies of spindle assembly. The answers will help us understand how the cell executes one of its most important duties: safeguarding genomic stability for future generations.These results, Rebollo et al. propose, strongly suggest that microtubules of noncentrosomal origin may significantly contribute to spindle assembly even in cells that contain active centrosomes. Moreover, by facilitating the nucleation of such noncentrosomal microtubules, the degraded nuclear envelope may play a previously unsuspected role in spindle assembly in


Acheta domesticus, with either attractive or unattractive males and measured a variety of direct and indirect fitness components. These fitness components were combined to give relative estimates of the number of grandchildren produced and the intrinsic rate of increase (relative net fitness). We found that females mated to attractive males incur a substantial survival cost. However, these costs are cancelled out and may be outweighed by the benefits of having offspring with elevated fitness. This benefit is due predominantly, but not exclusively, to the effect of an increase in sons' attractiveness. Our results suggest that the direct costs that females experience when mating with attractive males can be outweighed by indirect benefits. They also reveal the value of estimating the net fitness consequences of a mating strategy by including measures of offspring quality in estimates of fitness.The fitness consequences of mate choice are a source of ongoing debate in evolutionary biology. Recent theory predicts that indirect benefits of female choice due to offspring inheriting superior genes are likely to be negated when there are direct costs associated with choice, including any costs of mating with attractive males. To estimate the fitness consequences of mating with males of varying attractiveness, we housed female house crickets,  Experiments reveal that female crickets that choose an attractive mate have lower survival, but that this can be cancelled out or even outweighed by the increased fitness of her offspring Whether mate choice can be maintained by indirect selection when females incur direct costs by being choosy is the subject of ongoing theoretical controversy ,2,3,4,5.In order to understand how mate choice evolves, it is necessary to estimate the overall effect of mate choice on female fitness ,10,11,12Evidence from studies that have measured one or a few fitness components has been invoked to support direct benefits ,14, “viaHow fitness should be estimated is controversial ,28. MeasAcheta domesticus, mated to either attractive or unattractive males for the term of their adult life span. We present a female's total fitness as both a rate-sensitive (the intrinsic rate of increase) and a rate-insensitive estimate of fitness  in interpreting our findings.Here, we measured both direct and indirect fitness components of female house crickets, estr) than females mated with unattractive males . Of the constituent measures of week 1 reproductive effort, only egg width differed significantly between treatments .When we combined a female's egg number, egg width, and egg length (from the first week of egg laying) into a single index of reproductive effort, we found that females mated to attractive males exerted greater reproductive effort in the first week of the experiment than those mated to unattractive males  and may be outweighed by (when we use the rate-sensitive estimate of the intrinsic rate of increase) the benefits of having offspring with elevated fitness .Contrary to some theoretical predictions ,3,5, butDrosophila melanogaster mated to large (and thus presumably attractive) males incur a greater survival cost than females mated to smaller males [The costs of choice, including the costs of mating with attractive males, are of central importance to theoretic models of mate-choice evolution ,3,4,5,29er males ,22, and A. domesticus, males pass substances in their ejaculate that promote higher rates of short-term oviposition. Alternatively, it may be adaptive for females to invest more in the offspring of attractive males [While we do not know the exact mechanisms driving the survival cost seen in our experiment, our finding that females mated to more attractive males experience lower survival is consistent with sexual conflict between males and females over mating decisions ,35, and ve males ,40. Diffve males . The indSeveral studies have reported fitness benefits of mating with attractive males. Females mated to such males have been reported to have offspring that have greater longevity ,41, fastA number of studies have proposed the use of an aggregate measure of male attractiveness rather than a single morphological indicator ,45. Our A. domesticus, fighting ability has been shown to be positively correlated with attractiveness as we have measured it here [Due to the nature of our experimental design we were unable to measure all the costs and benefits associated with choosing and mating with attractive males. First, we did not measure sons' ability to compete with other males for access to females. However, in this population of  it here ,49. ThusThe fitness estimate of choice in empirical studies may depend on the importance of reproductive timing in the system in question . BrommerThere are several reasons why reproducing early and having short maturation times is likely to be advantageous in crickets. First, extrinsic mortality of crickets in the wild is likely to be high. Second, females become less choosy , lose coOur research constitutes one of the first attempts to directly and simultaneously test the combined direct and indirect effects of mating with males that differ in attractiveness. Only by following the effects of mating with attractive or unattractive males through at least two generations, and through both sons and daughters, is it possible to observe the combined direct effects on female lifetime fecundity and the genetic effects on offspring fitness ,12,25. AA. domesticus nymphs from a commercial cricket breeder . Nymphs were separated into single-sex culture tubs (4 × 80 l containers per sex) to ensure their virginity, and reared with constant access to food (Friskies Go-Cat senior) and water until eclosion. At eclosion, adults were maintained in single-sex cultures for a further 10 d to ensure sexual maturity.We obtained approximately 1,000 final-instar −3 and fed grain ad libitum. In these conditions males and females mate multiply. Males fight with other males and court females, and there is a positive relationship between male dominance and attractiveness [A. domesticus is generally sequential. That is, females choose males by either mating or rejecting males one at a time, rather than choosing between males simultaneously. We chose to work on cultured A. domesticus because our laboratory conditions closely resemble the culture conditions under which they have recently evolved. This similarity maximises the evolutionary relevance of our measures. Our experimental design, however, requires that females be kept alone, creating an important environmental difference from the culture conditions to which females have been adapted.In the cultures from which the insects have been derived, crickets are raised in densities ranging from 23,000–34,000 mtiveness . DespiteA. domesticus a female mounting a male is a reliable predictor of mating success . Also, To obtain males that were either attractive or unattractive to females we ran a two-round tournament. In round one, each male was placed in a clear plastic container (7 × 7 × 5 cm) with a single randomly assigned female, at night, under red lighting. When a female mounted a male, but before spermatophore transfer, they were separated. Once half of the females had mounted, all remaining pairs were separated. Round two commenced with a new female assigned at random to each male. The first half of first-round mounted males to be remounted became our “attractive” treatment males. The half of first-round unmounted males that remained unmounted longest in round two became the “unattractive” treatment males. Only males that courted females during the tournament were used. This biological assay of male attractiveness incorporates all traits that make a male attractive during short-range courtship, rather than a single trait correlated with attractiveness see ,11,44).,44.11,44Forty females were randomly assigned to each of three treatments: attractive, unattractive, and an unmated control. Females were weighed and placed individually in a small plastic container (as above) with food, water, and a petri dish of moist sand for egg laying. Males from the appropriate treatment were randomly assigned to a female. Every 7 d, or whenever a male died, a new male from the same treatment (but from a new tournament) was placed with the female. This allowed us to measure the fitness consequences of the strategy of mating with attractive or unattractive males, rather than the consequences of mating with a given individual male. Food, water, and sand were replaced every 7 d.Female survival was monitored daily, and the number of eggs laid was counted weekly. Hatching success was estimated as the proportion of eggs that hatched within 14 d of the first egg hatching in each collection. Hatchlings were collected every 3 d, and their mean weight was recorded. Each week, 50 hatchlings per female were separated into two boxes (20 × 13 × 13 cm), each containing 25 nymphs. We monitored offspring survival every 7 d and recorded the time to mature and sex and body weight at eclosion.If a female had fewer than 50 hatchlings in a given week these were discarded. For these females, the actual number of hatchlings was multiplied by the overall experimental mean for each subsequent offspring fitness measure, to predict the number of grandchildren produced. This is a conservative approach to missing values because it reduces the difference between the treatments.Offspring generation times were calculated from the time females were first placed with a male until the offspring matured. This takes into account not only the time it takes for the offspring to mature, but also the timing of egg laying. Mature offspring were housed individually, and their survival monitored daily. Ten days after eclosion each son's attractiveness was estimated by placing him with a stock virgin female in a small plastic container for 90 min. Mounted males were separated from females before spermatophore transfer occurred. We used the proportion of a female's sons that were successful in this assay as our measure of sons' average attractiveness .Ten days after eclosion daughters were placed with a stock male for 12 h to allow mating. Afterwards, survival of sons and daughters was again monitored daily, and sand was collected from daughters weekly. Eggs from daughters were counted to estimate lifetime fecundity.estg), and a rate-sensitive estimate, estr.We calculated two estimates of female relative net fitness when mating with either an attractive or unattractive male. A rate-insensitive estimate, the relative number of grandchildren produced by a female (estG), we added the number of grandchildren she had through daughters, estimated asTo estimate the absolute number of grandchildren each female had , by dividing each female's estR by the experiment-wide mean.where P-values are based on the proportion of randomisations in which the absolute value of the estimated difference was greater than that observed in the original data.Due to the non-normal distributions of many fitness components, we tested the significance of treatment differences for each fitness component using two-tailed randomisation tests. In each randomisation test the observed data was randomly assigned to the two treatments 10,000 times. estr to variation in each fitness component we used a model-building approach. We removed the variance of each fitness component from our full model, in turn, by assigning every female the overall experimental mean value of that component. We similarly excluded every combination of two fitness components. We then ran a randomization test  for each reduced model to test whether the treatment effect remained. We also obtained 10,000 jackknifed estimates of the difference between the treatments for each reduced model (by randomly omitting 20% of the sample in each estimate), to test whether the reduced model resulted in a significantly different effect size than the original full model. P-values are based on the proportion of jackknifed estimates in which the absolute value of the difference between the treatments was greater than the absolute difference in the full model.To explore the sensitivity of our estimates of We used principal components analysis to investigate the effects of mating with attractive or unattractive males on week 1 reproductive effort via egg number, egg width, and egg length. All three measures showed a strong positive loading on the first principal component, which explained 66% of the variation in the constituent measures. We then tested for differences in female reproductive effort between the treatments using a randomisation test.


Considerable variation exists in the protocols used to induce hyperresponsiveness in murine models of allergic sensitisation. We examined the effect of varying the number of antigen exposures at challenge on the development of methacholine responsiveness in systemically sensitised mice.aw) and lung parenchyma (tissue damping (G) and tissue elastance (H)). Responsiveness to inhaled methacholine (MCh), inflammatory cell profile and circulating IgE were assessed 24 and 48 hours after challenge. The threshold dose of MCh required to elicit a detectable response (sensitivity) and response to 30 mg.mL-1  were determined for each compartment.BALB/c mice were sensitised with ovalbumin (OVA), challenged with 1, 3 or 6 OVA aerosols. Lung function was measured using low frequency forced oscillations and partitioned into components representing the airways (RSensitivity; All three OVA protocols resulted in an increased sensitivity to MCh in Raw but not in G or H. These responses where present at 24 and 48 hrs, except 1 OVA aerosol in which changes had resolved by 48 hrs. Maximal response; 1 OVA aerosol increased maximal responses in Raw, G and H at 24 hrs, which was gone by 48 hrs. Three OVA aerosols increased responses in H at 48 hrs only. Six OVA challenges caused increases in Raw, G and H at both 24 and 48 hrs. Eosinophils increased with increasing antigen challenges. IgE was elevated by OVA sensitisation but not boosted by OVA aerosol challenge.The pattern of eosinophilia, IgE and MCh responsiveness in mice was determined by antigen dose at challenge. In this study, increased sensitivity to MCh was confined to the airways whereas increases in maximal responses occurred in both the airway and parenchymal compartments. The presence of eosinophilia and IgE did not always coincide with increased responsiveness to inhaled MCh. These findings require further systematic study to determine whether different mechanisms underlie airway and parenchymal hyperresponsiveness post antigen challenge. Persistent asthma is an allergic disease characterised by airway inflammation -5) and h and h5])et al.  adsorbed onto aluminium hydroxide (Alum), followed by airway challenge with the same antigen -22). How. How22])et al.  for 30 minutes on consecutive days starting at day 21  injection with 20 μg of OVA  suspended in 200 μL of Alum  on days 0 and 14. Mice were then challenged with either 1, 3 or 6 OVA (1% w/v in PBS) aerosols delivered with an ultrasonic nebuliser  and ketamine  at a dose of 0.01 mL.g-1. Mice were tracheostomised with a 10 mm section of polyethylene tubing (1.27 mm OD: 0.86 mm ID) and ventilated  at 450 b.min-1 with a tidal volume of 8 mL.kg-1 and a positive end expiratory pressure (PEEP) of 2 cmH2O. The lung volume history of the mice was standardised prior to measurement of lung mechanics using two deep inflations and three P-V curves. The respiratory system input impedance (Zrs) was measured during periods of apnea using a 16 s signal containing 19 mutually prime sinusoidal frequencies ranging from 0.25 to 19.625 Hz. The constant phase model ([aw), tissue damping (G), tissue elastance (H) and hysteresivity (η) ([Changes in Zrs were measured using a modification of the low frequency forced-oscillation technique (LFOT) as described previously . Brieflity (η) .®, Devilbiss, Somerset, Pennsylvania). Zrs was then measured every minute for the next 5 minutes. This aerosol procedure was repeated with 1/2 log10 incremental doses of MCh from 0.1 to 30 mg.mL-1 with Zrs measured every minute for at least 5 minutes after the aerosol until the parameters calculated from the constant phase model had peaked.Changes in respiratory mechanics following inhaled MCh were measured either 24 or 48 hrs after the last OVA aerosol. Following measurement of baseline Zrs, mice were exposed to a 90 s saline aerosol delivered with an ultrasonic nebuliser  and lidocaine  from the lungs three times. The BAL was then centrifuged at 2000 rpm for 4 mins. The supernatant was removed and the pellet resuspended in PBS. The cells were stained with trypan blue to determine viability and the total cell count (TCC) obtained by counting the cells with a haemocytometer. Differential counts were obtained from the cytospin sample, stained with Leishman's stain and examined using light microscopy. Three hundred cells were counted from each sample to determine the relative proportions of each cell type.Separate groups of mice, sensitised using the same protocol described above, were anaesthetised and tracheostomised 24 or 48 hrs after their last aerosol. BAL fluid was collected by slowly infusing and withdrawing a 1 mL aliquot of PBS containing BSA  was incubated at 1:500 for 30 minutes and plates washed eight times thereafter. Delfia enhancement solution  was added and the plates were agitated on a shaker for 10 minutes prior to reading the fluorescence on a Wallac Victor 2 counter . The detection limit of this assay is approximately 100 ng.mL-1.In a separate group of mice, serum samples were periodically collected for analysis of total IgE. An additional control group was included in the analysis of serum IgE consisting of mice sensitised with PBS/Alum. Sera were diluted 1:7.5 in Delfia Assay buffer . The diluted sera were analysed for the presence of total IgE by time-resolved fluorescence (TRF) assays. Briefly, 96-well plates  were coated overnight at 4° C with anti-mouse IgE . Plates were blocked with 200 μl of 0.5% BSA in TRIS-HCl pH 7.4 for 1 hour at room temperature on a plate shaker. For all subsequent steps a volume of 50 μl per well was used and incubations were performed for 1 hour at room temperature unless otherwise indicated. Between steps, plates were washed five times with wash buffer (TRIS-HCl pH 7.8 Tween20). Mouse anti-TNP IgE  was used as an interassay standard. Biotinylated anti-mouse IgE  was added to the wells at 2 μg.mL10 transformed inflammatory cell and immunoglobulin data were compared using ANOVA and Tukey's post-hoc test. Responses in Raw and G to inhaled MCh at the maximum dose used (30 mg.mL-1) were expressed as a percentage of the response to the saline aerosol and compared using non-parametric ANOVA on ranks and Dunn's post-hoc test. Responses in H were expressed as a percentage of the response to saline, log10 transformed and compared using ANOVA and Tukey's post-hoc test. The threshold dose of MCh where there was a detectable change in Raw, G or H (termed sensitivity hereafter) was interpolated from the raw dose response curve as the upper limit of the 99% CI of the 5 measurements taken following the saline aerosol  between treatment groups and naïve mice is presented in Table The degree and time of observed maximum MCh induced responses in Rnts Fig. . A summats Fig. - between A single OVA aerosol was sufficient to induce a significant increase in MCh responsiveness in the airways, seen as both a lower threshold dose of MCh required to induce a response (increased sensitivity) and increased response at the 24 hour time point Table . In the aw, G or H and only an increased response in H after 48 hours but not Raw and G.Three OVA aerosols resulted in significantly increased airway  sensitivity to MCh at both the 24 and 48 hour time points Table . HoweverSix OVA aerosols resulted in both significantly increased airway sensitivity and maximal responses to MCh at 24 and 48 hours post-challenge. Increased maximal responses, but not increased sensitivity, were also seen in the parenchymal compartment at both the 24 and 48 hour time points.Challenge with a single PBS aerosol following systemic sensitisation with OVA did not cause a significant increase in TCC in the BAL (p = 0.552) compared to naïve mice Fig. . There wdata not shown).The number of aeroallergen challenges also had a significant impact on the number of eosinophils (p < 0.001) and macrophages (p < 0.001) in the BAL. There were significant increases in the number of eosinophils in sensitised mice challenged with 1 (p = 0.032), 3 (p < 0.001) and 6 (p < 0.001) OVA aerosols Fig.  compareddata not shown).Total serum IgE was significantly increased at day 21 (p < 0.001), 7 days after the second injection of OVA/Alum, compared to naïve mice Fig. . In cont-1 MCh showed a variable pattern. A transient response was observed in both airway and parenchymal compartments after a single OVA aerosol. After 3 OVA aerosols significant increases were seen in the tissue compartment at 48 hours, while after 6 OVA aerosols an elevated response was seen in the airway and parenchymal compartments that persisted beyond 48 hours. There was a significant influx of inflammatory cells in the BAL in response to OVA aerosols, however, the presence of this inflammation did not always result in hyperessponsiveness to inhaled MCh.Varying the number of aeroallergen challenges in a systemically sensitised murine model of allergic bronchopulmonary inflammation altered the degree and timing of hyperresponsiveness to inhaled MCh. A single OVA challenge increased airway sensitivity to inhaled MCh 24 hours after the challenge, while sensitivity remained elevated for 48 hours after three and six challenges. OVA challenge did not increase parenchymal sensitivity at any level. In contrast to sensitivity measurements, the maximum response to 30 mg.mLet al.  a-31 a29-3et al. , which et al. . This w1. We have shown that it is possible to determine sensitivity to inhaled MCh in mice and that only the airway compartment shows heightened sensitivity following allergic sensitisation and challenge. While increased maximal responses can be seen in both airway and parenchymal compartments, depending on which model is used, no increase in parenchymal sensitivity is seen with any of the models we used. As such, these findings reinforce the value of using lung function techniques that are capable of assessing airway and parenchymal mechanics separately.Typically, most human studies measure MCh responsiveness in terms of sensitivity as they report the concentration of MCh required to produce a 20% fall in FEVTotal serum IgE was significantly elevated following systemic sensitisation but was not increased by aerosol challenge. There was, however, a tendency for total serum IgE to decline by day 27 in mice that were systemically sensitised but not challenged with OVA aerosols, compared to mice additionally exposed to 6 OVA aerosols. It is possible that if the study had been extended to include further exposure to antigen over subsequent days, a difference would have been detected between mice that were only sensitised and mice that were sensitised and challenged. Given that antigen specific IgE and other immunoglobulin subtypes were not measured in this study, further work is required to characterise the effect of dose of antigen at challenge on the development of antibody responses to OVA in mice.The protocol used in the present study induced significant eosinophilia after a single airway challenge. The degree of eosinophilia increased with increasing number of airway challenges. This finding is consistent with several previous studies using similar protocols to induce allergic inflammation in the lungs of mice ,29-31). -31. 29-3et al. ([et al. ([In recent studies there has been some focus on the association, or lack thereof, between indicators of systemic sensitisation, such as the levels of serum antibodies, airway inflammation and AHR . In a ret al.  conclud[et al. , show tThe findings of the present study demonstrate the significant impact of changing antigen challenge dose in a murine model of allergic bronchopulmonary inflammation. Given the variability of the inflammatory profile and characteristic responses observed in this study, it is clear that investigators must carefully characterise their allergen-driven murine models to ensure the model used contains the characteristic of interest. Future studies need to be directed at understanding the mechanisms that underlie airway and parenchymal hyperresponsiveness post antigen challenge.GRZ carried out the animal studies and drafted the manuscript. CvG carried out the IgE analysis and assisted in the interpretation of results and editing the manuscript. PAS assisted in the interpretation of results and editing the manuscript. PGH assisted in the conceptualisation of the study and interpretation of the results. PDS and DJT assisted in the conceptualisation of the study, interpretation of the results and editing the manuscript.


Two scientist members of the President's Council on Bioethics express their concerns about two recently issued reports by the Council in which the science is presented incompletely and myths are perpetuated We are two of the scientist members of the President's Council on Bioethics. In late 2001, we were invited by the President of the United States to serve on this Council. The Bioethics Council was appointed by the President to “monitor stem-cell research, to recommend appropriate guidelines and regulations, and to consider all of the medical and ethical ramifications of biomedical innovation…. This council will keep us apprised of new developments and give our nation a forum to continue to discuss and evaluate these important issues.”This was a difficult invitation to accept. On the one hand, the President's views on the use of human embryonic stem cell research and somatic cell nuclear transfer techniques were well-known and in conflict with our own beliefs about the costs and benefits of the use of progressive technologies to advance biomedical research. On the other hand, we were grateful that the President, despite his views in opposition to these therapies, was willing to invite serious biomedical scientists to help formulate advice to him—and ultimately to contribute to the development of national policy—on these critically important advances.We knew that on this originally 18-member (but for most of the past two years a 17-member) Council, as scientists we would be in the minority in our belief of the good to be gained through these and other areas of biomedical research. We were also aware that some others on the Council had strong opposing views. Thus, it was only with the assurances of the Council chairman, Leon Kass of the University of Chicago, and of the President of the United States himself that we were persuaded that our voices would be heard and integrated into the statements of the Council. Furthermore, we felt, and continue to feel, that bioethical issues are important not only to all biologists, but also to society at large, and thus especially worthy of engaging debate and discussion.http://bioethics.gov/reports/beyondtherapy/index.html) and “Monitoring Stem Cell Research” (http://bioethics.gov/reports/stemcell/index.html), are therefore of deep concern to us. We discuss them in turn below.Two recently issued reports of the Council, “Beyond Therapy: Biotechnology and the Pursuit of Happiness”  and “biological aging” (expected lifespan), only one view, a conservative one, of the supposed “best” way to raise children is presented. The report also suggests, with no clear reasoning behind it, that longer lives will somehow undermine human determination to contribute as much as one can during a lifetime. Despite requests for inclusion of material that would allow for a balanced treatment of these topics, the report minimized discussion of potential positive aspects of slowing biological aging, such as prolonged good health.lack of any necessary mechanistic linkage of the two.Finally, the report repeatedly emphasizes a “profound and mysterious” link between longevity and fertility, thereby leaving the reader with the distinct but erroneous impression that anything done to extend healthy life will be traded for decreased fertility, despite the fact that current scientific literature, which was made available for inclusion in the report, shows a With respect to the “Monitoring Stem Cell Research” report, we feel that some facts that would help the public and scientists better assess the content of the report were not brought out clearly or were omitted entirely. First, from the published scientific literature in peer-reviewed journals on stem cells, a major message can be distilled: namely, the vast difference that currently exists in our understanding of, and the potential utility of, embryonic versus adult stem cells as sources of material for research and clinical purposes. In brief, human stem cells have been isolated from a variety of embryonic, fetal, and adult tissue sources. However, enormous differences exist in purity, properties, data reproducibility, and understanding of cells from these different sources. Much of our ignorance is related to the relative paucity of funding for research using embryonic stem cells.Years of rigorous and careful research in animal models have documented that embryonic stem cells have great utility for scientific studies. This work has also rigorously and reproducibly established the great plasticity of these cells and supports the opinion that human embryonic stem cells possess the greatest broadest potential and promise for clinical applications. As well as therapeutic uses, important potential applications include studies of embryonic stem cells bearing complex genotypes susceptible to poorly understood common human diseases and testing and screening drug efficacy.The report does not make clear that the best-characterized adult stem cells are hematopoietic stem cells. Currently, major difficulties and inadequate understanding exist with most other types of adult stem cells reported to date. In addition, many experiments suggesting that adult stem cells have broad plasticity may be incorrectly interpreted owing to an error caused by an experimental artifact of cell fusion present in some unknown proportion of the experiments. Research on some of the reported adult stem cell preparations may conceivably in the future demonstrate that they, too, like hematopoietic stem cells, can also be prospectively identified, “single cell cloned,” expanded considerably by growth in vitro with retention of normal chromosome structure and number, and preserved by freezing and storage at low temperatures. But it should be strongly cautioned that this has not been done for most adult stem cell preparations, and, even if possible, it is not clear that any of the just-mentioned procedures will be accomplished in the near future, owing to the technically very demanding nature of such experiments.We feel it is important to emphasize a point that the report mentioned, that the reported isolation and properties of multipotent adult progenitor cells (MAPCs) must be reproduced in additional laboratories for any reliable interpretation of the results reported with these cells. After considerable effort, this has still not been achieved. Thus, in the reported results, the possible significance of the reported isolation and properties of human MAPCs is left unclear, as is their potential as a source of stem cells for clinical purposes. Hence, a strong overall caution is that many of the reports on the properties of cells differentiated from adult stem cell preparations are to date preliminary and incomplete. If results with any isolated and characterized adult stem cells are validated, it will then be very important to compare their properties—and those of any more differentiated cells that can be derived from them—with other stem cell sources, such as the well-characterized hematopoietic stem cells, and with human embryonic stem cell preparations.Two major considerations argue strongly for non-commercial, federal, peer-reviewed funding to be made available for this work. The first is the sustained effort this work will require. The second is the importance of reliable and unbiased design of experiments and of open, public availability of the complete findings.The New Republic. In it, an article about this report of the Council falls right into the trap: it states, “But changes come with longer life. Worms and mice that are altered for extended lifespans become sterile, or barely reproduce.” The public is done a disservice when science is presented incompletely; myths are then perpetuated.In being concerned about the content of these reports, neither of which makes any recommendations for legislative or policy actions, are we worrying too much? We think not. Indeed, already, sadly as a result of the way the sections on aging research in the report were written, the myth that longevity has an inevitable tradeoff of diminished fertility is now gaining a further foothold: witness the January 26, 2004, issue of the critical reading!” .This is but one example of the dangers that three of the Council members who are scientists  pointed out, in a Commentary within the edition of the “Beyond Therapy” report published by the Dana Foundation in November 2003. In that Commentary, we stated that “Our concern … is that, moving forward, the debate carry on with all of the scientific evidence—or as much as such a widespread public discussion can include—and take care not to leave an erroneous impression as to the nature of the potential problems at hand.” We ended the Commentary by saying “We urge both good reading and These reports had as their premise the aim of neutrality in the scientific analysis of the issues addressed. But our concern is that some of their contents, as in the few examples outlined above, may have ended up distorting the potential of biomedical research and the motivation of some of its researchers. Continuing discussions will form the basis for future decisions on these topics; keeping such discussion open and balanced is of paramount importance.The President's Council on Bioethics was created on November 28, 2001. Its mission includes: to “advise the President on bioethical issues that may emerge as a consequence of advances in biomedical science and technology. In connection with its advisory role, the mission of the Council includes the following functions:to undertake fundamental inquiry into the human and moral significance of developments in biomedical and behavioral science and technology;to explore specific ethical and policy questions related to these developments;to provide a forum for a national discussion of bioethical issues;to facilitate a greater understanding of bioethical issues; andto explore possibilities for useful international collaboration on bioethical issues.”From Executive Order 13237George W. BushThe White House, November 28, 2001Federal Register date: November 30, 2001Federal Register page: 66 FR 59851The members of the President's Council on Bioethics at the time these reports were written included Leon R. Kass, M.D., Ph.D. (Chair), American Enterprise Institute; Elizabeth H. Blackburn, Ph.D.*, University of California, San Francisco; Rebecca S. Dresser, J.D., M.S., Washington University School of Law; Daniel W. Foster, M.D., University of Texas, Southwestern Medical School; Francis Fukuyama, Ph.D., Johns Hopkins University; Michael S. Gazzaniga, Ph.D., Dartmouth College; Robert P. George, J.D., D.Phil., Princeton University; Mary Ann Glendon, J.D., M.Comp.L., Harvard University; Alfonso Gómez-Lobo, Dr. Phil., Georgetown University; William B. Hurlbut, M.D., Stanford University; Charles Krauthammer, M.D., syndicated columnist; William F. May, Ph.D.*, Southern Methodist University; Paul McHugh, M.D., Johns Hopkins Hospital; Gilbert C. Meilaender, Ph.D., Valparaiso University; Janet D. Rowley, M.D., University of Chicago; Michael J. Sandel, D.Phil., Harvard University; and James Q. Wilson, Ph.D., University of California, Los Angeles.* These members had their Council terms terminated by White House directive on February 27, 2004.


In order to confirm therapeutic effects of topiramate on posttraumatic stress disorder (PTSD) observed in a prior study, a new prospective, open-label study was conducted to examine acute responses in chronic, nonhallucinatory PTSD.Thirty-three consecutive newly recruited civilian adult outpatients  with DSM-IV-diagnosed chronic PTSD, excluding those with concurrent auditory or visual hallucinations, received topiramate either as monotherapy (n = 5) or augmentation (n = 28). The primary measure was a change in the PTSD Checklist-Civilian Version (PCL-C) score from baseline to 4 weeks, with response defined as a ≥ 30% reduction of PTSD symptoms.t-test, P < 0.001) with similar subscale reductions for reexperiencing, avoidance/numbing, and hyperarousal symptoms. The response rate at week 4 was 77%. Age, sex, bipolar comorbidity, age at onset of PTSD, duration of symptoms, severity of baseline PCL-C score, and monotherapy versus add-on medication administration did not predict reduction in PTSD symptoms. Median time to full response was 9 days and median dosage was 50 mg/day.For those taking the PCL-C at both baseline and week 4 (n = 30), total symptoms declined by 49% at week 4 (paired Promising open-label findings in a new sample converge with findings of a previous study. The use of topiramate for treatment of chronic PTSD, at least in civilians, warrants controlled clinical trials. Posttraumatic stress disorder (PTSD) is a difficult-to-treat condition that over a lifetime affects approximately 10% of the general population . PTSD is+ channel blockade [2+ channels [A receptors [Topiramate has a broad spectrum of pharmacologic properties, including Nablockade -11, inhichannels , enhanceeceptors ,14, gluteceptors ,16, and eceptors . These peceptors ,18, suggTreatment with topiramate has been reported to improve reexperiencing symptoms associated with civilian PTSD . ResultsAlthough the DSM-IV definition of PTSD lists hallucinations among reexperiencing symptoms , becauseBased on these earlier results of topiramate in PTSD, a fully prospective open-label study was begun to see whether the previously observed signal of a therapeutic benefit of topiramate for chronic civilian PTSD could be confirmed. None of the patients reported in the earlier study were included in the present study. In order to address methodological limitations of the prior study, the current study is modified in 4 major ways: (1) it excludes patients with hallucinations, (2) it limits the study to 12 weeks, (3) it prospectively employs the PTSD Checklist-Civilian Version (PCL-C) as a validated clinical assessment instrument in all patients, and (4) it determines a clinical response rate as of week 4, using a conventional, predetermined definition for positive response as a ≥ 30% improvement in a standardized clinical rating scale.This sample included all consecutive adults (n = 33) meeting the DSM-IV criteria for chronic civilian PTSD in an outpatient private practice who started open-label topiramate between January 2000 and November 2002 in the course of clinical care. The study excluded patients with concurrent auditory or visual hallucinations associated with either PTSD or a possible psychotic disorder. Topiramate, administered either as monotherapy (n = 5) or added to the patients' existing therapeutic regimen (n = 28) Table , was iniThe study was performed according to the Declaration of Helsinki. Each participant provided verbal informed consent based on disclosure of off-label usage, the availability of alternative medications for PTSD such as sertraline and paroxetine, and expected adverse events and risks. Verbal consent was deemed appropriate and sufficient because of the nonexperimental nature of the treatment. Because this was a descriptive study using aggregate data gathered in the course of treating patients in clinical practice for their own benefit and not an experimental design, there was no protocol that required Institutional Review Board approval. Patients were asked to measure their symptoms using standardized clinical instruments already routinely used as a part of individual care. The study included no features of clinical studies, such as randomization, concealment of treatment, or surrender of patient decision-making rights, as an integral part of conducting the study.k~0.90) with the Clinician-Administered PTSD Scale (CAPS), a structured clinical interview widely used in PTSD studies [The primary outcome was a change in PTSD symptoms calculated from change in PCL-C score from baseline through 4 weeks of treatment . The PCL studies . The PCLData on patient demographics, concomitant medication, comorbidities, primary trauma, and duration of PTSD were collected at baseline. Assessments were made through clinical interviews over a 12-week period. Cessation of reexperiencing symptoms was considered partial if subjects reported a definite reduction in intensity and frequency of nightmares or intrusive recollections or flashbacks. Full cessation was defined as complete elimination of nightmares and intrusions for a sustained period. Adverse effects were recorded if they resulted in treatment discontinuation.® Version 2.0 . A single paired t-test of before and after total PCL-C scores was used to analyze the data. Subscale scores for PCL-C Clusters B, C, and D were also evaluated.Statistical analyses were performed using Jandel SigmaStatBaseline patient characteristics and concomitantly administered medications are listed in Tables t-test, P < 0.001). In addition, subscale symptoms for reexperiencing (Cluster B), avoidance/numbing (Cluster C), and hyperarousal (Cluster D) decreased by 53%, 43%, and 48%, respectively, from baseline  with baseline PCL-C measurements completed a PCL-C at week 4. In those patients, the total score was 62.6 at baseline and 40.3 at week 4. In addition, the scores for reexperiencing, avoidance, and hyperarousal clusters were 18.2, 25.1, and 19.2 at baseline and 11.2, 17.2, and 12.3 at week 4, respectively. To calculate percentage reduction in PTSD symptoms, to correct for an absence of symptoms being scored as "1" for each item, the change in score between baseline and week 4 was divided by the baseline score minus the minimum score for no symptoms. Therefore, the change in total symptoms was calculated as (baseline – week 4)/(baseline – 17). Total symptoms declined significantly (49%) by week 4 , duration of symptoms (r = 0.37), age at onset of PTSD (r = -0.36), severity of baseline PCL-C score (r = 0.02), age (r = -0.02), female sex , and monotherapy versus add-on medication administration  found none significantly associated with reduction in PCL-C symptoms.Of the 33 patients who entered the study, 79% fully ceased having reexperiencing symptoms with a mean time to full cessation of 15 days. The onset of full cessation for all patients occurred at ≤ 200 mg/day and the onset of partial cessation for all patients at ≤ 50 mg/day. Ninety-four percent of patients with nightmares and 79% of patients with intrusions at baseline reported full cessation at the end of week 4.There were no serious adverse events during this study. Of the 33 patients who entered the study, 7 (21%) discontinued due to adverse events and 5 (15%) for other reasons. The single most common adverse event resulting in discontinuation was intolerable nervous system overstimulation (panic/nervousness/overstimulation/ shakiness), reported by 3 patients. Other adverse events resulting in discontinuation included clumsiness (n = 2), cognitive impairment (n = 1), and severe headache (n = 1). Additional reasons for discontinuation included personal choice (n = 1) and lack of relapse of PTSD symptoms after interrupting medication (n = 4). Of the 12 patients that discontinued, 54% (n = 7) fully ceased having reexperiencing symptoms of nightmares/intrusions and 15% (n = 2) partially ceased having reexperiencing symptoms of nightmares/intrusions. The median time to termination was 25.5 days .P = 0.05). No monotherapy patients discontinued because of overstimulation/panic-like responses, so the possibility of drug interactions or conditions such as comorbid panic disorder contributing to this reaction should be considered in future investigations.There were no statistically significant predictors of panic-like adverse events resulting in discontinuation from the study , although the coadministration of a benzodiazepine nearly reached significance , median time to full response (9 d versus 8 d), suppression of intrusions and nightmares (88% versus 89%), and discontinuation rate (39% versus 32%) were all similar. Overall, the open-label experience with topiramate, based on patient statements and self-rating reports, suggests that the drug may have a rapid rate of response with limited risk of clinically significant adverse events and no evidence of tolerance developing over time ,31. MoreLimitations of the present study include: (1) the absence of structured assessment data following week 4 to test the maintenance of response over time, (2) use of self-report instruments that, although validated and correlated with results of structured clinical interviews, may be less accurate than structured interviews, and (3) the absence of clinical assessment scores at the time patients discontinued the trial. From a broader perspective, the greatest limitation of this study is characteristic of all open trials: the lack of standard features of clinical trials such as placebo controls, randomization, and blinding of raters.Fundamentally, the findings of current and prior topiramate studies are convergent and consistently signal a potentially beneficial therapeutic effect for all 3 criteria clusters of chronic PTSD in civilian adults. In both studies, there is a positive response in a large proportion of patients, at dosages considerably below the usual anticonvulsant dosage levels of 200 to 600 mg/day, and a rapid onset. The effect appears independent of comorbidity with bipolar disorder, age, sex, duration of symptoms, baseline severity of illness, or administration alone or with other psychotropic medications. The purpose of this study was to prospectively test the results of an earlier open-label trial in a new sample; however, the limitations of those open studies will have to be addressed in double-blind, placebo-controlled studies, which are currently in progress.The author is a consultant to and a participant in the Speaker's Bureau for Ortho-McNeil Pharmaceutical, Inc. He also holds a licensing agreement with Ortho-McNeil based on issuance of a United States Government patent for topiramate therapy of PTSD.JLB conceived of the study and its design, coordination, statistical analysis, and drafted the manuscript.The pre-publication history for this paper can be accessed here: