The algorithm trains a regression model on sequence and contextual features extracted from miRanda-predicted target sites. In a large-scale evaluation, miRanda-mirSVR is competitive with other target prediction methods in identifying target genes and predicting the extent of their downregulation at the mRNA or protein levels. Importantly, the method identifies a significant number of experimentally determined non-canonical and non-conserved sites. These small (approximately 22 nucleotide) single-strand RNAs guide a gene silencing complex to an mRNA by complementary base pairing, mostly at the 3' untranslated region (3' UTR). The association of the RNA-induced silencing complex (RISC) to the conjugate mRNA results in silencing the gene either by translational repression or by degradation of the mRNA . ReliablEarly studies of target recognition revealed that near-perfect complementarity at the 5' end of the microRNA, the so-called "seed region" at positions 2 to 7, is a primary determinant of target specificity [in vivo microRNA targets found a significant number of non-canonical sites [Conversely, a number of studies have shown that some target sites with a mismatch or a G:U wobble in the seed region confer a noticeable regulatory effect -5, and aal sites ,7. ThereMost computational methods require sites to have perfect seed complementarity  -10, withet al. defined a four-class hierarchy of canonical seed types of differing efficiencies and identified additional "context" features of target sites that correlate (but only weakly) with reduced expression levels, in particular the AU content flanking the target site. Using univariate regression between feature scores and expression change, they developed a seed-class-dependent scoring system called "context score", which has been incorporated into the TargetScan prediction program. Nielsen et al. assessed the significance of similar features by the shift in the cumulative distribution of log expression ratios using the same four-class seed hierarchy. Recently, proteomics studies of protein expression changes in response to microRNA transfection and knockdown [Several studies have used genome-wide mRNA expression changes following microRNA transfection to elucidate microRNA target specificity rules ,9,16. Grnockdown ,18 corroHere we present a new algorithm called mirSVR for scoring and ranking the efficiency of miRanda-predicted microRNA target sites by using supervised learning on mRNA expression changes following microRNA transfections. mirSVR incorporates target site information and contextual features into a single integrated model, without the need to define seed subclasses. We use support vector regression (SVR) to train on a wide range of features, including secondary structure accessibility of the site and conservation.We first compared mirSVR against a number of existing target prediction algorithms using a large panel of independent microRNA transfection and inhibition experiments as test data. For a fair comparison, we limited consideration to sites with canonical seed pairing in this analysis. mirSVR performs as well as, and often better than, existing methods for the task of predicting the extent of downregulation of genes at the mRNA or protein level. The miRanda-mirSVR approach effectively broadens target prediction beyond the standard notion of seed hierarchy and strict conservation without introducing a large number of spurious predictions. In particular, we found that the mirSVR scoring model correctly identified functional but poorly conserved target sites, and that imposing a conversation filter results in a reduced rate of detection of true targets.http://www.microRNA.org.mirSVR downregulation scores are calibrated to correlate linearly with the extent of downregulation and therefore enable accurate scoring of genes with multiple target sites by simple addition of the individual target scores. Furthermore, the scores can be interpreted as an empirical probability of downregulation, which provides a meaningful guide for selecting a score cutoff. We found that the model can correctly identify genes that are regulated by multiple endogenous microRNAs - rather than transfected microRNAs whose concentrations are above physiological levels - by analyzing targets bound to human Argonaute (AGO) proteins as identified by AGO immunoprecipitation . We alsoThe mirSVR algorithm learns to predict target site efficiency by training on mRNA expression data from a panel of microRNA transfection experiments. Training examples consist of genes containing a single candidate target site for the transfected microRNA in the 3' UTR. Target sites are represented by a set of binary features of the predicted miRNA::site duplex as well as local and global contextual features Figure , togetheet al. [For all results reported below, we trained mirSVR on a set of nine microRNA transfection experiments performed on HeLa cells from Grimson et al. . We evalet al. ,9, the met al. [et al. [We first tested the canonical-only mirSVR prediction model, where we restricted consideration to genes with single canonical target sites, that is, sites with perfect complementarity to positions 2 to 7 of the microRNA. The test data consists of 17 independent microRNA transfection experiments followed by mRNA expression profiling from Linsley et al. , five mi [et al. -23.ranking performance of the algorithm. It is important to note that for this analysis, we did not filter the potential canonical target sites for conservation: mirSVR and comparison methods were required to rank all sites with seed matches, whether or not the sites are conserved. In this sense, we are not performing a typical method comparison of existing target prediction programs as they are implemented through various web servers. Instead, we are assessing the intrinsic value of different target site scoring systems to predict the extent of microRNA regulation.We compared the performance of the mirSVR model against well-known existing target prediction methods that were representative of the different methodologies, namely: TargetScan's context score , which iP < 0.002, signed rank test). The inclusion of a conservation measure into the mirSVR model does not account for the entire performance gain. After removing the conservation feature, mirSVR still outperforms context score in 18 out of the 25 test cases, suggesting that the learning algorithm - not just the inclusion of additional features - contributes to the performance gain.Our results show that when trained on canonical seed sites and using our full feature set, mirSVR strongly outperforms the alignment-based (miRanda) and energy-based (PITA) scores for the task of ranking single-site genes by their downregulation (upregulation) in response to microRNA transfection (inhibition), as shown in Figure P < 0.006, Figure In addition to the Spearman rank correlation, we compared the performance of mirSVR and context score by an ROC analysis where the true positive and true negative sets are defined as the top and bottom 20% of candidate target genes based on their expression changes following microRNA transfection (or inhibition) Figure . Consistet al. data set when evaluated in terms of rank correlation with extent of downregulation  [CT Pthreshold   and simiet al. data set containing either a single canonical target site or a single non-canonical site with at most a single G:U wobble or mismatch in the seed region. We confirmed that the "all-sites" mirSVR model performed similarly to our "canonical-only" mirSVR model for the task of predicting downregulation of canonical target genes  log expression reduction a < 0 and mirSVR score threshold S, we compute the empirical probability that a gene's expression change y is below or equal to a given that its score f(x) is smaller than or equal to S  log expression change of at least -1 (downregulation by at least a standard deviation in terms of log expression changes) and better than 50% probability of a log expression change of at least -0.5  > 7(A1) > 6 mer) . After rSo far we have measured mirSVR performance using expression data from microRNA transfection experiments. However, overexpression of microRNAs by transfection experiments may lead to stronger or more widespread downregulation than observed under physiological conditions and also appears to perturb endogenous microRNA regulation in the cell by out-competing the endogenous microRNAs for the silencing machinery . In addiWe included in our prediction set genes with target sites for any or all of the top six endogenously expressed microRNAs . An ROC analysis where the true sites are the 20% most AGO-IP enriched genes and false predictions are the top 20% most enriched in the washed lysate achieved an AUC of 0.72. Moreover, of the top 20% most enriched genes in the AGO-IP, mirSVR correctly detected approximately 85% of these genes as targets of one or more of the endogenous microRNAs using a gene-level mirSVR score threshold of -0.1. In addition, we compared the mirSVR canonical-only model to context score using this AGO IP test set. Similarly to the transfection experiments, we found that mirSVR improves over context score both when comparing the rank correlation of the prediction scores with the enrichment in the AGO IP and by ROC analysis . In each of the transfection experiments we used the mirSVR all-site model to score three sets of predictions: i) only canonical targets, ii) only non-canonical targets and iii) all target sites. True positives for all sets are defined as targets with a log expression change (Z-score) ≤-1 and false predictions are targets with log expression change ≥ 1. The results show that when considering only non-canonical sites, the AUC values are significantly above random  were Z-score transformed.Accessibility scores were computed using RNAplfold  with thes scores  for target al. 2007. Briefly, context score is composed of three regression values, which are specific to each seed class, that model the correlation between the AU composition, 3'-binding and distance from the nearer end of the UTR with mRNA downregulation. Target sites are first classified into one of the four seed classes: 6-mer, T1A 7-mer, m8 7-mer and 8-mer; the context score is computed as the sum of the three regression values specific to the seed class. The computed context scores are highly correlated with the scores downloaded from TargetScan release 5.0 }i = 1...m to learn a linear functionWe adopted a support vector regression (SVR) approach to model the degree of microRNA regulation given a set of numerical features representing the microRNA binding site and additional contextual information. SVR uses labeled training data {.so that the optimization problem only penalizes examples whose outputs fall outside an "epsilon tube" around the prediction function; here ϵ is a parameter chosen prior to training. SVR training was performed using the a, b, c parameters we performed five-fold cross-validation on the training data  and assembled the mirSVR prediction scores computed on each of the held-out sets. We then fit the parameters of the sigmoid function using MATLAB nlinfit function which performs a non-linear regression of mirSVR scores from the five-fold cross validation against their (Z-transformed) log expression changes. Finally, we retrained mirSVR on all the Grimson et al. data and transformed mirSVR prediction scores on test data using the sigmoid transfer function.mirSVR scores were transformed using the sigmoid function 3'UTR: 3' untranslated region; AGO: Argonaute protein; AGO-IP: AGO immunoprecipitation; AUC: area under the curve; CLIP: cross-linking and immunoprecipitation; PAR-CLIP: Photo Activatable Ribonucleoside enhanced CLIP; RISC: RNA-induced silencing complex; ROC: receiver operating characteristic; SVM: support vector machine; SVR: support vector regression.DB, CS and CL conceived the project. DB designed and implemented the algorithm and performed the analysis. AK and PA contributed to the computational analysis. DB and CL wrote the paper. CL helped to develop the algorithmic approach and supervised the research. All authors read and approved the final manuscript.Supplementary material and figures. Supplementary data and figures.Click here for file


The human papillomavirus (HPV) is the major etiologic agent in the development of cervical cancer and its natural history of infection is altered in persons infected with the human immunodeficiency virus (HIV). The prevalence of HPV infection and cervical dysplasia in the HIV sero-positive females in the Bahamas is not known. Finding out the prevalence would allow for the establishment of protocols to optimize total care of this population and help prevent morbidity and mortality related to cervical cancer.The Objective of this study is to determine the prevalence of high risk HPV genotypes and the prevalence of cervical dysplasia in the HIV sero-positive females attending the Infectious Disease Clinic at the Princess Margaret Hospital, Nassau, Bahamas.One hundred consecutive, consenting, non-pregnant, HIV-sero-positive females from the Infectious Disease Clinic at the Princess Margaret Hospital in Nassau, Bahamas were screened for high-risk HPV infections and cervical cytology abnormalities using liquid-based pap smear and signal amplification nucleic acid method for HPV detection. A questionnaire was also utilized to gather demographic information and obtain information on known risk factors associated with HPV infections such numbers of partners.-1 and in women with cervical cytology abnormalities (97%). The most common cervical abnormality was low-grade squamous intraepithelial lesions.The prevalence of high-risk HPV was 67% and cervical abnormalities were noted in 44% of the study population. High-risk HPV types were more likely to be present in women with CD4+ cell counts less than 400 μlFindings suggest that HIV-sero positive females should have HPV testing done as part of their normal gynecology evaluation and these patients should be encouraged and provisions be made for ease of access having regular PAP smears and HPV testing. The human papillomavirus (HPV) is the major etiologic agent in the development of cervical cancer  with knoThe goal of the study is to determine the prevalence of high-risk HPV infections and associated cervical abnormalities in HIV sero-positive females in the Bahamas.One hundred (100) consecutive, consenting, non-pregnant, HIV sero-positive females (18) years or older were recruited from the Infectious Disease Clinic at the Princess Margaret Hospital in Nassau, Bahamas. The criteria for eligibility required all of the following: Females 18 years and older, a positive HIV via ELISA and Western Blot methods and HIV infectivity via sexual intercourse. All participants were interviewed, provided informed written consent and had a liquid based pap smeared performed after receiving consent. The study received ethical approval from the Ethics Committee of the Public Hospital Authority and the University of the West Indies – Bahamas Campus.Each participant had a vaginal examination performed by an Obstetrics and Gynaecology Resident. Cervical samples were collected using a cytobrush (cytobroom) and stored in Sure Path vials. A cytotechnologist screened the prepared slides. All positive results and fifteen percent (15%) of all negatives results were reviewed by a board certified cytopathologist. The cytology was reported as either negative, atypical squamous cells of unknown significance (ASCUS), low-grade squamous intraepithelial lesion (LGSIL) or high-grade squamous intraepithelial lesion (HGSIL). Additionally co-infections as well as presence of HPV changes were noted. The Obstetrics and Gynaecology Oncologist referred all patients with abnormal cytology results to Colposcopy Clinic for further evaluation, treatment and follow-up as determined.Each patient was interviewed and a questionnaire completed by the interviewer. Information on associated HPV risk factors such as parity, number of sexual partners, and birth control method, risky behavior such as drug use, sex under the influence of drugs and condom usage as well as demographic were obtained via the questionnaire. The medical records were reviewed to obtain patient's date of HIV testing and confirm sero-positivity, most recent CD4+ lymphocyte counts and viral load, and use of high active anti-retroviral therapy.Samples collected from patients were stained with FlowCARE PLG CD4 Reagent. CD4+ lymphocytes counts were determined by flow cytometry. White blood cell counts were obtained with a COULTER STKS Instrument. The values were expressed in terms of percentage (%) of total lymphocytes and absolute count (cells/μL).The DNA HIV viral load was tested using a quantiplex HIV-1 RNA 3.0 Assay. This assay is a nucleic acid hydridization procedure to directly quantify HIV-1 RNA in human plasma. The HIV-1 is concentrated from the plasma by centrifugation then the genomic RRNA is liberated from the virions and captured on a microwell by specific synthetic oligonucleotide capture probes. The probes bind to different regions of the pol gene of the viral RNA which is them amplified.Multiple copies of the alkaline phosphatase-labelled probe are then hybridized to an immobilized complex to amplify the signal. Detection is achieved by incubating the complex with a chemiluminescent substrate. The light emission generated by the bound alkaline phosphatase reacting with the chemiluminescent substrate is recorded as relative light units (RLUs). The light emission is directly proportional to the amount of HIV-1 present in each sample. A standard curve is defined by light emission from standards containing known concentrations of beta proiolactone (BPL)-treated virus. Concentrations of HIV-1 RNA in plasma specimens are determined form this standard curve.HPV was detected using the HPV Hybrid Capture II assay which is a signal amplification nucleic acid method for the qualitative detection of eighteen types of human papillomavirus (HPV) DNA in cervical specimens. The assay differentiates between high risk and low risk groups. High HPV types detected are 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68.Clinical and laboratory risk factors for the study population were imported to a statistical software package for analysis. Statistical analyses were performed using the Intercooled STATA (version SE 10.0) software . Factors associated with high-risk HPV status were selected based on cross-tabulations and univariate logistic regression analyses. Cross-tabulations were analyzed using the Fisher's exact test. Logistic regression was used to perform the analyses of associations between demographic, behavioral and clinical variables with high-risk HPV status. Crude odds ratios were first calculated. Potential confounders were identified based on p-values = 0.05. Logistic regression was used to calculate the relative odds of high-risk HPV status after adjusting for the potential confounders (age (continuous), marital status, number of pregnancies, Tobacco use, duration of HAART therapy, CD4 counts, viral load and cervical cytology).One hundred females ranging from ages 20–71 years were interviewed and had a PAP smear performed. The mean and median ages were 40.1 years and 39 years respectively. Fifty-six participants (56%) were single and twenty-four (24%) were married (Table Sixty-seven women (67%) tested positive for high-risk HPV genotypes. The proportion of high-risk HPV-positive women was significantly higher than those who are HPV-negative (p = 0.001). Forty-four women (44%) were found to have abnormal cervical cytology with 95% testing positive for high-risk HPV. Seven patients (7%) had ASCUS, LGSIL in 31 (31%) and HGSIL in six (6%) participants.Adjusting for potential confounders , one hundred percent of participants with HGSIL were high-risk HPV positive . Of note, participants with greater than ten lifetime sexual partners were four times more likely to have high-risk HPV infections than those with less than 10 lifetime sexual partners . This finding was not statistically significant.-1, 38 (38%) had CD4+ counts between 200 and 499 μl-1, and 39 (39%) had CD4+ counts less that 200 μl-1. After adjusting for potential confounders, subjects with CD4+ counts greater than 400 μl-1 were less likely to test positive for high-risk HPV  (Table The mean and median CD4+ lymphocyte counts were 339.15 and 260 respectively. Twenty-three participants (23%) had CD4+ counts ≥ 500 μlHigh-risk HPV is prevalent in the HIV sero-positive females attending the Infectious Disease Clinic at the Princess Margaret Hospital. Unfortunately, a comparison with the HIV sero-negative population could not be done as there is no published HPV prevalence study for the general population. The high prevalence reported in this study is not unusual with HPV infections being quoted as anywhere from 33–47% in other published studies -12. The Subjects living with HIV infection for two to six years were three times more likely to carry high-risk HPV infections than those infected for less than two years, however this association was not significant (p > 0.1). Similarly, women who lived with HIV infections for more than 6 years were almost two times more likely to carry high-risk HPV infections however; this also was not significant.1 were more likely to test positive for high-risk HPV genotypes. As CD4+ count is a reflection of the state of the immune system, it reasonable to assume that the immune dysfunction noted at lower CD4+ levels is responsible for the increased detection of high-risk HPV in these women.Individuals with a CD4+ lymphocyte count of less than 400 μl-An increased number of lifetime sexual partners was associated with an increased likelihood of testing positive for high-risk HPV. Other behavioral variables such as cocaine use, alcohol use and non-barrier method did not show the expected increased risk. This lack of increased risk could be the result of a relatively small study population.The high prevalence of high-risk HPV genotypes and cervical cytology abnormalities demonstrates the need to screen and monitor HIV sero-positive women and to implement preventative measures to decrease the numbers of females with high risk HPV infections. Most participants reported not having yearly PAP smears and unfortunately, routine HPV screening is not conducted at the public facilities where most of these patients receive their PAP smears. Screening for HPV infections in HIV sero-positive females should be an integral component of their routine gynaecological evaluation and these patients should be encouraged and provisions be made for ease of access to having regular PAP smears and HPV testing.The authors declare that they have no competing interests.DND conceived of the study, participated in its design and coordination and drafted the manuscript. CR performed the statistical analysis and reviewed the manuscript. AGB performed the Pap smears and cervical examination. RB sourced the funding, helped with coordination of the study, provided further management for patients with detected abnormalities and reviewed the manuscript.


We report for the first time the frequency distributions of HLA-DRB1 and -DQB1 genes in 55 patients with testicular germ cell carcinoma (TGC) using the modified PCR-RFLP method and compare the results with those for 1216 healthy Japanese control subjects. The modified PCR-RFLP method produced accurate, reproducible cleavage patterns that are easily discriminated. HLA-DRB1*0410 was the susceptibility allele  and DQB1*0602 appears to be a candidate protective allele  for TGC in the Japanese. None of the HLA-DRB1 and -DQB1 alleles showed a specific tendency for histological type or clinical stage of the tumours. Previous studies based on serotyping methods failed to show these allelic associations. High-resolution genotyping is essential because the peptide-binding domain of MHC class II molecules is determined more precisely by their genotypes than by their serotypes. In addition, inherent technical difficulties and typing errors of up to 25% make serotyping inefficient. Our results suggest that high-resolution genotyping is a useful genetic marker to determine risk for TGC.


The growth inhibitory effects of interferons, IFN-alpha and IFN-gamma on human lung cancer cell lines were studied using both a tetrazolium (MTT) colorimetric assay and direct cell counting. Significant discrepancies between the two assays were observed, the MTT assay consistently underestimating the growth inhibitory effects of the IFNs. There was no direct chemical effect of the IFNs on the tetrazolium reduction process. IFN treated cells showed increased cell size compared with control cells, although there was little or no change in cell cycle distribution. Mitochondrial activity was 30-50% greater in IFN-gamma treated cells (COR-L23) than the controls. Reduced formazan production per cell was observed in medium which had supported cell growth for several days. Differential 'medium conditioning' led to a difference in formazan production per cell between IFN and control cells and this was the major basis of the observed discrepancy. This discrepancy was not due to the differences in the glucose concentrations between these media. However, differences in pH between the media proved to be the major contributory factor of the discrepancy.


To the Editor: Eleven years of molecular epidemiologic data allowed the Spanish Multidrug-resistant Tuberculosis (MDR TB) Surveillance Network to identify a specific MDR Mycobacterium tuberculosis strain that had been imported into Spain from Equatorial Guinea . The MDR TB isolates were collected within a 9-year period  and 561  was detected in the 10 MDR isolates. The presence of this uncommon mutation, Ile561Val, outside the rifampin resistance–determining region supports the hypothesis that the MDR isolates are clonal in origin. Furthermore, we demonstrated the absence of Ile561Val mutation in 3 drug-susceptible M. tuberculosis strains with an SIT177-LAM 9 spoligotype pattern, which ruled out a relationship between this spoligotype and the Ile561Val mutation.Analysis of drug resistance genes showed that all isolates harbored the M. tuberculosis and its West African sublineage, on the basis of polymorphisms in codons katG463 and gyrA95, the 7-bp pks15/1 deletion, and RD174 (Rio deletion confirmed that the strain belongs to the major RDRio sublineage of the LAM M. tuberculosis spoligotype family (Further analysis with phylogenetic markers assigned MDR-TBEG to the principal genetic group 2, the Euro-American lineage of M. tuberculosis RDRio sublineage isolated in Spain from Equatorial Guinean patients. Although the transmission of MDR-TBEG in Spain could not be conclusively ruled out, the fact that MDR TB developed in most patients within 3 months after their arrival, as well as the spatiotemporal distribution of the MDR TB cases and its clonal origin, strongly suggest that MDR-TBEG was imported into Spain and that active transmission of this particular clone could be occurring in Equatorial Guinea. However, additional molecular and epidemiologic studies should be conducted in this sub-Saharan country to ascertain its role in recent transmission of MDR TB. Greater international efforts should be made to provide appropriate tools to resource-limited areas for fighting against MDR TB and preventing development of extensively drug-resistant TB.We identified 1 MDR strain of Demographic and clinical characteristics of 10 patients from Equatorial Guinea infected with multiple drug resistant tuberculosis Equatorial Guinea strain*


The metabolism of dietary fatty acids in human has been measured so far using human blood cells and stable-isotope labeled fatty acids, however, no direct data was available for human peripheral tissues and other major organs. To realize the role of dietary fatty acids in human health and diseases, it would be eager to develop convenient and suitable method to monitor fatty acid metabolism in human.in situ for human lip surface lipids using the Fourier transform infrared spectroscopy (FTIR) – attenuated total reflection (ATR) detection system with special adaptor to monitor metabolic changes of lipids in human body. As human lip surface lipids may not be much affected by skin sebum constituents and may be affected directly by the lipid constituents of diet, we could detect changes of FTIR-ATR spectra, especially at 3005~3015 cm-1, of lip surface polyunsaturated fatty acids in a duration time-dependent manner after intake of the docosahexaenoic acid (DHA)-containing triglyceride diet. The ingested DHA appeared on the lip surface and was detected by FTIR-ATR directly and non-invasively. It was found that the metabolic rates of DHA for male volunteer subjects with age 60s were much lower than those with age 20s. Lipid hydroperoxides were found in lip lipids which were extracted from the lip surface using a mixture of ethanol/ethylpropionate/iso-octane solvents, and were the highest in the content just before noon. The changes of lipid hydroperoxides were detected also in situ with FTIR-ATR at 968 cm-1.We have developed the measurement system in situ non-invasively.The measurements of lip surface lipids with FTIR-ATR technique may advance the investigation of human lipid metabolism  In order to diagnose human body, especially the surface of skin or tissues non-invasively and easily, FTIR technique has a significant potential and it may be applied to detect important biomolecules, even though they are mixed in biological tissues and the depth profile in the skin may not be obtained. It could be used sophisticated statistical methods, such as partial least squares regression or other chemometrics , [3022], [3014], [2979], and [2960] indicate the intensities at 3010, 3022, 3014, 2979, and 2960 cm-1 and may contribute to the increase of DRI values. Smoking or non-smoking did not affect significantly the change of DRI values for age 60s group in the control diet.On the other hand, the increase of DRI values was detected at 3 to 4 hours of duration for both age 20s and 60s groups in the control diet using soy oil (0.6 g)-containing yogurt. In this case of control diet, the increase of arachidonic acid (C20:4) may affect the FTIR spectrum at around 3010 cmcis-alkene FTIR spectra of lip surface of human.These results suggest that dietary DHA metabolism may be detected non-invasively with measuring in situ, we tried to detect lipid hydroperoxides extracted from lip surface biochemically.Before measurement of lipid hydroperoxides (LOOH) by FTIR Fig. Next the lipid hydroperoxide near the spot of triglyceride hydroperoxide in the lip surface lipids was quantitated after charring through measuring spot intensities and compared with other neutral lipids.Fig. in situ. Fig. -1 (top figure) and at around 1740 cm-1 (bottom figure). It is well known that trans-alkene (HC = CH) in fatty acids of foods shows infrared absorption at 966~968 cm-1 /[TG] ratio was the highest at 12:00 clock time, just before lunch, and in this case the amount of triglyceride was decreased at 12:00. This biochemical profile of the time course was consistent with the spectral profile obtained from peak ratio in the FTIR spectra, where the ratio between trans-alkene peak at 968 cm-1 and the ester peak at 1740 cm-1 was the highest at 12:00 clock time.Fig. in situ could detect the change of lipid hydroperoxide on lip surface.These results indicate that the measurement of lip surface FTIR in situ non-invasively. Normally the metabolism of dietary fatty acids has been measured so far using human blood cells and stable-isotope labeled fatty acids [The present paper has described that the measurement of FTIR spectra of human lip surface could detect the metabolism of dietary fatty acids, especially docosahexaenoic acid (DHA), and the change of lipid hydroperoxides ty acids , and it ty acids . MoreoveWe found as shown in this paper that the metabolism of dietary DHA or other fatty acids/lipid hydroperoxides could be measured non-invasively through human lip. As lip surface contains thinner stratum corneum (SC) in the vermilion zone (red zone) with less barrier functions ,13 than in situ for human lips. Generally human lip has 4 portions anatomically, (1)appendage-bearing epidermis, (2)keratinized vermilion (red zone), (3)parakeratinized intermediate zone, and (4)labial mucosal epithelium [However, we have no data available so far to estimate the penetration and diffusion coefficient of chemicals ithelium . In the ithelium , and parAs shown in this paper, the dietary fatty acids, such as DHA, could be transported from blood vessels to the lip  surface. However, the appearance of dietary DHA on the face skin surface could not be detected by the same FTIR-ATR method (data not shown). Normal skin surface fatty acids may be originated mainly from sebum. Because it would take time to incorporate dietary lipids from blood and synthesize lipids in sebaceous glands of skin, it may be hard to detect the dietary DHA or other polyunsaturated fatty acids of blood on the skin surface. On the other hand, the lip vermilion surface lipids may not be from sebum, because sebaceous and sweat glands in the vermilion portion were rare histologically.The transport or movement of dietary DHA from blood vessels to the lip surface was unexpectedly rapid for younger men as shown in Fig. This difference in transport rate between DHA and other fatty acids from blood vessels to lip surface may not be simply explained by a passive penetration mechanism of DHA, but we may have to assume the presence of specific transport mechanism of DHA. The transport of DHA from blood vessels to neonate is reported to be dependent on the expression of fatty acid-transport proteins in placenta  and thatAlthough the role of such fatty acid-binding protein is not known in transport of lipids from blood vessels to lip, a facilitated transport mechanism of DHA in human lip may be present as shown in this paper and this transport mechanism may be different from that of other fatty acids, such as arachidonic acid. We demonstrated also that the transport of DHA to the lip vermilion surface was carried out mainly in the form of triglyceride (or less in diglyceride form). More detailed analyses may be needed using our newly developed FTIR-ATR apparatus.in situ by FTIR-ATR, and the LOOH/TG ratios were increased just before noon in our experiments. The lip lipid hydroperoxides were probably triglyceride form mainly as shown in Fig. ex., cigarette smoke). In any case we could not assume the lip lipid hydroperoxides were solely dietary origin, because the increase of lipid hydroperoxides was not observed in the afternoon even at 4 hours later after lunch. In both breakfast and lunch, these diets normally contained significant amount of linoleic acid (C18:2) even in bread or rice, a base diet. The drastic increase of LOOH/TG ratio just before noon on the lip may thus indicate a physiological phenomenon. As PUFA in triglyceride of lip surface may have a rhythm to be increased just before noon or 4 to 5 hours after breakfast under the normal diet as shown in Fig. Not only polyunsaturated fatty acids were transported to lip surface but also a significant amount of LOOH was detected on the surface. These lipid hydroperoxides were non-destructively measured It has been known that lipid hydroperoxides on the normal skin like face forehead is derived from mainly cholesterol  and squaWe have demonstrated that our newly developed FTIR-ATR system could detect non-invasively and easily the metabolic changes of polyunsaturated fatty acids and lipid hydroperoxides in human body through lip vermilion surface. It was observed that dietary DHA in triglyceride was transported to the lip surface more rapidly for men with age 20s than age 60s, and that lipid hydroperoxides of lip vermilion were increased just before noon with correlation to the increase of polyunsaturated fatty acids.p-phenylenediammonium dichloride; DRI: DHA-relative intensity; EPA: eicosapentaenoic acid; EPI: the solvent mixture of ethanol/ethylpropionate/iso-octane; FTIR-ATR: Fourier transform infrared spectroscopy with attenuated total reflectance; LN: linoleic acid; LOOH: lipid hydroperoxide; PVDF: polyvinylidenedifluoride; SC: stratum corneum; TG: triacylglycerol or triglyceride.DHA: docosahexaenoic acid; DMPD: N, N'-dimethyl-The authors declare that they have no competing interests.SY has made substantial contribution to conception, design, acquisition of data, analysis and interpretation of data, drafting the manuscript, and final approval of the version to be published. QZZ carried out TLC and GCMS analyses for fatty acids and lipids in lips. SS participated in the design of the skin and lip studies. SM carried out the TLC, GCMS, and FTIR measurements for lip lipids and fatty acids. All authors read and approved the final manuscript.


Interrogation of chromatographic data for biomarker discovery becomes a tedious task due to stochastic variability in retention times arising from solvent and column performance. The difficulty is further compounded when the effects of exposure  and biological variability result in varying numbers and intensities of peaks among chromatograms.We developed a software tool to correct the stochastic time shifts in chromatographic data through iterative selection of landmark peaks and isometric interpolation to improve alignment of all chromatographic peaks. To illustrate application of the tool, plasma peptides from Fischer rats exposed for 4 h to clean air or Ottawa urban particles (EHC-93) were separated by HPLC with autofluorescence detection, and the retention time shifts between chromatograms were corrected (dewarped). Both dewarped and non-dewarped datasets were then mined for models containing peptide peaks that best discriminate among the treatment groups using ClinproTools™. In general, models generated by dewarped datasets were able to better classify test sample chromatograms into either clean air or EHC-93 exposure groups, and 0 or 24 h post-recovery time groups. Peak areas of peptides in a model that produced the best discrimination of treatment groups were analyzed by two-way ANOVA with exposure  and recovery time  as factors. Statistically significant (p < 0.05) time-dependent and exposure-dependent increases and decreases were noted establishing these as biomarker candidates for further validation.Our software tool provides a simple and portable approach for alignment of chromatograms with complex, bi-directional retention time shifts prior to data mining. Reliable biomarker discovery can be achieved through chromatographic dewarping using our software followed by pattern recognition by commercial data mining applications. Biomarkers are of interest and value in many experimental population studies and in clinical assessments as biological indicators of environmental exposures or disease status -4. DetecVarious approaches of peak alignment such as local shifting and peak matching , target The objective of this work was to develop and validate a dewarping approach that is implemented in a simple and portable software tool, and that is not constrained by a need to specify or optimize dewarping parameters, to correct the stochastic variability in chromatographic data manifested as large complex bi-directional retention time shifts and to generate datasets better amenable to downstream data mining for biomarkers.Our dewarping approach consisted of a software assisted iterative selection and alignment of landmark peaks by interpolation or removal of data points between peaks for dewarping of all peaks across chromatograms. This was implemented as a Windows based software tool ("DewarpTool"). Dewarping using this approach was validated by the application of the tool to dewarp chromatograms from 12 successive runs of a mixture of standard peptides. An overlay of standard chromatograms Figure  illustraRetention time variations were also seen in complex plasma peptide chromatograms from animals exposed to clean air and to Ottawa urban particles (EHC-93) by inhalation. A representative chromatogram of the plasma peptide profile recovered for rat plasma across the entire run (2700 seconds or 45 min) is shown in Figure In order to examine if our dewarping approach generated chromatograms that are better amenable to data mining for generation of discriminatory models, both dewarped and non-dewarped chromatograms were mined using ClinproTools. While a number of non-dewarped chromatograms were not calibratable and therefore unusable for discriminatory model generation even with a large maximal peak shift parameter to accommodate large peak shifts Figure , the dewDiscriminatory models generated using both dewarped and non-dewarped datasets  were validated by their ability to classify experimental samples into air or EHC-93 exposure groups  and into 0 and 24 h post-recovery time groups . In general, models generated using dewarped datasets had a better discriminatory capacity when compared to the models generated by using non-dewarped datasets Table . Among tThe results of cross-validation of models using the approaches the One-Out, Random and K-Folds, wherein one or more of the chromatograms were randomly or sequentially excluded from model generation and were classified against models generated using the remaining data as the training data, are shown in Table The need to have well aligned chromatographic fingerprints for data mining and visualization approaches such as PCA has been underlined in many previous studies. Examples include: use of correlation optimized warping of GC-MS data followed by PCA for fingerprinting of petroleum biomarkers , correlaOur alignment approach consists of software-aided, visual matching of landmark peaks across chromatograms, followed by programmatic, isometric interpolation or removal of data points, to stretch or shrink regions between landmark peaks to achieve their alignment. Johnson et al  employedThe correction of retention time shifts in our plasma peptide chromatograms resulted in datasets amenable for averaging and differential comparisons using the differential analysis component of DewarpTool. Although this allowed a visual verification of chromatogram alignment and a qualitative assessment of treatment-dependent responses that may warrant detailed quantitative analysis, given the potential subjectivity of a visual peak selection approach and the availability of commercial data mining tools, we tested whether our dewarping approach can generate datasets that are more amenable than the non-dewarped chromatographic data for an objective analysis of biomarkers using ClinproTools.In order that our chromatographic data are treated appropriately within ClinproTools, a tool intended for mass spectral data mining, data preparation and peak selection parameters were appropriately adjusted for accurate definition of chromatographic peaks. The non-dewarped datasets were not as amenable to data mining as the dewarped datasets, as a larger number of non-dewarped chromatograms were not recalibratable and hence unusable for data mining even after using a large peak matching window to accommodate large retention time shifts. In addition, the choice of the peak shift parameter (0.5% or 4.0%) impacted the average peak profile generated by non-dewarped datasets, but not that of dewarped datasets, suggesting the possibility of ambiguous peak alignments by ClinproTools when using non-dewarped datasets. If this is true, it can be expected that the mining of dewarped data would generate better discriminatory models than those generated using non-dewarped datasets. This is supported by our analysis using the genetic and support vector machine algorithms, the two multivariate model-building methods provided by ClinproTools, and by examining the ability of the generated models to classify experimental chromatograms into appropriate exposure  and post-exposure recovery time  groups. Models generated by dewarped datasets had, in general, better discriminatory capacity than those generated by non-dewarped datasets. In addition, non-dewarped datasets resulted in insufficient number of recalibratable and hence useable chromatograms for cross validation.In short, our dewarping approach implemented in DewarpTool allowed generation of chromatographic datasets that were more suited for data mining resulting in models with better discriminatory capacity for classification of treatment groups. The peaks contained in the best discriminatory model showed both significant particle and time dependent responses and non-significant pollutant-time interactions, pointing to their potential involvement in pathways relating to air pollution related biological changes. These peptides may provide interesting marker candidates and mechanistic insights into adverse health impacts of low ambient levels of air contaminants, upon their identification. Generation of models comprising of candidate peptides showing significant treatment effects resulting in greater reliability for discrimination of treatment effects validated the models and reaffirmed the utility of our dewarping approach in generating chromatographic datasets for biomarker mining.Reduction of retention time variations of peptide peaks in rat plasma chromatograms by using DewarpTool resulted in datasets that were better amenable for data-mining and that, after mining, resulted in models of peptide peaks showing high discriminatory capacity between treatment groups (types of air pollutant exposures and times of recovery after exposures), and therefore of potential value as biomarkers after mechanistic validation. Overall, our approach involving dewarping by DewarpTool followed by mining for discriminatory biomarker patterns provides a simple, novel, portable and useful approach for the mining of liquid chromatographic data that may be characterized by large, complex peak shifts. DewarpTool installation files (Windows operating system) are provided as supplementary material to this communication [Additional files The following HPLC-grade standards were used: angiotensin II acetate (human), endothelin-1 , endothelin-2 (human), enodothelin-3  and big-endothelin-2 (human) ; big-endothelin-1 (human) ; big-endothelin-3 (rat) .ad libitum. All experimental protocols were reviewed and approved by the Animal Care Committee of Health Canada. Animals were exposed for 4 hours to clean air or 50 mg/m3 Ottawa urban particles (EHC-93) in nose-only exposure chambers as described previously [Pathogen-free Fischer-344 male rats (180–250 g) obtained from Charles River  were housed in individual plexiglass cages on wood-chip bedding under HEPA-filtered air and held in a 12 h dark/light cycle. Food  and water were provided eviously . Plasma HPLC analysis of plasma peptides was conducted as described by Kumarathasan et al. . BrieflyWe developed a dewarping software (DewarpTool), using Visual Basic , and deployed the software in a Windows XP environment. The analysis workflow consists of baseline correction, data smoothening, landmark peak selection, peak alignment, average chromatogram calculation and differential visualization  for biomarkers. Prior to data import, data preparation and peak selection parameters within ClinproTools were defined as follows: resolution = 100; baseline subtraction by convex hull method; baseline flatness = 0.80; mass range (retention time range) for analysis = 0 – 1200; recalibration with 0.5% or 4.0% maximal peak shift and 30% match to calibrant peaks; signal to noise threshold = 5.0; peak area calculation by zero level integration. Data preparation workflow within ClinproTools consists of baseline correction, spectral normalization based on total ion count (peak count), calculation of a recalibration function for each spectrum to correct for peak shifts, calculation of a total average spectrum using recalibrated peaks, and calculation of areas from individual spectra for peaks represented in the total average spectrum. Recalibration corrects for peak shifts by a process of selection of a number of reference peaks in the average chromatograms and confirmation of occurrence of these peaks in individual chromatograms within a window, as defined by the maximal peak shift parameter. Chromatograms that do not contain a minimum number of reference peaks even after adjusting for the peak shifts are dubbed 'non-recalibratable'. Peaks represented in the total average spectrum are then detected in the individual spectra, their areas are integrated, and the resulting data matrices are used in generation of discriminatory models. Although the chromatographic datasets  were 2700 seconds (45 min) in length, we illustrated the application using the first 1200 seconds (20 minutes) of data that corresponded to the period where the peptides eluted at sufficient peak resolution.Discriminatory models were built by using the genetic algorithm and support vector machine approaches of the ClinproTools software. Genetic algorithm was applied with a mutation rate = 1.0 (peak substitutions within a model) and recombination rate = 1.0 (peak exchanges between models). Each model was tested for efficiency of classification of experimental samples into correct exposure groups (clean air or EHC-93) and time of recovery groups (0 h and 24 h). The reliability of the models for classification of unknown samples (future predictive capacity) was tested by cross validation using the One-Out, the Random and the K-fold methods. The One-Out method consists of generating a model after leaving one of the chromatograms out and classifying this left-out chromatogram against the model, and repeating this until all chromatograms have been used in classification. For the Random method, 20% of the total chromatograms, randomly selected, are omitted during model building and are then classified against the model generated. This process was repeated 10 times and classification abilities of the generated models were averaged. For the K-fold cross-validation method, the dataset was divided into 12 equal parts, and a part was left out during model building and then classified against the model. The process was iterated until all K parts were used as test data.Exposures  and duration of recovery  as factors were tested for statistical significance of effects by two-way ANOVA, followed by Holm-Sidak's procedure to elucidate the pattern of significant effects (p = 0.05) using Sigma-Stat .RV and SK conceived the dewarping approach described here. SK authored DewarpTool to implement the approach. RV planned and directed animal inhalation exposures. PK developed the chromatographic method for fluorescence analysis of plasma peptides and directed the analysis. SK conducted chromatographic dewarping using DewarpTool and data mining by ClinproTools. SK, PK and RV co-authored the manuscript, while SK led and coordinated the writing of the manuscript. All authors approved the final manuscript.DewarpTool Setup Package. Zip file containing DewarpTool installation files.Click here for fileDewarpTool Installation Instructions. This contains instructions for installation of DewarpTool using the DewarpTool Setup Package (Additional File Click here for fileDewarpTool User Manual. This is a guide to the use of DewarpTool for dewarping chromatograms.Click here for file


An automated analyser for total hydrocarbon contents and hydrocarbon composition (from methane to pentanes) was constructed and tested in both laboratory and field exploitation. It used two-channel analysis: continuous measurements of total hydrocarbon contents and periodic (90 or 150 s) composition analysis after separation of hydrocarbons on a gas chromatographic column. Flame ionization detectors were used in both channels. A simple 16-bit analogue-to-digital converter was used , while the full measuring range (six orders of magnitude) was ensured by automatic dilution of the sample (or standard) with clean air. Full control of the operating  cycle was performed by microcomputer. An external programme, based on a computer provided with full information on the instrument operating conditions, presents the results of calibrations/analyses and enables them to be archived in a standard database used in the oil/gas drilling industry (N-LAB) by providing a suitable link. The instrument measuring range was 1 ppm to 100% with precision not worse than 5% at the detection limit. The analyser can operate autonomously for two months, recalibrating itself daily.


Cells expressing high levels of functional P-glycoprotein  are highly responsive to Pluronic treatment, while cells with low levels of P-glycoprotein expression  are much less responsive to such treatment. Cytotoxicity studies suggest that Pluronic acts as a chemosensitizer and potentiates cytotoxic effects of doxorubicin in MDR cells. The ability of Pluronic to inhibit P-glycoprotein and sensitize MDR cells appears to be a result of ATP depletion. Because many mechanisms of drug resistance are energy dependent, a successful strategy for treating MDR cancer could be based on selective energy depletion in MDR cells. Therefore, the finding of the energy-depleting effects of Pluronic P85, in combination with its sensitization effects is of considerable theoretical and practical significance. © 2001 Cancer Research Campaign


Diabetes specific emotional problems interfere with the demanding daily management of living with type 2 diabetes mellitus (T2DM). Possibly, offering direct feedback on diabetes management may diminish the presence of diabetes specific emotional problems and might enhance the patients' belief they are able to manage their illness. It is hypothesized that self-monitoring of glucose in combination with an algorithm how and when to act will motivate T2DM patients to become more active participants in their own care leading to a decrease in diabetes related distress and an increased self-efficacy.Six hundred patients with T2DM (45 ≤ 75 years) who receive care in a structured diabetes care system, HbA1c ≥ 7.0%, and not using insulin will be recruited and randomized into 3 groups; Self-monitoring of Blood Glucose (SMBG), Self-monitoring of Urine Glucose (SMUG) and usual care (n = 200 per group). Participants are eligible if they have a known disease duration of over 1 year and have used SMBG or SMUG less than 3 times in the previous year. All 3 groups will receive standardized diabetes care. The intervention groups will receive additional instructions on how to perform self-monitoring of glucose and how to interpret the results. Main outcome measures are changes in diabetes specific emotional distress and self-efficacy. Secondary outcome measures include difference in HbA1c, patient satisfaction, occurrence of hypoglycaemia, physical activity, costs of direct and indirect healthcare and changes in illness beliefs.The IN CONTROL-trial is designed to explore whether feedback from self-monitoring of glucose in T2DM patients who do not require insulin can affect diabetes specific emotional distress and increase self-efficacy. Based on the self-regulation model it is hypothesized that glucose self-monitoring feedback changes illness perceptions, guiding the patient to reduce emotional responses to experienced threats, and influences the patients ability to perform and maintain self-management skills.Current Controlled Trials ISRCTN84568563 Diabetes specific emotional problems can interfere with the strict regime type 2 diabetes mellitus (T2DM) demands. Emotional problems, such as not accepting diabetes, fear for hypoglycaemia and worrying about complications, might impact aspects of quality of life, for example, increase diabetes related distress, which in turn might affect self-care behaviours and glycaemic control ,2. DiabeThe hypothesis that self-monitoring of glucose empowers the patient by its feedback is based on the principles of Leventhal's self-regulation model ,7. This To date, research in self-monitoring of glucose has primarily focused on reaching and maintaining glycaemic control. Therefore, up to now the evaluation of success or failure of self-monitoring has been based upon its ability to decrease HbA1c to normal values.Self-monitoring of glucose has been proven successful in regulating HbA1c in patients with type 1 diabetes  and T2DMBefore the widespread extent of SMBG, self-monitoring of urine glucose (SMUG) was used to indicate episodes of hyperglycaemia during the corresponding urine excretion time. Even though urine glucose excretion is influenced by renal threshold, SMUG can provide basic information on glycaemic status. Furthermore, feedback derived from SMUG can motivate T2DM patients to become actively engaged in self-management of T2DM , irrespeWhether self-monitoring of glucose can help patients to better understand their diabetes and to an increased self-efficacy and less diabetes related distress is not known and has not been investigated as a primary objective yet. Information from qualitative research suggest that self-monitoring of glucose may have a negative impact regarding aspects of quality of life and patient-satisfaction -20. PossIn this trial the effect of self-monitoring of glucose regarding diabetes-specific distress and self-efficacy in combination with education in self-monitoring and an algorithm what to do is evaluated.The aim of the study is to assess the effects of SMBG and SMUG in patients with type 2 diabetes who are not using insulin compared to usual care. Primary outcome measures are changes in diabetes specific emotional distress and self-efficacy. Secondary outcomes are changes in glycaemic control, patient treatment satisfaction, physical activity, health status, status of depression, occurrence of hypoglycaemia, cost-effectiveness and cost-utility. As part of a process evaluation changes in illness perceptions, perceived severity and social support are determined as well."IN CONTROL" entails a 3-armed randomized clinical trial performed in the province of Noord-Holland, The Netherlands. The Medical Ethical Committee of the VUmc approved the study design, protocols, information letters to the patients and informed consent form.The participating diabetes care systems provide and manage regional diabetes care in addition to the care of general practitioners and their nurse practitioners. All care systems are based on the chronic care model -23 and cIn total 600 consenting participants will be recruited. Patients are considered eligible if they are diagnosed with T2DM and meet the following criteria:❑ known disease duration of over 1 year❑ recent HbA1c ≥ 7.0%❑ treated with diet and/or oral hypoglycaemic agents❑ do not require insulin at inclusion❑ aged between 45 and 75 years❑ used SMBG or SMUG less than 3 times in the previous yearParticipants will be randomly assigned to one of the following 3 groups figure :❑ intervention group A, performing SMBG with specific SMBG education, in addition to usual diabetes care provided by the regional diabetes care system.❑ intervention group B, performing SMUG with specific SMUG education, in addition to usual diabetes care provided by the regional diabetes care system.❑ control group receiving usual diabetes care provided by the regional diabetes care system.For intervention allocation a randomization list is drawn up using a computerized randomization computer program (Random Allocation Software version 1.0.0). As different effects may be expected of prescribed drug treatments that are likely to cause hypoglycaemia, for example sulfylureas or glinides (SU), randomization will be pre-stratified by treatment (using SU or not (Non-SU)). A research-manager, who is not involved in the patients care, allocates the patient to one of the 3 groups by using the randomization list. Intervention allocation will be concealed for the principal investigator who is responsible for the data analyses. Participants may withdraw from the study at their own request and without providing reasons. Date of withdrawal will be recorded and effort will be made to contact participants lost to follow up. Only participants withdrawn prior to randomization will be replaced.Following randomization participants allocated to one of the intervention groups receive a training in SMBG or SMUG delivered by trained research assistants and a flowchart with instructions when to perform and how to interpret self-monitoring. After this training participants should be able to perform SMBG or SMUG and interpret the results, identify factors that can influence glucose level and identify when additional tests are needed.® Ultra®2). Participants in the SMUG group are requested to test their urine twice a week  in the evening after dinner (Urispec™ plus urine glucose in vitro reagent test strips). Both groups will receive a diary in which they have to record the obtained values for blood or urine glucose. To avoid extra psychological burden we allow participants to adjust self-monitoring to a frequency 'they feel comfortable with' starting from 2 months after baseline until the end of the study.The acquired skills in self-monitoring are checked and if necessary corrected in a control visit of maximal 30 minutes, 7 to 14 days after randomization. During this visit a stepwise script outlining the self-monitoring skills needed is used. All participants in the SMBG group are requested to measure their blood glucose twice a week  at 6 different time points a day  with a glucose-meter  ,25 and sA process evaluation will be performed to evaluate our theories of the processes underlying the effect of self-monitoring. For this evaluation changes in mastery are assessed by the subscales personal control and treatment control of the Revised Illness Perception Questionnaire (IPQ-R)  and chanA script outlining the topics to be covered per visit is used to support the nurses and research assistants in their tasks. Before inclusion all research assistants attend a training in self-monitoring (SMBG and SMUG) and before the start of the intervention their self-monitoring skills are tested in a pilot. During the inclusion period the research assistants are audited. Directly after the training the research assistants are asked about their opinions about their acquired skills in self-monitoring and their capability to train and instruct participants following protocol . This will also be done after the inclusion period. The participants of the intervention groups are asked to give their opinion on 6 items regarding skills, importance and motivation for self-monitoring at baseline and 2 months after baseline .The proposed trial is designed to detect a clinically relevant change of self-monitoring of glucose on diabetes specific emotional distress and self-efficacy. No consensus exists about minimal important differences (MID) of diabetes specific distress measured with the PAID and self-efficacy measured with the CIDS. Therefore we set the MID at half a standard deviation . In diabOn the basis of an intention-to-treat analysis, differences between the intervention groups and usual care group are calculated with 95% confidence intervals. In addition per protocol analyses will be performed. The subgroups SU and non-SU will be analysed separately for all outcome measures. In case there is no effect modification by treatment the data of both subgroups will be pooled. If there are any relevant differences in baseline measurements between the 3 arms, we will adjust the outcomes for these factors . Linear and logistic regression will be used to determine the effect of the intervention on each of the outcome measurements. The effect of the intervention will be estimated for sub-groups defined by duration of diabetes, socio-economic status, perceived social support and perceived severity of diabetes, glycaemic control and self-monitoring frequency in order to gain a better understanding as to who benefits most from the intervention. Analyses of moderators and mediators will be performed for the process-evaluation.For the economic evaluation from both the societal and the health insurer perspective all costs related to diabetes, and diabetic complications are considered relevant. Costs of the intervention  are considered direct healthcare costs. The costs of i.e. absence from paid and unpaid work are considered indirect healthcare costs. Differences in mean costs between the groups will be presented with 95% confidence intervals. An incremental cost-effectiveness ratio (ICER) will be given both for PAID and CIDS by dividing the incremental mean costs by the incremental mean reduction in PAID or CIDS score. A cost utility ratio will estimate the incremental costs per Quality Adjusted Life Year gained for the study period. Cost-effectiveness planes and acceptability curves will be presented and 95% confidence intervals will be estimated for the ICERs.This article presents a detailed description of a RCT, designed to explore the effectiveness of SMBG and SMUG on diabetes specific emotional distress and self-efficacy in non-insulin dependent T2DM patients. The proposed trial is sufficiently powered and, to the best of our knowledge, the first to allow for conclusions on the effect of self-monitoring of glucose on both diabetes specific emotional distress and glycaemic control in a large sample. In addition, due to a pre-stratification in oral treatments likely to cause hypoglycaemia or not, a possible interaction with type of treatment can be discovered. With the use of a theoretical model as a base for our hypothesis the evaluation of processes theoretically underlying the (in)effectiveness of our trial will be allowed as well. Therefore, this trial will make an important contribution to the evidence of self-monitoring in non-insulin requiring patients with T2DM.Franciosi et al.  reportedAs glycaemic control is an important issue in diabetes care most research regarding self-monitoring has focused on clinically relevant changes in HbA1c ,37-40. TThis trial will deliver important insights in the effects of self-monitoring of glucose in T2DM patients not using insulin on diabetes related distress and self-efficacy and the role of possible barriers and facilitators underlying its (in)effectiveness.BMI: Body Mass Index; CIDS: Confidence In Diabetes Self-care questionnaire; DTSQ: Diabetes Treatment Satisfaction Questionnaire; GP: General Practitioner; ICER: Incremental Cost-Effectiveness Ratio; IPAQ-7: short International Physical Activity Questionnaire; IPQ-R: Revised Illness Perception Questionnaire; MDQ: Multidimensional Diabetes Questionnaire; MID: Minimal Important Difference; PAID: Problem Areas In Distress scale; PHQ-9: Patient Health Questionnaire; RCT: Randomized Controlled Trial; SMBG: Self-Monitoring of Blood Glucose; SMUG: Self-Monitoring of Urine Glucose; SU: Sulphonyl-Urea or Glinides; T2DM: Type 2 Diabetes Mellitus.The authors declare that they have no competing interests.ULM is responsible for the data collection and wrote the manuscript. SDMB, JMD and GN developed the original concept for the study. The study design was further developed by ULM, SDMB, PJK, FJS, JMD and GN. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In normal-hearing subjects, monaural stimulation produces a normal pattern of asynchrony and asymmetry over the auditory cortices in favour of the contralateral temporal lobe. While late onset unilateral deafness has been reported to change this pattern, the exact influence of the side of deafness on central auditory plasticity still remains unclear. The present study aimed at assessing whether left-sided and right-sided deafness had differential effects on the characteristics of neurophysiological responses over auditory areas. Eighteen unilaterally deaf and 16 normal hearing right-handed subjects participated. All unilaterally deaf subjects had post-lingual deafness. Long latency auditory evoked potentials (late-AEPs) were elicited by two types of stimuli, non-speech (1 kHz tone-burst) and speech-sounds (voiceless syllable/pa/) delivered to the intact ear at 50 dB SL. The latencies and amplitudes of the early exogenous components (N100 and P150) were measured using temporal scalp electrodes.Subjects with left-sided deafness showed major neurophysiological changes, in the form of a more symmetrical activation pattern over auditory areas in response to non-speech sound and even a significant reversal of the activation pattern in favour of the cortex ipsilateral to the stimulation in response to speech sound. This was observed not only for AEP amplitudes but also for AEP time course. In contrast, no significant changes were reported for late-AEP responses in subjects with right-sided deafness.The results show that cortical reorganization induced by unilateral deafness mainly occurs in subjects with left-sided deafness. This suggests that anatomical and functional plastic changes are more likely to occur in the right than in the left auditory cortex. The possible perceptual correlates of such neurophysiological changes are discussed. Unilateral deafness represents a particular model for the investigation of functional auditory plasticity mechanisms in humans. In normal hearing subjects, the cortical activation pattern is characterized by shorter and larger neurophysiological responses over the hemisphere contralateral to the stimulated ear in response to monaural stimulation -3. This In adult humans, previous data have revealed that auditory plasticity mechanisms also occur within the first weeks after the onset of unilateral deafness ,10 and cThis research was performed in compliance with the Helsinki Declaration and the protocol was approved by the local ethics committee . Written consent was obtained from all participants.Thirty-four,  right-handed, French-native speakers aged from 27 to 59 years participated in the study. No subject reported any history of neurological impairment or language disorder. All patients had a profound unilateral deafness with bone conduction hearing thresholds on the deaf side worst than 65 dB HL for all frequencies tested between 250 and 8000 Hz .Scalp-EEG activity was recorded from 29 electrodes embedded in an electrode cap and placed in accordance with the international reference system : a non-speech sound -1 kHz tone-burst, total duration = 375 ms- and a speech-sound -voiceless consonant-vowel (CV) syllable/pa/, Voice Onset Time = +35 ms, total duration = 260 ms-. The stimulus intensity for EEG recordings was set at 50 dB SL , delivered either to the healthy ear of deaf patients or the tested ear of control subjects. This intensity level was used to avoid any transcranial transfer effect to the contralateral ear in the control groups. The inter-stimulus interval (ISI) varied from 800 to 1200 ms. Six series of 100 stimuli  were repeated three times in a single session; as a result, each individual set of EEG data contained 1 800 responses (900 responses relative to each stimulation condition). Testing took place in a sound attenuated booth with the subjects seated in a comfortable reclining chair while watching a video tape (mute-subtitled mode). The neurophysiological recordings lasted for 90 minutes per subject.® software . Offline data were digitally band-pass filtered  and subjected to an automatic artefact rejection algorithm wherein sweeps containing activity exceeding ± 150 μV in any channel were excluded from subsequent analyses. Mean averaged waveforms for each eliciting stimulus were obtained separately for each subject. The neurophysiological approach focused on the early exogenous waves N100 and P150 of late-AEPs which are determined primarily by the physical properties of the stimuli contrary to the endogenous components associated with more cognitive processes [Each individual's AEP data were analyzed with ELANAnalyses of variance (ANOVAs) were carried out to test the effect of group on the parameters measured  for each stimulus. An additional post-hoc Bonferroni-adjusted t-test was performed on any significant ANOVAs. All mean values are presented with the standard error in brackets (+/- SEM).Figure In order to simplify presentation of the results, data are shown separately for each group of subjects. Figure The comparison between ipsi and contralateral responses for both groups of normal hearing subjects (NH-r and NH-l) revealed shorter contralateral N1 mean latency with positive IHLD Table . These rWhatever the stimulation condition, there was no difference between the RD-l group and normal hearing subjects for all measured parameters: the RD-l group exhibited shorter N1 mean latency over the contralateral cortex with positive IHLD mean values , i.e. the normal pattern of asynchrony over the temporal lobes. Moreover, the RD-l group showed larger mean amplitudes of N1-P2 complexes over the contralateral cortex with positive IHAD mean values (reduced but preserved compared to the controls (non significant)), reflecting a normal pattern of asymmetry over the temporal lobes.3.30 = 1.508; p = 0.232). In spite of a significant group effect for contralateral N1 latency , the post-hoc multiple comparison procedure also did not show any significant differences between groups. However, the one-way ANOVA revealed a main group effect on the N1 latency ipsilateral to the stimulated ear . The post-hoc multiple comparison test revealed a significantly shorter N1 ipsilateral latency in the LD-r group compared to both the NH-r  and RD-l  groups. With amplitude, the ANOVAs did not show any group effect either for N1-P2 mean amplitude values or the corresponding IHAD mean values , the mean value of N1 IHLD which tended towards 0 was lower than the values obtained in the other groups : the N1 IHLD mean value of the LD-r group was negative, significantly reversed compared to both RD-l  and the controls NH-l . Furthermore, whereas the groups did not significantly differ for contralateral N1 latency, a one way ANOVA revealed a significant group effect on ipsilateral N1 latency . Multiple comparisons revealed a significantly shorter ipsilateral N1 latency in the LD-r group compared to the RD-l group . In response to speech-sound, the ANOVA revealed a group effect on the N1-P2 complex IHAD mean value : the LD-r group showed a negative IHAD mean value, significantly reversed compared to the NH-l group . With amplitude mean values, the healthy-side dominance of the N1-P2 complex exhibited by the LD-r group seemed to result from a combined contralateral decrease/ipsilateral increase in activity compared with controls stimulated in the same ear (NH-r) (non significant).In response to speech-sound (unvoiced syllable/pa/), neurophysiological changes exhibited by the LD-r group were more pronounced. The ANOVA revealed a significant group effect on N1 IHLD , monaural stimulation with non-speech and speech sounds produced a normal pattern of asynchrony and asymmetry over the temporal lobes in favour of the contralateral cortex. This result agrees with data in the literature regarding the cortical activation pattern of normal hearing subjects in response to monaural stimulation -3. Late-In unilaterally deaf subjects, the modifications of the activation timing over auditory areas depended on the side of deafness. In the RD-l group, stimulation of the healthy ear with non-speech and speech sounds produced a normal pattern of asynchrony over the auditory areas; asynchrony in favour of the contralateral cortex was even more pronounced (non significant) in both conditions compared with the NH-l control group. In contrast, stimulation of the healthy ear in the LD-r group produced an abnormal activation pattern characterized by synchrony over the auditory areas in response to non-speech sound and even a significant reversal pattern in favour of the ipsilateral cortex in response to speech sound by a significant shortening of the N1 ipsilateral latency. Although this was not the key result of their studies, the MEG data from Vasama and Makela  and FujiAlthough Khosla et al., using click stimuli delivered at an intensity level close to the one we used (70 dB HL), did not report any influence of the deafness side on late-AEP latencies, they showed clear differential ear effects when it came to late AEP amplitudes. Stimulating the healthy ear, they reported that inter-hemispheric amplitude differences of Root Mean Square, N1b-P2 and Ta-Tb complexes were the same in both controls and subjects with right-sided deafness; in contrast, subjects with left-sided deafness showed significantly reduced inter-hemispheric differences for all these parameters, i.e. a more symmetrical activation pattern over hemispheres . In our Overall, our profiles of neurophysiological responses elicited in subjects with RD did not differ from those of normal hearing subjects: a "normal" activation pattern over auditory areas may suggest a weak degree of cortical reorganization following RD. Conversely, important modifications of both timing and degree of activation in auditory areas were reported in subjects with LD: the more symmetrical activation pattern in response to pure tone and especially the reversal pattern in favour of the ispilateral hemisphere in response to speech sound may reflect a high potency of cortical reorganization following LD. These results are in agreement with those reported by Khosla et al.  who suggWhereas unilateral deafness is well known to significantly alter the perceptual performances of the intact ear in spatial sound localization -29 and sNumerous studies have reported cortical reorganization following unilateral deafness in both mammals and humans. However, the influence of the deafness side on auditory cortical plasticity remains unclear in humans. The results of the study show that cortical reorganization induced by unilateral deafness mainly occurs in subjects with left-sided deafness, consistent with previous data from Khosla et al. . MoreoveLC, HTV and EV conceived of the study, participated in its design and coordination and have been involved in drafting the manuscript. KA made substantial contributions to acquisition and analysis of data. JB helped substantially in the analysis and interpretation of data under ELAN software. JH, first author, was involved in acquisition, analysis and interpretation of data and wrote the manuscript


Mucosa associated lymphoid tissue (MALT) lymphoma is the third most common non-Hodgkin's lymphoma subtype. Clinical presentation is often insidious as a low-grade lesion and disease tends to remain localised for a long period of time. Ileal involvement is rare and presentation within an area of focal anti-mesenteric ileal wall dilation simulating a large diverticulum has not been reported.A 59-year-old man of Caucasian origin presented to a general surgical outpatients clinic with an 18-month history of intermittent upper abdominal pain following meals. Following normal gastroscopy and abdominal ultrasound, a focally dilated segment of ileum was seen on computed tomography and further clarified by barium investigation. Histology of this segment demonstrated MALT lymphoma of the small bowel.A solitary focally dilated segment of ileal wall may be neoplastic in nature and surgical resection needs to be considered. Mucosa associated lymphoid tissue (MALT) lymphoma otherwise known as extra-nodal marginal zone lymphoma was first described by Isaacson and Wright in 1983. The term MALT lymphoma was originally used to describe lymphoid tissue seen to histologically closely resemble that of the terminal ileum . The gasThis report describes the case of a 59-year-old Caucasian man presenting to a general surgical outpatients clinic with an 18-month history of intermittent upper abdominal pain following meals. Clinical examination, blood tests and an ultrasound examination were all unremarkable and no justification for symptoms was found at upper gastrointestinal endoscopy. A computed tomography (CT) scan of the abdomen following an acute admission with abdominal pain and vomiting demonstrated a 4.6 cm dilated segment of ileum containing both contrast and debris Figure . DiffereAt laparoscopy, a focally dilated anti-mesenteric segment of ileum was found 140 cm from the ileocaecal valve and displaying an abnormal serosa Figure . A segmeA thorough literature review has failed to identify a similar case of MALT lymphoma presenting within an area of focal anti-mesenteric dilation simulating a large diverticulum. Other differentials include MALT within a non-Meckel's diverticulum, ileal duplication or Meckel's diverticulum. Small bowel diverticula (non-Meckel's) most commonly arise in the duodenum and emerge on the mesenteric border where mesenteric vessels penetrate the bowel wall. Intestinal duplication is a rare condition which tends to present in early life and is closely associated with the mesenteric border. Isolated adult cases of ileal duplication, some with malignant change, have however been reported . IntestiMALT lymphoma of the GI tract often presents with non-specific symptoms. Additionally, traditional investigative techniques often miss the early stages of the diseases process making timely clinical diagnosis challenging. In patients with unexplained gastrointestinal symptoms, video capsule endoscopy may need to be considered as it is the diagnostic test with the highest yield for small bowel malignancies .A solitary focally dilated segment of ileal wall may be neoplastic in nature and surgical resection needs to be considered.CT: computed tomography; MALT: mucosa associated lymphoid tissueWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.RS, MG and IB were all involved in the drafting and re-editing of the manuscript. The final manuscript was read and approved by all three authors.


Different phylogenetic methods have been developed to predict secondary structures in RNA viruses, for high-resolution thermodynamic scanning and for detecting SSSV. These approaches have been successfully in predicting cis-acting signals in different members of the family Picornaviridae and Caliciviridae. In order to gain insight into the identification of cis-acting signals in viruses whose mechanisms of replication are currently unknown, we performed a phylogenetic analysis of complete genome sequences from 49 Human Norovirus (NoV) strains.Discrete RNA structures such as cre motif at positions 240 through 291 of the RNA dependant RNA polymerase (RdRp) sequence (relative to strain [EMBL:EU794713]). This sequence region has a high probability to conform a stem-loop.The complete coding sequences of NoV ORF1 were obtained from the DDBJ database and aligned. Shannon entropy calculations and RNAalifold consensus RNA structure prediction identified a discrete, conserved, invariant sequence region with a characteristic AAACG Hepatovirus cre stem-loop.A new predicted stem-loop has been identified near the 5' end of the RdRp of Human NoV genome. This is the same location recently reported for  The results of these studies are shown in Fig. cre sequence motif is embedded in a sequence region with a very high probability to conform a stem-loop structure cre sequeRNA structure predictions are consistent with previous analyses based on the thermodynamic folding of individual sequences ,17,18. ACaliciviridae, and they are non-enveloped viruses with positive, single-stranded RNA genomes. They also share other important features with picornaviruses, like having a VPg protein covalently linked to the 5' of the genomic RNA [NoV belong to the family omic RNA . Neverthomic RNA . NoV areomic RNA .cis-acting signals in the 5' and 3' regions as well as at the start of the sg RNA transcript of NoV [Despite the importance of these outbreaks, our understanding of the RNA structures or sequences required for NoV replication has been limited. Previous reports have identified the poly-pyrimidine tract-binding protein (PTB), poly-A binding protein (PAB) and La autoantigen to interact with the 3' untranslated region of the Norwalk virus genome . Very ret of NoV .Caliciviridae, NoV are thought to replicate in a manner typical of positive-stranded RNA viruses, through the synthesis of a full-length anti-genomic strand (reverse complement copy) using the viral RdRp translated initially from the RNA genome entering the cell [As a member of the family the cell . The minthe cell .in vitro due to the lack of a standard cell culture to grow these viruses, the probability that this predicted structure will acts as a functional element may open new avenues to our understanding of molecular mechanisms of NoV replication.Although the presence of this new putative cis-acting signal predicted in this study was not yet investigated Picornaviridae, like Poliovirus (PV) and Human Rhinovirus 14 (HRV-14), revealed a critical conserved AAACA/G cre sequence motif in the 5' half of the loop sequence that is essential for its function [cre elements of other picornaviruses and are important for RNA replication [Extensive mutagenesis studies performed in members of the family function . Similarlication ,24,25.cre act as the template for VPg uridylylation through a "slide-back" mechanism catalyzed by the 3Dpol (RdRp) [Paul and colleagues (2003) have shown that the PV l (RdRp) ,26,27. Tl (RdRp) .pol (RdRp) generates VPg-poly(U) and that this uridylylated VPg can prime the replication of polyadenylated RNA [de novo [de novo initiation of replication of antigenomic RNA [Interestingly, recent studies have shown that incubation of VPg with NoV 3Dated RNA . In cont[de novo . These fomic RNA . Besidesomic RNA .cre-like sequence motif with members of the family Picornaviridae [It is possible that the predicted stem-loop identified near the 5' end of the NoV RdRp coding region, which share a aviridae , will beaviridae .The authors declare that they have no competing interests.JC conceived the study. MV and JC designed, performed and revised the phylogenetic analysis. MV, RC, MPM and JPL contributed to the interpretation and discussion of the results found. JC wrote the manuscript. All authors read and approved the final manuscript.Origins of Norovirus strains. The table provides the strain names and accession numbers of their respective sequences of ORF1 of Norovirus strains included in this study.Click here for file


Extraskeletal Ewing's sarcoma is a rare soft tissue tumor that is morphologically indistinguishable from Ewing's sarcoma of bone. We report a case of extraskeletal Ewing's sarcoma with several systemic problems. A 69-year-old man presented with a 5-month history of a rapidly enlarging mass in the right thigh. Because preoperative radiotherapy with sanazole (AK-2123) contributed the tumor mass reduction down to 40% in size, the tumor was successfully resected with clear surgical margins and repaired with a musculocutaneous flap. The high efficacy of pre-operative low-dose radiotherapy with sanazole was histologically confirmed that the resected tumor specimen involved no viable tumor cells and showed 100% necrosis. Based on clinical outcomes in this case, the combined modality of pre-operative low-dose radiotherapy with hypoxic cell radiosensitizer and adequate surgical resection might provide for the useful clinical application of extraskeletal Ewing's sarcoma treatment. Extraskeletal Ewing's sarcoma (EES) is a rare type of Ewing's sarcoma that arises in the soft tissue at any location and is now regarded as a member of a family of small cell neoplasms of bone and soft tissue, including primitive neuroectodermal tumors (PNETs) , 2.MultiEfficacy of RT largely depends on tumor radiosensitivity and the tolerance of normal tissues. To improve therapeutic outcomes, RT is often combined with chemotherapeutic drugs that are themselves cytotoxic and/or radiosensitizing. Sanazole (AK-2123) as a radiosensitizer with irradiation in various tumors showed encouraging results –9. Here,A 69-year-old man presented at our hospital with a 5-month history of a rapidly enlarging mass in the right thigh. His past history was pulmonary tuberculosis treated with thoracoplasty and hepatitis C. Physical examination revealed the presence of a large irregular mass with multiple ulcers and necrotic tissues, measuring approximately 10 × 10 cm in the lateral side of the proximal thigh . AdmissiBased on clinical findings and imaging characteristics, the tumor was diagnosed as a primary soft tissue tumor and an incisional biopsy was performed. Histopathology of the permanent sections showed the nodular or sheets-like proliferation pattern of small round cells with a high nuclear/cytoplasmic ratio in the background of sparse extracellular collagen . Immunoh2  × 10 days), the tumor volume was reduced to approximately 40% of the pre-treatment condition (Considering patient's age, hepatic dysfunction, and patient's disagreement with chemotherapy, we precluded the choice of the neoadjuvant chemotherapy. To define surgical margins especially at subcutaneous tissues and to increase tumor resectability, we determined to treat the patient with pre-operative low-dose RT and additional administration of sanazole before surgery . As a reondition . On thalondition . The hugondition . There wondition . Based oEES is a tumor of unknown mesenchymal origin predominantly affecting men in the second and third decades of life , 11. TheDespite the role of RT in the management of bone sarcoma is limited, its primary application appears to be in Ewing's sarcoma . The impDespite the limitation of this case including its short follow-up period, our clinical outcomes in radiosensitive EES suggested the high efficacy of pre-operative low-dose RT with sanazole. Therefore, the combined modality of pre-operative low-dose RT with hypoxic cell radiosensitizer and adequate surgical resection might provide for the useful clinical application of EES treatment.


An 83-year-old man underwent electrophysiological testing for focal atrial tachycardia (AT) exhibiting narrow P waves with negative deflections in the inferior leads. Catheter ablation at the cavo-tricuspid isthmus (CTI) successfully eliminated the AT. The propagation map during AT and pacing study from the successful ablation site demonstrated that the atrial activation throughout the CTI did not produce significant P wave deflections. Consequently, during AT, the left atrial activation time determined the P wave duration. This case demonstrates that AT originating from the CTI may exhibit narrow P waves which can be misinterpreted as AT originating from the inter-atrial septum. Focal atrial tachycardias (ATs) originating from either the right atrium (RA) or left atrium (LA) have been proved to be good candidates for radiofrequency (RF) catheter ablation. Although electroanatomic mapping may be helpful for identifying the exact site of an AT origin, the P wave morphology is a useful clue during electrophysiological study because it can predict the site of the AT origin easily and non-invasively ,2. HowevAn 83-year-old man with coronary artery disease underwent an electrophysiological study (EPS) and catheter ablation for supraventricular tachycardia (SVT). Echocardiography revealed no evidence of structural heart disease. Written, informed consent was obtained, and the EPS was performed after all antiarrhythmic drugs had been discontinued for at least five half-lives prior to the study. At baseline, the SVT was incessant. During sinus rhythm the 12-lead electrocardiogram exhibited normal P waves. The SVT was a long R-P' tachycardia (cycle length = 520 ms) with narrow P waves that were negative in the inferior leads and biphasic (+/-) in lead V1 . MultipoIt is known that the sites of AT foci are usually associated with the specific anatomic locations ,2. In thAlgorithms using the P wave morphology to predict the site of the AT origin should be consistent with the anatomical considerations. In general, ATs with an origin in the atrial free wall should exhibit broader P waves than those with an origin in the inter-atrial septum (IAS) because activation from the AT origin in the IAS can propagate throughout the atrium faster than that from the AT origin in the atrial free wall. However, in this case P waves appeared to be narrower during AT and pacing from the site of the AT origin than during pacing from the CS ostium. A previous study demonstrated that during cavo-tricuspid isthmus dependent atrial flutter, the atrial activation which goes through the cavo-tricuspid isthmus represents the isoelectrical segment between the negative saw-tooth waves in the inferior leads while the atrial activation in the LA and IAS represents the negative saw-tooth waves in the inferior leads . In fact