Peg3, Snrpn and Lit1 genes associated with imprinted loci, and at two repetitive elements in the Line-1 and IAP-LTR genes in the various tissues. In this population of mice, methylation at several loci was sex-associated and intra-tissue correlations among the studied loci were observed for brain and spleen. Inter-tissue correlations were rarely observed. To investigate method-dependent experimental variability, we re-analyzed the same spleen and tongue samples using SIRPH and pyrosequencing methods and reconfirmed intra-tissue correlations for spleen and sex-associated correlations for DNA methylation for tongue. When we repeated DNA methylation measurements for a second mouse population raised under similar conditions three months later, we did not detect sex-associated or intra-tissues correlations. Additional studies that examine large numbers of loci may be required to further understand the factors that influence stability of DNA methylation.In humans, considerable variation in methylation at single loci and repetitive elements in various cells and tissues is observed. Recently, several inter- and intra-tissue correlations for DNA methylation have been reported. To investigate the extent and reproducibility of such correlations, we investigated inter- and intra-tissue methylation correlations among seven different loci in 9 different tissues in a population of 100 healthy seven-week-old CD1 outbred mice. We used a highly quantitative approach to measure methylation levels to high accuracy at two single loci in the alpha-actin and myosine light chain promoters, at three differentially methylated regions of the  DNA methylation plays an essential role in the regulation of gene expression. It modifies the 5-carbon position to yield 5-methylcytosine or 5-hydroxymethylcytosine Understanding the influence of different environmental conditions on the methylation levels in normal populations and for changes in normal environmental conditions is essential when considering DNA methylation as markers for detecting abnormal physiological states associated with disease. Furthermore, the presence of reliable and reproducible inter-tissue correlations for DNA methylation exclusively associated with healthy or disease conditions might allow the use of methylation, for example in blood cells, as surrogate markers for other less accessible tissues. However, only sparse data on intra- and inter-tissue correlations for DNA methylation exists.IGF2R, APOC1, LEP and CRHWe recently reported some correlations in DNA methylation between adenocarcinomas tumor tissue and peripheral blood cells For one population of mice and a limited number of CpG sites in multiple tissues, our data suggest that sexes differ in methylation levels at several loci and in many tissues. Moreover, intra-tissue correlations were not widely seen and were mainly limited to spleen, brain and bone marrow in our study. Inter-tissue correlations were rarely detected. However, sex-associated differences and intra-tissue correlations for DNA methylation were not found for a separate mouse population. Our data suggest that sex-associated differences and inter- as well as intra-tissue correlations might be affected by several factors including variability in experimental procedures, stochastic variation of epigenetic markers or by unknown environmental factors. Accordingly, our results suggest that more populations and genetic loci will need to be included in future studies to determine if DNA methylation correlations can form the basis of surrogate disease marker diagnosis methods.Mice were raised and sacrificed according to institutional, national and international guidelines. Animals were raised and sacrificed without any special clinical treatment or invasive procedure. The sacrifice of animals for scientific studies without any clinical handling (prior to killing) is not part of the German animal protection law (paragraphs §8b and §9). This procedure lies within the responsibility of the local animal protection officer at the Institute of Experimental Therapy  where the animals were raised. Therefore, no ethics approval was received, as this was not necessary.2, organs were isolated within minutes and snap frozen in liquid nitrogen before storing at −80°C for later use.Two populations of outbreed CD1 mice, each consisting of 100 mice , were included in this study. All mice were about seven weeks old when sacrificed. The two populations were sacrificed roughly three months apart. From the first population, complete organs including brain, skin (ear lobe), tongue muscle, lungs, heart, liver, spleen, kidneys, bone, bone marrow, skeletal muscle, and testis were isolated. From the second population, brain, lung, spleen and tongue muscle were isolated. After the animals were sacrificed by asphyxiation with COPeg 3 , Snrpn   and Lit1 (long intronic transript 1), which are all methylated on the maternal allele and paternally expressed. The third group included repetitive elements that were amplified using degenerate primers targeting high CpG-density regions within the Line-1 (long interspersed element 1) and IAP-LTR  sequences. We studied two CpG sites within each region in the first mouse population, while only one CpG was studied in the second population.As targets for the methylation analysis we selected three groups of loci. The first group included two single loci with low CpG density: the cytoskeletal α-actin and myosin light chain genes. Both are expressed to different degrees in skeletal, smooth and cardiac muscles. The second group of loci included three genes with differentially methylated regions (DMR) that have high density of CpGs and associated with imprinted genes 260nm/A280nm ratio was >1.8 for all DNA used.The extraction of DNA was performed using the DNeasy blood and tissue kit . Prior to the extraction procedure, the manufacturer-recommended amount of starting tissue was homogenized in a 1.5 ml Eppendorf tube using a single-use plastic hand-held mortar by grinding until no tissue particles were visible. For more mechanically resistant tissues, like bone and cardiac muscle, an electric homogenizer was used. To access the quality and quantity of extracted DNA, concentration of the DNA samples was measured by spectrophotometer and aliquots were loaded on 1% agarose gel to ensure presence of high molecular weight DNA. The Aet al. Peg 3, Lit1, Line-1 and IAP-LTR were screened according to Hajkova et al. Snrpn was screened according to El-Maarri et al. Bisulfite treatment of the DNA was performed according to the EpiTect bisulfite conversion protocol using 1 µg of DNA . Primers used in this study are listed in et al. Site-specific methylation measurements at two selected CpG sites in every region were carried out using the SIRPH protocol as previously described by El-Maarri Analyses were performed using the SAS 9.2 package  or the GraphPad Prism version 5. Normal distribution of the data was tested by D’Agostino & Pearson omnibus normality test. Significance of the sex-associated difference in methylation at each locus was calculated by t-test and non-parametric Mann-Whitney test. Pearson and Spearman correlations were performed on the different groups of samples to determine the significance and degree of correlations. All P values were corrected for multiple testing using the Bonferroni method. The significance difference between the levels of methylation at a given locus in different tissues was calculated by the non-parametric Krustal-Wallis test. The power calculation and estimated needed sample size was performed using GPower v.3; the effect size was calculated based on the values in the first population of mice Myc. The least methylation was measured for α-actin, while imprinted loci exhibited a measured range of 40–60% as expected. Very low methylation levels were observed at the three paternal imprinted genes in testis since male gametes are not methylated at these differentially methylated regions (DMRs).Each CpG of the seven studied loci was studied in 8 or 9 different tissues. The tissue specificity of the methylation is evident for each locus . In facti.e., the tissue is not homogeneous with respect to degree of cellular differentiation). We also noted that the variance of methylation level for α-actin was relatively greater for testis than for all other studied somatic tissues. The degree of variability showed no obvious correlation to a given tissue or to a specific locus. Both sexes showed similar variability at a given locus. This is illustrated by the fact that the locus/tissue combination with the greatest and the least variance in methylation values is the same in sexes. The lowest was at Line-1 in skin, while the highest was at α-actin in brain (excluding testis here) (The accurate quantitative methylation measurement provided by the SIRPH reaction (precision of 1–2%) allowed us to study variability at unprecedented high precision at each locus across individuals. All loci showed variable methylation levels in different tissues with variance values ranging from as low as 0.49 for Line-1 in skin to as high as 59.35 for IAP-LTR in muscle in males, and from 0.58% for Line-1 in skin to 90.15 in alpha actin in brain in females . Of noteis here) .For the first mouse population, we analyzed two CpG sites at every region. The detail of the locations and distances between the two studied CpGs are shown in For the first mouse population, a sex-association comparison of the methylation values at all loci in all tissues allowed 56 comparisons with 26 of them exhibiting statistical significance . We noteMYC, PEG3, LIT1, SNRPN, IAP and L1, but was not reproducible for the single non-imprinted loci probably due to the smaller measured differences between sexes. The comparison between the first SIRPH and pyrosequencing methods, however, exhibited good reproducibility at CpG-1 for α-actin, PEG3, and SNRPN. When considering the average of two CpGs in the first SIRPH measurement and the average of all studied CpGs by the pyrosequencing method, we found good reproducibility for α-actin and SNRPN. No comparision was possible for PEG3 and L1 because SIRPH CpG2 data was missing. In summary, a high degree of reproducibility was found between the two SIRPH measurements, but to a less extent between SIRPH and pyrosequencing methods. The later result is due to the intrinsic differences between the two methods and the fact that pyrosequencing includes additional experimental steps that require the isolation of single stranded DNA and, thus, is more prone to artifacts than the SIRPH method. The SIRPH method is more sensitive and has higher resolution for small differences in DNA methylation than pyrosequencing and thus may not be effective for measuring small differences in methylation.To establish confidence for the above results, we reanalyzed all seven loci for the same tongue tissue again using the same SIRPH procedure at both CpG-1 and CpG-2 and using an alternative method based on the pyrosequencing methylation assay Lit1 (muscle)- Lit1 (heart) in females at CpG-1, α-actin (spleen)- α-actin (bone marrow) in males at CpG-1 and SNRPN (lung) and PEG3 (Skin) in Females at CpG-1  did not reveal the same intra-spleen correlations except for the L1-SNRPN correlations in males  and IAP-From the above comparisons, we can also conclude that the correlations between single CpGs, although reproducible when the same measurement method is used, loose significance if methylation of a larger number of CpGs are averaged. This highlights the necessity to consider such data at single CpG resolution when highly accurate methylation measurement methods are used such as SIRPH.Next, we used another population of 50 males and 50 females mice to analyze just four tissues, including brain, lung, spleen and tongue, using the SIRPH method. In these four tissues, nine loci showed significant differences between sexes in the first mouse population. In addition, spleen was the only tissue with five observed intra-tissue correlations. However, in the second population, only α-actin in lung was significantly higher methylated in females (data not shown), although this was not the case for the first population. Moreover, no statistically significant intra-tissue correlations were observed for the second population. The sample size needed to replicate the first finding regarding the sex-associated influence on DNA methylation was adequate based on the previously observed effect size observed in for the first population .e.g., brain tissue) and a locus in an accessible tissue , a diagnostic test could be performed on the correlated accessible tissue to establish the condition of the less accessible tissue. Yet, extensive research is still required to identify and establish inter-tissue correlations for site-specific DNA methylation between both healthy physiological and pathological conditions. In this study, we take a first step in presenting results of an investigation in which we analyzed a small sample of seven genetic loci in nine different tissues in a large population of mice. We have also reanalyzed the same loci, for the tissues that showed the most statistically significant correlations for site-specific DNA methylation in the first population, in a second, equal-sized mouse population. However, if such correlations exist, we were not able to detect them with certainty, possibly due to natural epigenetic variability or due to the effects of unknown environmental factors affecting DNA methylation that we were unaware of and, therefore, could not hold constant for both sample populations of mice. Thus, future studies would likely benefit from including inter-tissue DNA methylation data for more populations to extend our results presented here.Examples of highly accurate quantitative assays for methylation analysis are the widely used pyrosequencing-based method and our previously described SIRPH method Previous reports form several groups, including ours, have demonstrated that in human leukocytes In accordance with this, we saw no systematic sex-associated bias for all tissues. The sex-associated differences observed for tongue are remarkable; where males are significantly more methylated at most loci . ReanalyLit-1 between muscle and heart and between SNRPN in lung and PEG3 in skin) were confirmed  and two inter-tissue correlations in females , and spleen tissues (in both sexes) . The facThe differences in the sex-associated effects and the intra-tissue correlations, between the two mouse populations, clearly indicates variability in methylation levels affecting sex-associated influence and intra-tissue correlations. We suggest three possibilities, of which any one or a combination of the three could explain these variations.The first possibility is the experimental variability as several factors or artifacts have previously been reported to affect bisulfite based quantitative methylation measurements. DNA extraction and bias in the PCR towards either methylated or unmethylated DNA strands were previously considered et al. also concluded that increasing the methyl donors in the diet increased the number of loci that exhibit variability of methylation Arabidopsis thaliana−4 per CG site per generation The second possibility is the presence of stochastic variation in dynamic methylation in living cells. Variability in methylation in mice has been shown to occur in studied liver and brain tissues and these relatively hypervariable methylation sites were overrepresented in genes related to development, morphogenesis and organogenesis 2 inhalation during sacrifice of the mice. This can be associated with activation of several responses in the body of the mouse including activation of anaerobic metabolism resulting in a decrease in alpha-ketoglutarate. The latter is an essential cofactor, together with Fe++, for TET enzymes  transforming 5-methyl-cytosine to 5-hydroxymethyl-cytosine into 5-formylcytosine and into 5-carboxylcytosine in sequential oxidations steps 2  and resulting variability in the TET enzyme activities that leads to variability in demethylation among different individual mice could be a possible mechanism underlying the differences in intra-tissue correlations. This hypothesis can be directly tested in future studies.The third possibility explaining the presence of correlations and the much more pronounced sex-associated differences in one population of mice, but not in the other, might be differences in environmental conditions. Presumably, such differences over time could impose pressure to select variations evolving in a more uniform direction at specific loci. This would result in detectable intra-tissue correlations as observed for spleen tissue in one of our cohorts. Both mouse populations were housed at the same facility under similar, standardized conditions, but at two different points in time separated by nearly four months. Although we are not aware of any major environmental differences in the rearing of the two populations, we cannot exclude that these may have substantially contributed to the DNA methylation differences we observed. For example, the first population was raised in August/September, while the second was raised in December/January. Variation in methylation due to uncontrolled seasonal variables, for example, could contribute to the interpopulation differences we found. Another possible environmental variable could stem from COWhile we do not have experimental evidence for any of the above possibilities, our data nevertheless clearly demonstrate that methylation levels are not uniform. Instead, they show considerable variability possibly linking environmental conditions with the genome, thus, possibly providing an adaptation mechanism to a given environmental condition. Our results for a very small number of CpGs at seven loci suggest that at least some intra-tissue correlations for locus-specific DNA methylation can be detected using high sensitivity methods as described, and that there is likely an absence of widespread inter-tissue correlations in healthy mouse populations. However, the correlations found in another population of healthy mice were not the same. Therefore, until further studies investigate intra-and inter-tissue correlations in site-specific DNA methylation, results from single sample populations should be interpreted with caution. Accordingly, we recommend verification of correlations for DNA methylation including at least one independent sample population before considering this correlation as indicative of any specific associated condition. An important aspect of our study that has not previously been included in other studies of intra- and inter-tissue correlations of DNA methylation is the focus on comparison of results between large (n = 100) populations, However, limitations of this study include comparison of results for only two sample populations and the relatively small number of genetic loci studied. Future studies that investigate larger numbers of loci and larger number of sample populations for both healthy and pathological population models conditions will be required to establish the extent of correlated DNA methylation sites with an aim towards developing either direct or indirect, surrogate tissue disease-specific diagnostic screening methods.Figure S1Details of the studied regions. Both wild type (upper panel for each region) and bisulfite sequences (lower panel for each region) are shown. The amplification forward and reverse primers as well as the SNuPE SIRPH primers are labeled with yellow and red respectively. The studied CpG sites in every region are labeled in blue. The pyrosequencing primers correspond to SNuPE primer 1. The CpGs covered by the pyrosequencing are highlighted in red.(PDF)Click here for additional data file.Figure S2Methylation correlation between CpG-1 and CpG-2 for all regions and all tissues of the first population of mice (SIRPH experiments). Heatmaps of both Pearson (right) and Spearman (left) correlation values are shown in part A. In the lower part B the detailed values together with the p value for every correlation are given.(PDF)Click here for additional data file.Figure S3Spearman correlation between the first and the second SIRPH experiment in spleen tissue of the first population of mice for males (A) and females (B). Heatmap of all possible intra-tissue correlations are shown in the upper part Click here for additional data file.Figure S4Spearman correlation between the first SIRPH and the pyrosequencing of the spleen tissue in the first population of mice. A) data for males and B) female data. Heatmap of all possible intra-tissue correlations are shown in the upper part  and blue  boxes correspond to the position of the significant correlations that was detected by the first SIRPH experiment in the first population of mice as shown in (PDF)Click here for additional data file.Table S1List of amplification primers, SIRPH oligos and pyrosequencing primers used in this study.(XLS)Click here for additional data file.Table S2Detailed methylation data for both populations of mice at the first CpG site (SIRPH data). Average (Ave.), median, highest and lowest values, 25% percentile (Q1) and 75% percentile (Q3), standard deviation (SD), variance (Var.) and differences between sexes together with uncorrected and corrected p values of the t-test are given in this table. The values of skewness (Skew.) and kurtosis (Kur.), K2 and P values of the D’Agostino and Pearson omnibus normality test for estimation of normal distribution are also given. Conclusion of the normal distribution test is given in the ND column. The Z-power, the % power and the sample size required to replicate the results are also given. The Blue highlight corresponds to the statistically significant male-female difference after Bonferoni corrections.(XLS)Click here for additional data file.Table S3p values of male-female differences by t-test or the Mann-Whitney test. Blue highlight correspond to the significant differences detected by t-test at CpG-1 in the first mice cohort. Both tests are 100% concordant for the significance.(XLS)Click here for additional data file.Table S4Statistical significance of sex effect on methylation in first cohort tongue tissue. Results of two SIRPH and one pyrosequencing measurements are shown.(XLS)Click here for additional data file.


Introduction. Paraplegia and kyphotic deformity are two major disease-related problems of spinal tuberculosis, especially in the early age disease. In this study a 2-year-old boy who underwent surgical decompression, correction, and 360° instrumented fusion via simultaneous anterior-posterior technique for Pott's disease was reported. Case Report. A 2-year-and-9-month-old boy presented with severe back pain and paraparesis of one-month duration. Thoracic magnetic resonance imaging demonstrated destruction with a large paraspinal abscess involving T5-T6-T7 levels, compressing the spinal cord. The paraspinal abscess drained and three-level corpectomy was performed at T5-6-7 with transthoracic approach. Anterior instrumentation and fusion was performed with structural 1 autogenous fibula and rib graft using screw-rod system. In prone position pedicle screws were inserted at T4 and T8 levels and rods were placed. Six months after surgery, there was no weakness or paraparesis and no correction loss at the end of follow-up period. Discussion. In cases of vertebral osteomyelitis with severe anterior column destruction in the very early child ages the use of anterior structural grafts and instrumentation in combination with posterior instrumentation is safe and effective in maintenance of the correction achieved and allows efficient stabilization and early mobilization. Spinal tuberculosis, the most common pattern of extra pulmonary tuberculosis, also has increased rapidly around the world in recent years, especially in the undeveloped and developing countries . ParapleA 2-year-and-9-month-old boy was admitted to our department with back pain that had developed over a 1-month period. Neurological examination revealed a paraparesis, which was evaluated as grade C according to Frankel Scoring System. Roentgenograms of chest and thoracic spine showed destruction at the T5-T6-T7 levels and the local kyphosis angle was 37° at this level. Thoracic magnetic resonance imaging (MRI) demonstrated a large paraspinal abscess involving these three levels, extending to the spinal canal and compressing the spinal cord . There wThe surgery was performed under general anesthesia with the transthoracic approach after removing the fifth rib in right lateral decubit position. After exposure, the paraspinal abscess drained spontaneously. Because of the bone destruction and cord compression, three-level corpectomy was performed at T5-6-7. Sequestered bone fragments of affected vertebrae were removed through corpectomy. Decompression was carried out until duramater was visible through anterior approach, so all affected vertebrae were extracted. The deformity was flexible; the reduction of kyphosis performed until physiological angels and anterior fusion was performed with structural 1 autogenous fibula and 1 autogenous rib graft. Periost of the fibula was prevented and closed anatomically after resection. Anterior instrumentation with one screw to each vertebra at T4 and T8 was performed to increase the primer stability . The sysMicrobiological examination of tissue and abscess samples obtained during operation was Mycobacterium Tuberculosis. No complication of bleeding persisted and the drainage tube was taken after 72 hours. One week after surgery, the patient could sit on the bed or walk around under the effective support of thoracolumbosacral orthoses, and this orthoses support was maintained for 3 months. The patient received antituberculosis chemotherapy for 9 months . 6 weeks after surgery, neurological status of the patient was Frankel E and there was no weakness or paresthesia which was presented before the surgery. Kyphosis angle showed a correction of a 4° (89.2%) in the postoperative period and there was no correction loss at the end of follow-up period.Tuberculosis of the spine is an unstable lesion that tends to progress until having bony fusion at the spine. Bone fusion of the vertebrae is thought to be the most reliable evidence of healing of spinal tuberculosis . To achiThe aims of spinal tuberculosis treatment are to eradicate the disease, to prevent the development of paraplegia and kyphotic deformity, to manage the existing deformity and neurological deficit, to allow early ambulation, and to return the patient back to daily life , 18–21.There are many options for surgical treatment of spinal tuberculosis including anterior debridement and arthrodesis , 23, plaIn the literature, there are few reports about surgical treatment of spinal tuberculosis in early child ages. To our knowledge this is the first report that shows the surgical treatment which is applied with anterior debridement, anterior instrumentation with structural autogenously fibula, and rib graft and posterior transpedicular instrumentation in early childhood.In conclusion, in cases of vertebral osteomyelitis with severe anterior column destruction in the early child ages the use of anterior structural grafts and instrumentation in combination with posterior instrumentation is safe and effective in maintenance of the correction achieved and allows efficient stabilization and early mobilization. Further follow-up and series with more account of patients are necessary to confirm these early results.


Distributing a multiple computer-tailored smoking cessation intervention through the Internet has several advantages for both provider and receiver. Most important, a large audience of smokers can be reached while a highly individualized and personal form of feedback can be maintained. However, such a smoking cessation program has yet to be developed and implemented in the Netherlands.To investigate the effects of a Web-based multiple computer-tailored smoking cessation program on smoking cessation outcomes in a sample of Dutch adult smokers.Smokers were recruited from December 2009 to June 2010 by advertising our study in the mass media and on the Internet. Those interested and motivated to quit smoking within 6 months (N = 1123) were randomly assigned to either the experimental (n = 552) or control group (n = 571). Respondents in the experimental group received the fully automated Web-based smoking cessation program, while respondents in the control group received no intervention. After 6 weeks and after 6 months, we assessed the effect of the intervention on self-reported 24-hour point prevalence abstinence, 7-day point prevalence abstinence, and prolonged abstinence using logistic regression analyses.Of the 1123 respondents, 449 (40.0%) completed the 6-week follow-up questionnaire and 291 (25.9%) completed the 6-month follow-up questionnaire. We used a negative scenario to replace missing values. That is, we considered respondents lost to follow-up to still be smoking. The computer-tailored program appeared to have significantly increased 24-hour point prevalence abstinence , 7-day point prevalence abstinence , and prolonged abstinence  rates reported after 6 weeks. After 6 months, however, no intervention effects could be identified. Results from complete-case analyses were similar.The results presented suggest that the Web-based computer-tailored smoking cessation program had a significant effect on abstinence reported after a 6-week period. At the 6-month follow-up, however, no intervention effects could be identified. This might be explained by the replacement of missing values on the primary outcome measures due to attrition using a negative scenario. While results were similar when using a less conservative scenario , the results should still be interpreted with caution. Further research should aim at identifying strategies that will prevent high attrition in the first place and, subsequently, to identify the best strategies for dealing with missing data when studies have high attrition rates.Dutch Trial Register NTR1351; http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=1351 (Archived by WebCite at http://www.webcitation.org/67egSTWrz) Worldwide, the smoking of tobacco is the most preventable cause of illness and premature death . TherefoThe Internet has been discovered to be a popular gateway for delivering health behavior change interventions in general  and compAlthough a key element of computer tailoring is that the intervention materials are adapted to specific respondent characteristics, some smokers might benefit more than others from particular smoking cessation interventions. For example, the level of nicotine dependence has previously been suggested to moderate the effectiveness of smoking cessation interventions . MoreoveAs Web-based multiple tailored smoking cessation feedback has not yet been offered to the Dutch general public outside scientific studies, our research group developed a Web-based multiple computer-tailored smoking cessation program and offered Dutch adult smokers the opportunity to participate in this program. The present study investigated the effectiveness of this program on smoking cessation outcomes reported after 6 weeks and 6 months. To imitate a natural situation in which smokers who do not participate in a smoking cessation program do not receive the intervention, the control group did not receive any of the intervention’s components. Nevertheless, both the intervention and control group were free to use other smoking cessation aids during the study period. In addition, we investigated whether the effect of the intervention was different for specific subgroups of smokers and whether we could detect a dose–response relationship between the number of feedback messages received and abstinence at the last follow-up.The Web-based multiple computer-tailored smoking cessation program was based on a previously developed effective single computer-tailored intervention , while t All respondents in the experimental condition received at least one tailored feedback letter . At the 6-week follow-up, respondents could have received at most two tailored feedback letters , and at the 6-month follow-up, they could have received a maximum of three tailored feedback letters .This study was approved by the Medical Ethics Committee of Maastricht University and the University Hospital Maastricht , and is registered with the Dutch Trial Register (NTR1351). A full description of the study protocol is provided elsewhere .We recruited adult smokers from December 2009 to June 2010 by advertising our study in the mass media and on the Internet. We sent several press releases to regional newspapers in the Netherlands. Most of these newspapers subsequently mentioned our study on their website, included an item about the project in the print version of their newspaper, or mentioned our study on their local radio station or television channel, or both. We also used a Dutch online social network website (Hyves) and several online smoking cessation forums to disseminate our recruitment text. In addition, we advertised our study in a free national newspaper obtainable at all Dutch train stations and several other public places throughout the Netherlands.After 12 months, we expected a 10% point prevalence abstinence rate in the control condition. Based on results from previous projects, we expected the multiple tailoring program to lead to a 20% point prevalence abstinence rate. To be able to detect this difference significantly , according to a 2-tailed Fisher exact test, 281 respondents per arm were required at the end of the trial  [Interested smokers could sign up for the study on the study website (http://www.persoonlijkstopadvies.nl) and were eligible to participate if they were 18 years of age or older, were motivated to quit smoking within 6 months, and had access to the Internet. On the study website, participants were informed that the study was financed by the Dutch Cancer Society and conducted by researchers from Maastricht University in cooperation with the Dutch Expert Center on Tobacco Control (STIVORO). Additionally, the website included information about the objectives of the study, the randomization procedure and the incentive provided when respondents completed all questionnaires . Respondents could choose their own username and password and were informed that no one but the research team was able to retrieve these passwords. As respondents had to report their email address when signing up for the study, we could easily flag respondents with multiple identities and remove them from further analyses. After providing online informed consent, participants were randomly assigned to the intervention group or the control group by a computer software randomization device, allocating approximately 50% of all respondents to each group. Blinding of respondents was not possible, as they had to take notice of whether they were receiving tailored feedback.All questionnaires used in the present study were previously used and tested among Dutch smoking adults and were self-administered online ,28,29.We measured six demographic variables: age, gender , educational level , nationality , and the occurrence of cardiovascular and respiratory diseases .Exclusion criteria were based on current smoking behavior and motivation to quit smoking: current smoking behavior was measured by 1 item asking whether the respondent had smoked during the past 7 days . Motivation to quit smoking was measured by an adapted version of the Stage of Change algorithm . We askeWe measured overall tobacco consumption using five open-ended questions regarding the number of cigarettes, hand-rolled cigarettes, cigars, cigarillos, and pipes smoked per day. Subsequently, the answers on these five questions were converted into an overall score for tobacco consumption (expressed as number of cigarettes), whereby 1 hand-rolled cigarette or cigarillo equaled 1 cigarette and 1 cigar equaled 4 cigarettes . As no cWe measured addiction level by the abbreviated Fagerström Test for Nicotine Dependence  .We assessed the number of past quit attempts with 1 item, asking the respondents how often they had tried to quit smoking in the past.At the 6-week and 6-month follow-ups, we assessed prolonged abstinence by 1 item asking whether the respondent had refrained from smoking since the previous measurement . At the 6-week follow-up, prolonged abstinence referred to abstinence since the questionnaire that respondents received 2 days after their set quit date . At the 6-month follow-up, this measure referred to abstinence since the 6-week follow-up . In addition, at both follow-ups we assessed 24-hour and 7-day point prevalence abstinence, each by 1 item asking whether the respondent had refrained from smoking during the past 24 hours or 7 days .t tests and chi-square tests. Additionally, to determine whether selective dropout had occurred, we compared those remaining in the study versus those lost to follow-up after 6 weeks and 6 months using 2-sided t tests and chi-square tests.First, we conducted descriptive analyses to determine the sample’s characteristics. To check for differences between the intervention and control groups, we conducted 2-sided Second, we conducted logistic regression analyses to determine whether the intervention had an effect on the outcome measures assessed after follow-up periods of 6 weeks and 6 months. A negative scenario was used to replace missing values. That is, respondents lost to follow-up were considered to still be smoking. To test the robustness of the results, these analyses were also conducted with complete cases only.Third, to determine whether the effect of the intervention was different for specific subgroups of smokers, we investigated whether we could identify interaction effects between the study condition and baseline demographic or behavioral measures using logistic regression analyses.P < .05.Data were analyzed using SPSS 17.0 . The significance level used was 22 = 6.11, P = .047). Therefore, educational level was included in subsequent analyses as a potential confounder. As no data concerning level of education were missing for any of the respondents, we included all 1123 in further analyses. Respondents included in the analyses had a mean age of 49.5 years; 535 (47.6%) were male; and 513 (45.7%) had a medium level of education. Respondents in the experimental group significantly differed from those in the control condition in their level of education (χP = .02) and significantly more addicted (P = .01) than those who remained in the study.As Of the 552 respondents in the intervention group, 91 (17%) reported that they had refrained from smoking during the past 24 hours, 74 (13%) reported that they had not smoked during the past 7 days, and 60 (11%) reported that they had not smoked since the previous measurement 2 days after their quit date. In the control group (n = 571) these numbers were 55 (10%), 38 (7%), and 33 (6%), respectively. The intervention had a significant effect on all outcome measures, even when controlling for the baseline difference between the intervention and control groups with regard to their level of education . SignifiAfter 6 months, a total of 51 (9%) respondents in the intervention group reported having refrained from smoking during the past 24 hours, 45 (8%) reported not having smoked during the past 7 days, and 23 (4%) reported not having smoked since the previous measurement. In the control group these numbers were 36 (6%), 34 (6%), and 19 (3%), respectively. We investigated interaction effects between condition and baseline demographic or behavioral measures, although none of these turned out to have a significant influence on any of the abstinence measures reported after 6 weeks or 6 months (data not reported).In the present study we investigated the effects of a multiple computer-tailored smoking cessation program delivered through the Internet. The results presented suggest significant effects of the intervention on short-term abstinence: at the 6-week follow-up, respondents who received the intervention were more likely to report being abstinent for the past 24 hours, for the past 7 days, and since the previous measurement  than those who did not receive the intervention. Despite incorporating goal and relapse prevention strategies (action and coping plans), however, we found no effect of the intervention on abstinence measures assessed after 6 months.A potential explanation for not finding any suggestion of intervention effects on long-term abstinence might be that more than 70% of the values on the primary outcome measures had to be replaced, as our study had relatively high levels of attrition, as have many previously developed Web-based interventions ,33-37. IAnother possible explanation for the lack of intervention effects on long-term abstinence may be that Web-based smoking cessation programs are not sufficiently tailored and adapted to the long-term wishes of recent ex-smokers to prevent relapse to smoking. Respondents received feedback only at fixed points in time; it was not possible to obtain additional personal feedback or support at times when smokers might have needed it most. The integration of ecological momentary assessment, by collecting real-time data through, for example, palmtops, personal digital assistants, or electronic diaries, might be promising. Studies using palmtop computers showed that a decrease in self-efficacy, an increase in positive smoking outcome expectancies, and an increase in negative affect predicted the occurrence of a lapse to smoking on the next day ,43. InteWe found no support for different intervention effects for specific subgroups of smokers. Based on the results, it could thus be argued that the intervention was equally effective for all smokers who participated in the program. However, respondents who dropped out of the study were relatively more addicted and relatively younger than those who remained in the study, which is in line with previous research ,50. A poMajor strengths of the present study were the large sample of smokers who initiated participation in the smoking cessation program and the relatively long follow-up period. However, as mentioned previously, the study had relatively high dropout rates. In the present study, we applied several strategies previously suggested to prevent attrition ,39: usinThis Web-based computer-tailored smoking cessation program had a significant effect on abstinence measured after a 6-week follow-up period. However, this effect had entirely disappeared after 6 months. To prevent relapse, future studies should focus on the possibility of applying an ecological momentary assessment or combining the present Web-based intervention with the use of smoking cessation medication. Moreover, further research should aim at identifying strategies to prevent smokers from dropping out of Web-based smoking cessation interventions. As complete-case analyses and the replacement of missing values using a negative scenario both have their limitations, alternative strategies should be identified and tested.


Insect pests are responsible for human suffering and financial losses worldwide. New and environmentally safe insecticides are urgently needed to cope with these serious problems. Resistance to current insecticides has resulted in a resurgence of insect pests, and growing concerns about insecticide toxicity to humans discourage the use of insecticides for pest control. The small market for insecticides has hampered insecticide development; however, advances in genomics and structural genomics offer new opportunities to develop insecticides that are less dependent on the insecticide market. This review summarizes the literature data that support the hypothesis that an insect-specific cysteine residue located at the opening of the acetylcholinesterase active site is a promising target site for developing new insecticides with reduced off-target toxicity and low propensity for insect resistance. These data are used to discuss the differences between targeting the insect-specific cysteine residue and targeting the ubiquitous catalytic serine residue of acetylcholinesterase from the perspective of reducing off-target toxicity and insect resistance. Also discussed is the prospect of developing cysteine-targeting anticholinesterases as effective and environmentally safe insecticides for control of disease vectors, crop damage, and residential insect pests within the financial confines of the present insecticide market. Soybean aphids (Aphis glycines) cost U.S. farmers more than US$1 billion in yield losses and insecticide purchase and application. Bed bugs, which suck human blood and cause acute discomfort, mental stress, and social stigma, are now common in many residential areas. Insect resistance to current insecticides and growing concerns about insecticide toxicity to humans have contributed to a resurgence of insect pests.New and environmentally safe insecticides are urgently needed worldwide to contend with the problems of disease-carrying, crop-destroying, and residential insect pests. For example, African malaria mosquitoes  active site and the hypothesis that this residue is a novel target site for insecticide development. With these data, we discuss the differences between targeting the insect-specific cysteine residue and targeting the ubiquitous catalytic serine residue of AChE from the perspective of reducing off-target toxicity and insect resistance. We also discuss the prospect of developing cysteine-targeting anticholinesterases as effective and environmentally safe insecticides for controlling disease transmission, crop damage, and residential pests within the confines of the current insecticide market. 22.1Culex pipiens L.) transmit St. Louis encephalitis , the electric ray [Torpedo californica], the African malaria mosquito, the northern house mosquito, the greenbug, the cotton aphid, the green peach aphid, and the honeybee) identified a cysteine residue, Cys289 in the greenbug AP-AChE, that is absent in human AChE but conserved in insect and lancelet AChEs  fragment of AMTS13 to the insect-specific cysteine residue of AP-AChE. The time course and reactivation studies show that the slow, partial, and irreversible inhibition of human AChE by short- and medium-chain AMTS7–AMTS16 at low concentrations or by long-chain AMTS17–AMTS20 at high concentrations is caused by different inhibition mechanisms. Collectively, the results of the reactivation studies using aphid and mosquito samples demonstrate that the inhibition of greenbug or mosquito AChEs by 5.2AMTS13 appeared oracular and initially led to incorrect speculation about a reaction of AMTS13 with the catalytic serine residue of AChE [Although the reported slow, partial, and irreversible inhibition of human AChE by  of AChE , further of AChE . AMTS13 is initiated by a reversible interaction between the ammonium group of AMTS13 and Trp84 placing the methanethiosulfonate group at the rim of the active site. This positioning puts the reactive group close to the insect-specific cysteine residue and facilitates a rapid conjugation of the inhibitor with the mosquito AP-AChE. This disulfide bond formation mechanism is primarily supported by the reactivation of the AMTS13-inhibited insect AChE by 2-mercaptoethanol and data from the crystal structure of AMTS13 in complex with mouse AChE.The fast, nearly complete, and irreversible inhibition of African malaria mosquito AChE by 6 µM AMTS13 is caused by partial denaturation of the enzyme induced by AMTS13 and/or micelles of AMTS13. Evidence of this mechanism comes primarily from far-UV circular dichroism spectra that show a reduction of secondary structures in recombinant mouse AChE treated with AMTS13 for 45 hours as well as from atomic force microscopy results indicating that AMTS13 has a high propensity for micelle formation. This tendency is consistent with the report that alkyltrimethylammonium halides with 12–16 methylenes—in particular dodecyl-trimethylammonium halides—are effective surfactants that can denature α-lactalbumin or β-lactoglobulin [AMTS13 is analogous to that of dodecyl-trimethyl-ammonium halides. The slow, partial, and irreversible inhibition of human AChE by 6 µM globulin , 78. Alt66.1AMTSn together with the reactivation of AMTSn-inhibited AChEs by 2-mercaptoethanol unequivocally show that the insect-specific cysteine residue is, as shown in independent 3D models of the AP-AChEs, indeed accessible for conjugation with sulfhydryl agents [Despite the complication of surfactant-like inhibitors causing slow, partial, and irreversible inhibition of human AChE and a report that iodoacetamide-containing AChE inhibitors do not bond covalently to African malaria mosquito AP-AChE , the fasl agents , 76. In AMTS18 irreversibly inactivated >80% of the total AChE activity in greenbug and mosquito extracts [only AO-AChE [AMTS18. It is therefore conceivable from the reported data that inhibition of AP-AChE can deplete total insect AChE activity in greenbug and mosquitoes. This means that selectively inhibiting AP-AChE is at least theoretically adequate to incapacitate insects with cholinergic nerves.At this point, it is fair to question whether selectively inhibiting AP-AChE but not AO-AChE can deplete total insect AChE activity and lead to insect incapacitation. Consistent with the reported preponderance of AP-AChE over AO-AChE , 37, 51,extracts , 76. Thi AO-AChE —the idenN-ethylmaleimide, for conjugation with Florida lancelet AP-AChE and (2) an active-site phenylalanine residue that hampers the reactions of these two reagents with the insect-specific cysteine residue. Neither 5,5′-dithiobis(2-nitrobenzoic acid) nor N-ethylmaleimide is designed for insect AP-AChEs, and their reaction rates with the lancelet enzyme and the steric hindrance experienced in the lancelet enzyme appear to be irrelevant to cysteine-targeting insecticides. What is relevant is the reaction rates of sulfhydryl agents tailored for insect AP-AChEs. As shown in Fig. (9A), 6 µM AMTS13 causes rapid irreversible inhibition of African malaria mosquito AChE achieving 50% irreversible inhibition within 10 minutes. This fast conjugation with insect enzymes demonstrated by a primitive cysteine-targeting inhibitor indicates that sufficient rates for the reaction of sulfhydryl agents with the insect-specific cysteine residue should not be a theoretical or practical concern.To support the cysteine-targeting hypothesis further, it is necessary to address the reported reservations about targeting the insect-specific cysteine residue . If the 6.2Mechanistically, targeting cysteine and serine residues uses the same chemical approach—irreversibly modifying an active-site residue leading to physical blockage of the active site and subsequent inactivation of the enzyme catalysis. In addition, the two approaches share the same enzyme. These commonalities can lead to the incorrect perception that targeting the cysteine residue of AP-AChE is the same as targeting the serine residue of AChE and that cysteine-targeting insecticides are as same as current anticholinesterase insecticides. It is worthy of noting the fundamental differences between these two approaches, however. First, cysteine is chemically more reactive than serine. This increased reactivity means that the cysteine-targeting chemicals can be made less reactive and should, therefore, have less off-target toxicity than serine-targeting chemicals such as organophosphates or carbamates. This disparity in chemical reactivity also means that cysteine-targeting chemicals are structurally different from those that target the serine residue, which magnifies their difference from current serine-targeting insecticides because cysteine-targeting insecticides will, at least initially, face no insect resistance conferred by the established metabolic detoxification.Second, the locations of the two residues in the AChE active site are different, meaning that resistance to current serine-targeting insecticides conferred by mutations in the active site of AChE—for example, the Gly119Ser mutant of African malaria mosquito AP-AChE (GenBank ID: AJ515149 )—will noDespite some commonalities with serine-targeting insecticides, cysteine-targeting insecticides have lower off-target toxicity and decreased likelihood for insect resistance than serine-targeting insecticides owing to their relative chemical reactivity and the location of the insect-specific cysteine residue.6.3Given the discovery process of the insect-specific cysteine residue and its prospects as a new target for insecticide development, it may be tempting to use genomics and structural genomics to discover insecticide targets other than AChE. One should not overlook at the current fact that limited financial resources are available for insecticide development, however. Although new insecticide targets can be discovered using genomic and structural genomic studies that are now low-cost and “off-the-shelf” commodities, the expenses involved in developing a new chemical entity as an effective and environmentally safe insecticide can be formidably high and potentially impractical. Conversely, AChE is a well-studied enzyme and abundant literature details the development of effective AChE inhibitors, some of which are used currently as insecticides or others as clinical drugs for treating Alzheimer’s disease. Developing insecticides that target a different residue in AChE should be less costly than developing chemicals that target proteins that are not as well studied as AChE. For the financial and scientific reasons described in Sections 2–5, we advocate the pursuit of generic cysteine-targeting insecticides for effective and broad control of disease vectors and crop and residential insect pests.Presently no reports on cysteine-targeting chemicals that effectively incapacitate insects without measurable mammalian toxicity have been published, partly because much of the current research effort is still directed toward proof of concept. Additionally, unlike current anticholinesterase insecticides that are low-hanging fruits stemming from World War II research on nerve gas, cysteine-targeting chemicals are high-hanging fruits for the following reasons. First, most known alkylating agents or electrophiles cannot be used to target the cysteine residue because these agents, such as Michael acceptors , can cauIt has been recognized that honeybees and silkworms also have AP-AChEs with the insect-specific cysteine residue . This faATMSn, do not exceed 8-fold [Fortunately, honeybees and silkworms do not share habitat with mosquitoes or residential pests. A generic cysteine-targeting insecticide has to be developed first, and the deployment of this type of insecticide must be carefully managed to avoid toxicity to honeybees, silkworms, and other beneficial insects. A recent study shows that the observed differences in inhibitory potency by the prototypic irreversible inhibitor, d 8-fold . This re7Unlike mammals, most disease-transmitting, crop-damaging, and residential insects have AO- and AP-AChEs, each of which has an insect-specific cysteine residue. The cysteine of AP-AChE, located at the entrance to the active site, is accessible for rapid conjugation with sulfhydryl agents and represents a new opportunity to develop cysteine-targeting insecticides. Because cysteine is more reactive than serine—with which current anticholinesterase insecticides are designed to react—cysteine-targeting insecticides have lower off-target toxicity and a lower propensity for insect resistance than serine-targeting insecticides. Although the development of cysteine-targeting insecticides is not easy and the small insecticide market places financial constraints on research and development, the abundant literature on AChE inhibitor design and the cost-effectiveness of the development process make the insect-specific cysteine residue a viable target for developing effective and environmentally safe insecticides to control disease vectors, crop damage, and residential insect pests. Asian tiger mosquito , bed bug , booklice , human body louse , human head louse , oriental tobacco budworm , southern house mosquito , springtail , soybean aphid , striped riceborer , and whitefly .The cysteine-containing AP-AChE list now includes those of


Palivizumab has been shown to decrease the incidence of hospitalization due to respiratory syncytial virus (RSV) in infants at risk of severe RSV disease. We examined the association between compliance with palivizumab dosing throughout the RSV season and risk of RSV-related hospitalization in clinical practice.Subjects who were born and discharged from the hospital before the RSV season and received ≥1 palivizumab dose during their first RSV season were identified from a large US commercial health insurance database between 01/01/03 and 12/31/09. Subjects were deemed compliant if they received ≥5 palivizumab doses without gaps (>35 days) and their first dose was received by November 30. RSV-related hospitalizations were identified using ICD-9-CM diagnosis codes and examined over 2 observation periods: post-index dose and RSV season. A Cox proportional hazard model was used to evaluate the association between non-compliance and RSV-related hospitalization.Of the 5,003 subjects who received palivizumab, 62% were deemed non-compliant. Non-compliant subjects had significantly higher unadjusted rates of RSV-related hospitalizations compared to compliant subjects during both observation periods . In multivariate analyses, non-compliance was significantly associated with higher risk of RSV-related hospitalization . Of the 225 RSV-related hospitalizations observed during the RSV season, 61 (27%) occurred before the first dose of palivizumab.Subjects who did not receive monthly dosing of palivizumab throughout the RSV season had significantly higher rates of RSV-related hospitalizations. The RSV-related hospitalizations prior to the first dose of palivizumab suggest some dosing was started too late. Respiratory syncytial virus (RSV) is recognized as the leading cause of serious lower respiratory tract disease in infants and children -6. CondiPalivizumab is a humanized murine monoclonal antibody that is approved by the US Food and Drug Administration (FDA) for the prevention of severe respiratory tract disease caused by RSV in children with BPD, infants with a history of premature birth (≤35 wGA), and children with hemodynamically significant CHD. The FDA-approved dose is 15 milligrams per kilogram administered via intramuscular injection every 28–30 days throughout RSV season. Randomized controlled clinical trials of palivizumab versus placebo have shown a reduction in the incidence of hospitalization due to severe RSV disease by approximately 40%-80% in high-risk premature infants and certain children with CLD or CHD ,10. The The key objective of this analysis was to examine the association between compliance with palivizumab and risk of RSV-related hospitalization among commercially insured infants.The study used the Optum Research database, which contains de-identified medical and pharmacy claims for a geographically diverse and representative US privately insured population. Enrollees’ claims covered the period from May 01, 2003 through April 30, 2009. All data were accessed using HIPAA -complianCommercially insured members with medical and pharmacy benefits who received at least 1 dose of palivizumab  between October and April 2003–2009 were eligible for inclusion. Eligible subjects were in their first year of life with continuous enrollment in the health plan from their date of birth through April 30 of the following year. Inpatient claims do not capture hospital-administered drugs including doses of palivizumab that would be provided at discharge to eligible infants during the RSV season. To ensure the ability to clearly identify all doses, infants born during the RSV season (October 1 – April 30) were excluded from the study. Claims were analyzed from birth through April 30 of the following year.The date of the first palivizumab claim after October 1 was defined as the index date. The pre-index period was defined as the date of birth to the index date. We defined two observation periods for RSV outcomes. The first was the post-index period, defined as the index date through April 30 of the following year. The second was the RSV season, defined as October 1 through April 30 of the following year  diagnosis code 765.2x. Subjects with CLD were identified through medical claims with a diagnosis code for CLD and either a pharmacy claim for CLD medications (bronchodilators and/or diuretics), or a claim for oxygen use during the pre-index period. Subjects with CHD were identified through a medical claim with a diagnosis code for CHD and a pharmacy claim for a CHD medication during the pre-index period. High-risk categories were not mutually exclusive groups.The total number of palivizumab doses received during the RSV season was recorded for each subject along with the calendar month of the index dose and the average and maximum number of days between each dose received. Subjects were deemed compliant if they received ≥5 doses of palivizumab with no gaps (>35 days) between doses, and they received their index dose by November 30.The major outcome of interest was RSV-related hospitalizations, identified using RSV-specific diagnosis codes  and RSV-like diagnosis codes defined as unspecified bronchitis (466.0), bronchiolitis (466.19), viral pneumonia (480.9), bronchopneumonia (485), and pneumonia (486) that occurred during the RSV season -14. RSV-Pre-index clinical characteristics and healthcare utilization were identified to control for potential confounding variables. To account for variable pre-index and post-index periods, all healthcare utilization was calculated per subject per month.All study variables were analyzed descriptively. Numbers and percentages are provided for categorical variables. Means, medians, and standard deviations are provided for continuous variables. RSV-related hospitalizations for the post-index period were calculated as rates per 100 infant seasons to adjust for the variable length of follow-up time for each individual. Infant seasons were defined as the number of days in the follow-up (end of the season – date of first in-season injection +1) divided by 212 (the number of days in the season). Comparisons were made using t-tests for continuous variables and chi-squared tests for categorical variables.Multivariate analysis of RSV-related hospitalizations in the post-index period was conducted using a Cox proportional hazard regression. Parametric regression survival models were based on maximum likelihood estimation. Covariates were entered into the model in variable blocks; if one variable from the block was included, then all variables in the block were included. The first model examined only non-compliance vs. hospitalization. Chronic lung disease, CHD, and gestational age were added to subsequent models. All variables in the final model were tested for collinearity to ensure the accuracy of the parameter estimates. The results of Cox proportional hazard regression are presented as hazard ratios associated with each independent variable.To understand the stability of the results, the final model was run in population subsets to examine whether the direction or magnitude of the relationship between non-compliance and RSV-related hospitalizations changed in specific population cohorts. The subsets examined were: subjects with CHD, subjects with CLD, subjects whose index dose was received in either October or November, subjects without a pre-index palivizumab dose, subjects categorized by RSV season , and subjects categorized by birth month.A total of 5,003 subjects met study inclusion criteria. Over half 57.1%) were single births, with slightly more males (54.7%) than females (Table % were siApproximately two-thirds (66.7%) of the total sample had low birth weight (<2500 g), and a third 36.1%) had at least 1 prespecified comorbidity. The most common comorbidities were retinopathy of prematurity (24.1%), intraventricular hemorrhage (7.9%), and congenital airway anomalies (3.8%)  number of days between doses was 31.6 (8.5). Eighty-seven percent of the index palivizumab doses were given in October (58.7%) or November 28.2%)  and had low birth weight . There was no significant difference in the proportion of patients with at least 1 pre-existing comorbidity (p = 0.603). While uncommon, a significantly higher proportion of non-compliant subjects had a diagnosis of immunodeficiency state , while a significantly higher proportion of compliant subjects had a diagnosis of cystic fibrosis , 164 subjects (3.3%) had at least 1 RSV-related hospitalization during the post-index period. Non-compliant subjects had significantly higher unadjusted rates of RSV-related hospitalizations  had at least 1 RSV-related hospitalization during the entire RSV season. Non-compliant subjects also had significantly higher unadjusted rates of RSV-related hospitalizations . This difference was seen for both RSV and RSV-like hospitalizations  or during the RSV season .To control for collinearity in the model, a correlation matrix was created to examine the relationship between all of the covariates. There was a weak correlation between low birth weight and unknown gestational age, as well as weak associations between NICU stays and multiple births. No other significant associations were noted; therefore, no covariates were excluded from the model due to collinearity.After controlling for potential confounders, the Cox proportional hazard model demonstrated that non-compliance was significantly associated with a higher risk of RSV-related hospitalization .The aim of this study was to examine the association between compliance with palivizumab and RSV-related hospitalizations in commercially insured infants. In our study, non-compliant subjects had higher unadjusted rates of RSV-related hospitalizations than compliant infants. The multivariate analysis found that, after adjusting for potential confounding factors, non-compliant subjects were significantly more likely to have an RSV-related hospitalization than compliant subjects. While our analysis cannot determine causality, it identifies an association that should be further explored.This is one of the largest published studies in a commercially insured population to examine the association between compliance with palivizumab and RSV-related hospitalizations. Previous studies have examined the predictors of compliance with palivizumab, RSV-related hospitalization rates among high-risk infants, and costs and resource use associated with RSV-related hospitalization and/or palivizumab -22. HoweIn a retrospective claims analysis, Diehl et al.  examinedDespite a large number of subjects receiving 4 or more doses, our study showed significant differences in RSV-related hospitalization rates between compliance groups. On average, subjects had 1.5 dosing gaps (time between doses >35 days) with a mean gap length of 9.4 days or 44.4 days since the previous dose, suggesting that dosing gaps do not need to occur often or be lengthy to be associated with an increased risk of RSV-related hospitalization. Although a relatively small time period, gaps of approximately 15 days would be expected to lead to significantly lower serum levels of palivizumab . ResultsRSV-related hospitalizations prior to the index palivizumab dose occurred in both groups, with significantly more hospitalizations occurring before the index dose in the non-compliant group. The timing of the RSV season varies considerably by region , and theInterpretation of our study results must take into account the limitations of the study design and the use of healthcare claims. Administrative claims data are collected for payment purposes and not research; therefore, the degree to which claims accurately describe an individual’s medical history is limited. Claims data also do not contain information on behavioral or social risk factors. Information regarding the subjects’ race, ZIP code level data, and the parents’ socioeconomic status was also unavailable. The association between compliance and RSV-related hospitalization may be confounded by risk factors that we cannot account for in our model.We relied on diagnosis codes to determine high-risk cohorts. For gestational age, the ICD-9-CM codes are defined in 2-week segments . We used a combination of diagnosis codes and medication use to define CLD. CLD is broadly defined as any pulmonary condition resulting from a neonatal respiratory disorder. BPD is a form of CLD, but not all CLD is BPD. CLD is often used interchangeably with BPD. Therefore, some of the identified high-risk cohorts may include infants outside of the labeled indication for palivizumab .Given hospital coding practices, palivizumab doses administered during a hospitalization were not captured. Therefore, infants born during the RSV season, who are often at highest risk of serious RSV disease, were excluded from the study. Omission of this group may limit the ability to generalize study results to all infants receiving prophylaxis.RSV is often not tested for during the season; therefore, claims may result in a non-specific ICD-9 diagnosis code. A recent analysis  found thOur main analysis focused on RSV outcomes that occurred after the first palivizumab dose, defined as the post-index period. In this observation period, there may be differences in RSV exposure time between the compliant and non-compliant cohorts. For example, compliant infants could have the number of days at risk inflated due to adding days early in the season with a lower exposure risk. While non-compliant infants have fewer days at risk, they could have a higher percentage of days in the “peak” RSV period. One of the limitations to using administrative insurance claims is the inability to determine when RSV began circulating in the communities of the subjects included in our analysis. In order to address the potential issues related to the variable exposure/follow-up, we also looked at RSV hospitalizations for a uniform observation period (October 1 to April 30). In both analyses, we saw a statistically significant increased risk/odds of RSV-related hospitalizations associated with non-compliance. Future analyses examining the association between compliance and RSV hospitalization may want to consider collecting data on RSV circulation to adjust for exposure.In the full RSV season analysis, over a quarter of all RSV-related hospitalizations occurred prior to the first dose of palivizumab. Starting dosing late contributes to non-compliance, so we felt it was important to include hospitalizations that occurred prior to initiation of palivizumab prophylaxis in the analysis. However, hospitalizations prior to the first dose of palivizumab do not tell us the impact of partial prophylaxis on RSV hospitalizations once dosing has been started. For this reason, we chose the post-index observation period as our primary analysis. In the post-index period, we only examined RSV-related hospitalizations that occurred after infants had received their first dose of palivizumab and excluded hospitalizations (n = 61) that occurred prior to the first dose. We did not censor or exclude infants who had an RSV hospitalization prior to the first dose of palivizumab as children can become infected and hospitalized with RSV multiple times in one season. In both the full season and post-index period, we saw a statistically significant increased risk/odds of RSV-related hospitalizations associated with non-compliance.Finally, our data pertain to a commercially insured population. Therefore, the results are primarily applicable to commercially insured infants and may not be generalizable to other populations such as Medicaid or uninsured infants.To our knowledge, this is one of the largest studies in a commercially insured population to examine the association between palivizumab compliance and RSV-related hospitalizations in a clinical setting. In our study, non-compliant subjects had higher unadjusted rates of RSV-related hospitalization than compliant subjects and non-compliance was associated with a significantly higher risk of RSV-related hospitalization after adjusting for potential confounders.The association between compliance and RSV-related hospitalization and the occurrence of hospitalizations prior to the first palivizumab dose suggest the importance of getting a first dose early in the RSV season and monthly dosing throughout the season. Future analyses should consider other predictors of compliance such as behavioral and social risk factors when examining the association between compliance and RSV-related hospitalization.JS, BP, and LB are employees of Optum; KR is an employee of MedImmune and has received stock/stock options from MedImmune and AstraZeneca. DS and MF have served as consultants to MedImmune and have participated as members of MedImmune advisory boards. DS is on the speakers’ bureau for MedImmune. The authors have indicated that they have no other conflict of interest with regard to the content of this article.DS and MF collaborated in the design the study, interpretation of results, and drafting and review of the manuscript. KR conceived of the study, collaborated in the design of the study and interpretation of the results, participated in drafting and review of the manuscript. LB developed the programming and conducted the statistical analysis. BP collaborated in the design of the study, directed the analysis, participated in interpretation of the results, and helpt to draft the manuscript. JS participated in drafting of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/334/prepub


T-cells play an important role in the immune response and are activated in response to the presentation of antigens bound to major histocompatibility complex (MHC) molecules participating with the T-cell receptor (TCR). T-cell receptor complexes also contain four CD3 (cluster of differentiation 3) subunits. The TCR-CD3 complex is vital for T-cell development and plays an important role in intervening cell recognition events. Since microRNAs (miRNAs) are highly stable in blood serum, some of which may target CD3 molecules, they could serve as good biomarkers for early cancer detection. The aim of this study was to see whether there is a relationship between cancers and the amount of miRNAs -targeted CD3 molecules.Bioinformatics tools were used in order to predict the miRNA targets for these genes. Subsequently, these highly conserved miRNAs were evaluated to see if they are implicated in various kinds of cancers. Consequently, human disease databases were used. According to the latest research, this study attempted to investigate the possible down- or upregulation of miRNAs cancer patients.We identified miRNAs which target genes producing CD3 subunit molecules. The most conserved miRNAs were identified for the CD3G gene, while CD247 and CD3EAP genes had the least number and there were no conserved miRNA associated with the CD3D gene. Some of these miRNAs were found to be responsible for different cancers, following a certain pattern.It is highly likely that miRNAs affect the CD3 molecules, impairing the immune system, recognizing and destroying cancer tumor; hence, they can be used as suitable biomarkers in distinguishing cancer in the very early stages of its development. More than 30% of the human genome is controlled by miRNAs, regulating important biological processes There is strong evidence that has verified the effectiveness of miRNAs in the immune system. Accordingly, miRNAs can affect the development of B cells Since, the thymus is the principal organ responsible for T cell maturation The immune system can recognize and omit tumors which express antigens that are not observed in normal cells. So such antigens are considered as foreign by the immune system, which ultimately attacks and destroys them The TCR heterodimer is responsible for ligand recognition. When it assembles with four invariant proteins , it holds a characteristic sequence motif associated with the immunoreceptor tyrosine-based activation motifs (ITAMs), which are important for signal transduction in immune cells. The TCR_CD3 complex interaction plays an important role in intervening cell recognition events The key role of miRNAs in the immune system has been implicated by scientists; for example, miR-150 affects some mRNAs which are important for development of pre- and pro-B cells. So every change in these miRNAs may obstruct the development of the immune system miRNAs target mRNAs at specific sites to cause translational repression and inhibit translation or induce cleavage of the massage In accordance with the stability of miRNA in serum and the importance of the role of CD3 molecules in recognition of antigens, this study attempted to focus on the miRNAs that target CD3 proteins which could be ultimately used as reasonable biomarkers in the detection of cancers.http://asia.ensembl.org/Homo_sapiens/Info/Index/) was used http://www.targetscan.org/(updated -june 2012)) There are four CD3 subunits including CD3-Gamma, CD3-Delta, CD3-Epsilon and CD3-Zeta that bind to the TCR, leading to the formation of the TCR.CD3 complex, which plays an important role in T cell receptor function and T cell development http://202.38.126.151/hmdd/mirna/md/(September 2012)) http://mircancer.ecu.edu/browse.jsp (January 2012)) Subsequently, every miRNA of the conserved microRNA family has been investigated separately. In order to find out in which type of cancer, these miRNAs play an important role, we used the Human miRNA & Disease Database(HMDD)  was used Each of the predicted miRNAs were then reviewed in related research papers to see if these miRNAs were down-regulated or up-regulated in tumor tissues and the blood serum of cancer patients.The results showed that has-miR-378, which was a miRNA predicted for CD3G, had increased in all blood sera belonging to patients suffering from castration resistant prostate cancer (CRPC) The only miRNA predicted for targeting the CD3EAP gene, involved in CD3E expression, was hsa-miR-138. Studies showed that levels of this miRNA had decreased in tumour tissues of patients suffering from colorectal cancer Studies on the predicted miRNAs targeting CD247, which were involved in the expression of the CD3-zeta molecule, showed that there were three miRNAs which targeted the gene. Except for hsa-miR-214, no reports have been cited so far regarding the levels of miR-761 and miR-3619-5p in tumor tissues. Most of the data obtained in this study showed that miR-214 levels decreased in tumor tissues when compared to its relative normal tissue . miR-214miRNAs can act as tumor oncogenes and supressors and negatively regulate gene expression Since miR-378, miR-422a, miR-593, miR-494, miR-138 and miR-214 could target the CD3 subunits, some of which have been studied in different cancers and have been considered as biomarkers to detect cancer at the early stages, it is then highly likely that miRNAs damage the immune system so that it cannot distinguish cancer cells. Hence, their presence in the blood serum as biomarkers for early diagnosis of cancer could help us to understand the disability of immune system. Although this study looks bioinformatically reliable, more laboratory studies are needed to support these findings.


Multiplex experimental assays coupled to computational predictions are being increasingly employed for the simultaneous analysis of many specimens at the genome scale, which quickly generates very large amounts of data. However, inferring valuable biological information from the comparisons of very large genomic datasets still represents an enormous challenge.As a study model, we chose the NFI/CTF family of mammalian transcription factors and we compared the results obtained from a genome-wide study of its binding sites with chromatin structure assays, gene expression microarray data, and in silico binding site predictions. We found that NFI/CTF family members preferentially bind their DNA target sites when they are located around transcription start sites when compared to control datasets generated from the random subsampling of the complete set of NFI binding sites. NFI proteins preferably associate with the upstream regions of genes that are highly expressed and that are enriched in active chromatin modifications such as H3K4me3 and H3K36me3. We postulate that this is a causal association and that NFI proteins mainly act as activators of transcription. This was documented for one member of the family (NFI-C), which revealed as a more potent gene activator than repressor in global gene expression analysis. Interestingly, we also discovered the association of NFI with the tri-methylation of lysine 9 of histone H3, a chromatin marker previously associated with the protection against silencing of telomeric genes by NFI.Taken together, we illustrate approaches that can be taken to analyze large genomic data, and provide evidence that NFI family members may act in conjunction with specific chromatin modifications to activate gene expression. The size of the control dataset can then be chosen to parallel that of the experimental set, to simplify the statistical analysis [High-throughput assays are being widely employed in various fields of biology. For example in genomics, DNA microarrays are used to simultaneously measure the expression levels of nearly all genes of a genome ,2. Recenanalysis ,15.in vitro, the specificity of this family of proteins may be different from that observed in the cell, where interactions with other transcription factors may take place and DNA accessibility may be restrained by chromatin. Here we assessed the in vivo binding preferences of NFI/CTF, its global functional properties regarding the regulation of gene expression and the relationship of NFI binding sites with different histone methylation markers typical of either an open or closed chromatin structure.As a study group of proteins, we chose NFI/CTF family of mammalian transcription factors. NFI/CTF represents a family of transcription-replication factors comprising polypeptides encoded by four paralogous genes located on different chromosomes in mammals  ,17. NFI Statistical analysis in genomics often relies on the sub-sampling of datasets, which requires random sampling algorithms. We devised a random sampling algorithm that can be conveniently applied to large genomic datasets. The random sampling algorithm C++ source code is available as a text file online  and 5 kb of upstream sequences.We first used this random sampling method to compare data from a ChIP-Seq experiment performed on primary mouse embryonic fibroblasts for the NFI transcription factor family relative to the in silico predictions of its binding sites. The mouse genome (NCBI build 37 or mm9) was found to contain a set of 61,492 NFI predicted sites that were defined using a previously established position weight matrix ,28. The in vivo binding site occupancy was estimated using the average ChIP-Seq tag count covering each subset of NFI predicted sites. When tag counts occurring at TSS-proximal binding sites were compared to those that do not occur near known TSS, a higher tag counts was noted at the predicted sites, but also in regions extending several kb away from the known binding sites . Thus, we conclude that NFI preferentially occupies promoters or upstream regulatory regions of genes that exhibit high expression levels in the cell.Next, we assessed whether genes that bind NFI may have some common features regarding their gene expression levels. We selected 39,807 roblasts . Again, To test whether the preferential binding of NFI to upstream regions of expressed genes may be associated with specific chromatin modifications, we used ChIP-Seq data for 4 different histone H3 methylations, as previously obtained from mouse embryonic fibroblasts , to be awt-Eko) were determined for each gene within defined groups and plotted as an absolute value. The group of genes activated by NFI-C showed the highest median ΔE value, implicating this factor as a potent gene activator , the group of NFI-C suppressed genes had levels of expression that did not differ significantly from the control subgroups , again indicating that NFI-C acts as a moderate inhibitor of gene expression . These results suggest that NFI-C member of the NFI family acts globally as an effective gene activator.The preferential association of NFI with expressed genes prompted us to test whether NFI family members may have the potential to activate gene expression, or whether their binding is rather the consequence of histone modifications and chromatin accessibility at promoters. For this purpose, we used gene expression microarray data from mouse embryonic fibroblasts in which one of the NFI factors (termed NFI-C) was knocked out by insertional mutagenesis . We focur Figure . NFI-C sn Figure . OverallIn this study, we used a random sampling procedure as a general method to obtain reliable control datasets in the analysis of high-throughput genomic assays. We find that datasets of more than 100 individual values can be used without decreasing the robustness of statistical analysis, and that independently generated random subsets of data have statistically indistinguishable global properties. Thus, subsampling can provide a convenient way to display and compare the noise and signals from experimental and control datasets of the same size.First, we showed that NFI binds preferentially those predicted sites that are located upstream of the initiation sites of transcription Figure . SeveralWe also found that NFI occupy promoters or upstream regions of the group of genes that are significantly more expressed than the representative randomly selected control groups. Since correlation does not necessarily imply causal relationship, this observation does not allow the conclusion that NFI-family members actually activate the expression of these genes. For instance, NFI might bind highly expressed genes to suppress in part their expression, but still leaving relatively high transcription levels. However, taken together with previous observations that NFI activates the expression of many genes in higher eukaryotes ,32,34-37in vivo NFI binding sites are located up to 5 kb from their TSS, which is a relatively large distance, NFI might act as well through some of the types of remote regulation, for instance by the establishment of a chromatin domain boundary that would prevent the propagation of a silencing chromatin structure towards the promoter [The hypothesis that NFI family members may directly activate genes appears to be true for at least one of the member of the family (i.e. NFI-C), as mRNA profiling analysis performed on wild-type and NFI-C knock-out cells revealed that NFI-C is a more potent gene activator than a repressor. The 1000 genes that are most up-regulated by NFI-C had significantly higher change in their expression levels than the top 1000 down-regulated genes. In addition, up-regulated genes showed significantly higher expression levels than representative control gene samples selected from the total gene population, implicating again that this factor is a potent activator of gene expression. Since the selected promoter ,38.Histone H3 methylations such as the H3K4me3 and H3K36me3 modifications were found to be enriched around the TSS of NFI-occupied genes when compared with control gene groups. This finding is consistent with the model that NFI acts predominantly as an activator of transcription, since H3K4me3 and H3K36me3, but not H3K27me3, were proposed as markers of active gene transcription . This in2, DMEM , Supplementary 10% FBS , 1% v/v nonessential amino-acids , 1% v/v L-glutamine .Mouse embryonic fibroblasts (MEF) were extracted from mouse embryos of 14.5 days. Cells from WT (wild-type) and NFI-C knock-out embryos were cultured under the following conditions: 37°C, 5% COChromatin was extracted from approximately 20,000,000 primary mouse embryonic fibroblasts grown in culture and cross-linked using 11% formaldehyde. Extracted chromatin was fragmented to the average fragment size of 1000 bp using high-frequency sound sonication on VibraCell-75455 (Bioblock Scientific). ChIP was performed as described before  using thChIP DNA was processed using the contents of the ChIP-Seq Sample Prep Kit (Illumina). Size range of templates was selected by loading the entire processed sample on a 2% agarose gel and excising the gel region of 50-400 bp. PCR amplification of the gel-extracted DNA was performed for 18 cycles using the adapter-specific primers. Each sample was loaded into 3 separate flow cell channels of the Illumina Cluster Station and then subjected to sequencing-by-synthesis on the Illumina Genome Analyzer sequencing system. For each of the samples, sequence reads from independent channels were pooled together.In vivo NFI sites were defined using the ChIP-peak tool and applying the following parameters: window width - 300 bp, vicinity range - 300 bp, peak threshold - 5 tags, count cut-off - 1 tag, repeat filtering-on. Correlation analyses were made using ChIP-cor tool and applying the following parameters: window width - 50 bp, count cut-off - 1, normalization - global. Galaxy tools were used to operate on the genomic intervals [Clustering and correlation analyses of mapped reads were performed using ChIP-peak and ChIP-cor tools available on the ChIP-Seq Analysis Server ,41. In vntervals ,43.Random sampling algorithm was written in C++ language and compiled in Microsoft Visual Studio as detailed in the Additional file ChIP-Seq data for the wild type and NFI-C knock-out mouse embryonic fibroblasts were deposited at the Gene Expression Omnibus (GEO) repository under the accession number GSE15844. Gene expression microarray data for the wild type and NFI-C knock-out mouse embryonic fibroblasts were taken from the GEO repository under the accession number GSE15871. ChIP-Seq data for histone methylations in mouse embryonic fibroblasts  were taken from GEO repository under the accession number GSE12241.NFI: Nuclear Factor I; NFI-C: Nuclear Factor I - C; CTF: CAAT box transcription factors; ChIP: Chromatin immuno-precipitation; ChIP-Seq: Chromatin immuno-precipitation sequencing; MEFs: Mouse embryonic fibroblasts; TSS: Transcriptional start site; TFs: Transcription factors; NFI-C KO: Nuclear Factor I - C knock out; WT: Wild typeMP conceived the study, carried out the ChIP-Seq experiments, participated in the data analysis and drafted the manuscript. PP developed the random sampling algorithm and participated in the data analysis. CS, GA and PB developed ChIP-Seq analysis tools and participated in the data analysis. AG, GP made substantial contributions to the data collection and interpretation of data. CM, PB and NM participated in the design of the study, made substantial contributions to the interpretation of data, provided essential data revisions, and helped to draft the manuscript. All authors have read and approved the final manuscript..Click here for file.Click here for file


However, when generated from viral populations current mapping software is inadequate for dealing with the high levels of variation present, resulting in the potential for biased data loss. In order to apply the 454 Life Sciences' pyrosequencing system to the study of viral populations, we have developed software for the processing of highly variable sequence data. Here we demonstrate our software by analyzing two temporally sampled HIV-1 intra-patient datasets from a clinical study of maraviroc. This drug binds the CCR5 coreceptor, thus preventing HIV-1 infection of the cell. The objective is to determine viral tropism (CCR5 versus CXCR4 usage) and track the evolution of minority CXCR4-using variants that may limit the response to a maraviroc-containing treatment regimen. Five time points (two prior to treatment) were available from each patient. We first quantify the effects of divergence on initial read k-mer mapping and demonstrate the importance of utilizing population-specific template sequences in relation to the analysis of next-generation sequence data. Then, in conjunction with coreceptor prediction algorithms that infer HIV tropism, our software was used to quantify the viral population structure pre- and post-treatment. In both cases, low frequency CXCR4-using variants (2.5–15%) were detected prior to treatment. Following phylogenetic inference, these variants were observed to exist as distinct lineages that were maintained through time. Our analysis, thus confirms the role of pre-existing CXCR4-using virus in the emergence of maraviroc-insensitive HIV. The software will have utility for the study of intra-host viral diversity and evolution of other fast evolving viruses, and is available from  Due to high data volumes, error rates, and short sequence lengths, new sequencing technologies present a new challenge for computational biology. In addition, high-depth (or ultra-deep) datasets, for example from pathogens, contain exceptionally large amounts of variation over short genomes or genomic regions. Here we present software for the processing and downstream analysis of such short-read viral sequence data. We apply the software to the analysis of two HIV-1 infected individuals who did not respond optimally to the drug maraviroc. For each patient, pyrosequence data was available for five time points. In both cases we detect distinct clusters of low-frequency drug-insensitive variants that were present prior to maraviroc treatment and effectively unmasked by the removal of the drug-sensitive HIV. Sequencing platforms, such as the 454 Life Sciences' GS-FLX pyrosequencing system, has greatly parallelized the determination of nucleotide order within genetic material, resulting in the ability to produce extremely large datasets envTo date, software has focused on eukaryotic and prokaryotic genome-scale sequencing with its associated megabase reference genomes and vast quantities of read data Prior to any evolutionary study reads must be accurately mapped and aligned. Our software performs these tasks as well as subsequent tropism testing, phylogenetic tree inference and visualization . We demoWe apply our software to temporally sampled 454 datasets from two HIV-1 infected individuals in order to characterize the emergence of low frequency CXCR4-using variants following treatment with an HIV entry-inhibitor drug, the CCR5 antagonist maraviroc. As the drug will not directly impact on viruses using the CXCR4 coreceptor Samples for pyrosequencing were obtained from two HIV-1 infected males, patients D and E, both of whom were treatment naïve and enrolled in the QD arm of the A4001026 study env was performed. The amplicons were subjected to nebulization to generate fragments of approximately 600 nucleotides. These were amplified as described in Margulies et al.et al.http://www.ncbi.nlm.nih.gov/Traces/sra) under accession number SRA023641.1.For each sample, RNA extraction and amplification from the gp120 region of Although RNA extraction and amplification was carried out across the entire gp120 gene, the region that is required for the coreceptor prediction of HIV-1 variants is V3 To explore the effects of using a consensus template on k-mer mapping, for each dataset, we compared the number of reads mapped to the data-specific template for that dataset to the number of reads mapped using HXB2 – the latter being the pre-mapping stage prior to consensus template generation. Next, we took our patient D screening dataset and introduced random variation into the consensus template in sequential steps of 2, 4 up to 26% (50 repetitions for each). After each introduction of random variation, the k-mer mapping was performed and the number of reads successfully mapped recorded. It should be noted that no precise pairwise alignment to the template sequence is generated during k-mer mapping. Instead the reads within our datasets covering the portion of the HIV-1 genome between the coordinates 6900 to 7305 are identified along with their approximate start positions. The k-mer mapping approach implicitly allows for a higher degree of tolerance in identifying such reads when compared to approaches that limit the number of mismatches Following k-mer mapping, reads are pairwise aligned to the consensus template using Smith and Waterman env were extracted, truncated, identical reads removed (frequencies were stored) and multiply aligned using Muscle approximate likelihood ratio testReads spanning the V3 region of The pipeline used for processing the initial read data is available within our software . ImplemeFor each dataset, following k-mer mapping to the consensus template, high read coverage was observed across the V3 region . In eachReads were pairwise aligned to the appropriate region (identified from the k-mer mapping step) of the data-specific consensus templates and those spanning both the start and end of the V3 region were extracted and truncated. Between 17 and 33% of reads contain at least one insertion event across the V3 region in comparison to the consensus template . The vasFor both patients a high coverage of in-frame reads across the V3 region was observed at each time point  and 4 wiapproximate likelihood ratio test  used in the coreceptor prediction It is also important to consider how much CXCR4-using virus is acceptable in the context of combination therapy. At present a 2% threshold has been proposed by RH In conclusion, our results demonstrate that, in conjunction with appropriate software, pyrosequencing data has utility for the evolutionally analysis and detection of low frequency variants within viral populations. In our analysis we have provided a high-resolution snapshot, through temporally sampled data, of intra-patient viral diversity and evolution associated with the CCR5-antagonist maraviroc. We have also quantified the effects of viral diversity on the initial k-mer mapping of read data in relation to the correction of platform dependent insertion error. The features of the software used here can be applied to other drug susceptibility and resistance studies, within other genomic regions of HIV-1 or to other pathogen genomes.Figure S1Example of frame correction to a dataset specific template and HXB2. Pairwise alignment of the V3 region of the consensus template  to that of HXB2 (A). Correction of the V3 sequence based on HXB2, and subsequent loss of a complete codon (B). Correction of the V3 sequence based on the data-specific template, and no codon loss (C).(0.46 MB EPS)Click here for additional data file.