A combination of social marketing, retail marketing, and donor subsidies may be necessary to extend the health benefits of household-level water treatment to populations most at risk.Household-level water treatment products provide safe drinking water to at-risk populations, but relatively few people use them regularly; little is known about factors that influence uptake of this proven health intervention. We assessed uptake of these water treatments in Nyanza Province, Kenya, November 2003–February 2005. We interviewed users and non-user controls of a new household water treatment product regarding drinking water and socioeconomic factors. We calculated regional use-prevalence of these products based on 10 randomly selected villages in the Asembo region of Nyanza Province, Kenya. Thirty-eight percent of respondents reported ever using household-level treatment products. Initial use of a household-level product was associated with having turbid water as a source (adjusted odds ratio [AOR] = 16.6,  The vasversus traditional untreated water handling methods and a statistically significant 25% reduction in diarrhea among children < 2 years in compounds using flocculent-disinfectant compared to traditional untreated water handling methods [Products for household-level treatment of drinking water are available in the area. Locally-produced sodium hypochlorite solution  has been socially marketed since May, 2003 and a flocculent-disinfectant product was introduced later that year. Developed and manufactured by the Procter & Gamble Company  when the flocculent-disinfectant is mixed with highly turbid water, debris quickly settles and the water becomes visibly clear and disinfected. Highly turbid water has high chlorine demand, and previous research has demonstrated the ability of the flocculent-disinfectant to render such water potable . The loc methods . Despite methods , studies methods ; clearlyWe hypothesized that the immediate reinforcement of visibly clear water would be a strong motivator for use of flocculent-disinfectant product. Understanding motivators and identifying successful distribution models for water treatments products could enhance uptake of household-level water treatment and increase the numbers of persons receiving the benefits of safe water worldwide. In 2003, a local non-governmental organization, the Society for Women and AIDS in Kenya , began selling the flocculent-disinfectant in Asembo and Gem . Social marketing of sodium hypochlorite solution began in the area in 2000 and SWAP began campaigns for the flocculent disinfectant beginning in 2003 after the health outcomes study introduced the product to the community. Campaigns included training and mobilization with community groups, presentations, distribution of educational materials, and micro-finance projects. In one pilot micro-enterprise project, local individuals and community groups had the opportunity to purchase quantities of the product and sell it in their villages at a small margin over wholesale cost. SWAP intensified activities starting in May 2003 and integrated the flocculent-disinfectant into its community education campaigns that already promoted the sodium hypochlorite solution with safe water storage and other health-related behaviors. From November 2003–February 2005, we conducted three studies to assess usage patterns of these two water treatment products and document use-prevalence in Asembo, Kenya. The three studies included: (1) a baseline utilization study (November 2003); (2) a follow-up utilization study (January 2005); (3) a use-prevalence survey for water treatment products in the study area (February 2005). Campaigns related to household water treatment products were ongoing throughout this time period.2.2.1.2.1.1.The study assessed characteristics of persons who used the newly-available flocculent-disinfectant water treatment product. We defined a user as a person living in Asembo who purchased and used any quantity of the flocculent-disinfectant product to treat water for the family compound. Users were identified by review of records from SWAK and flocculent-disinfectant vendors. Non-user controls were randomly selected from family compounds in Asembo within 1 kilometer of one of the eight flocculent-disinfectant vendors using spatial mapping and census data from the CDC/KEMRI Demographic Surveillance System (DSS). After consent was obtained from the head of the family compound, interviews were conducted at each compound with the mother of the youngest child in the compound. All respondents answered questions about beliefs concerning water and diarrheal diseases, drinking water sources, water treatment and storage practices, familiarity with water treatment products, and indicators of socioeconomic status such as educational level, cash spending on hygiene products, housing characteristics and household goods. Researchers documented the presence or absence of soap, toothpaste, and water treatment products in each compound. Stored household water was tested for residual free chlorine using the N, N-diethyl-phenylenediamine colorimetric method .2.1.2.p < 0.05 were included in a multivariate model. We developed a multivariate logistic regression model to identify independent associations with use of the flocculent-disinfectant. Covariates and interaction terms were tested for significance and goodness-of-fit. Model checking was performed using likelihood ratio testing.Data were entered into an Access® database with the Cardiff TELEform® image scanning system . Analysis was performed using STATA10 . A socioeconomic status index was constructed using principal components analysis in the manner described by Vyas and Kumaranayake . Bivaria2.2.2.2.1.Family compounds of flocculent-disinfectant users who participated in the 2003 utilization study were revisited. Efforts were made to locate the same person who was interviewed in 2003. Participants answered 64 standard questions regarding drinking water sources, water storage and treatment, and socioeconomic indicators for the household. Observers documented the presence of nine water treatment and hygiene items such as soap. Stored household water was tested for the presence of chlorine using a standard pool test kit . We defined reported consistent use based on number of sachets purchased relative to water consumption and conducted a separate analysis on the sub-group with confirmed use based on the presence of chlorine in the household drinking water at the time of the interview.2.2.2.Statistical analysis was completed using STATA10 and the methods described for the baseline study.2.3.2.3.1.This study documented use-prevalence for household-level water treatment products in the study area. We randomly selected 10 villages from Asembo. Flocculent-disinfectant had been available for sale since November 2003 in each of these villages and the surrounding area. An interviewer and a village health worker, using the most recent DSS census, visited all compounds in these villages. A person in the compound with responsibility for water handling answered four questions regarding household-level water treatments in the previous 7 days and since the short rains of 2003 (November–December 2003).2.3.2.Use-prevalence was calculated for flocculent-disinfectant and sodium hypochlorite during the two time periods.3.3.1.versus non-users . The mean socioeconomic status index was significantly higher for users than non-users (p = 0.001).We enrolled 117 persons who met the definition of flocculent-disinfectant user and 193 control-persons who had never used the flocculent-disinfectant . Flocculp = 0.004). Those who used flocculent-disinfectant remained less likely to express the belief that diarrhea is a serious problem in the community .After adjustment for economic status index, spending on soap and toothpaste, and knowledge of the previous CDC/KEMRI study, two factors remained significantly associated with flocculent-disinfectant use. Use of turbid water sources was strongly associated with flocculent-disinfectant use . In contrast, of the 74 (71%) respondents who reported use of sodium hypochlorite in that time same period, 37 (47%) reported purchase from a duka and 20 (26%) reported purchase from a market.Of the 117 users in the baseline utilization study, 104 (89%) completed questionnaires for the follow-up study.  Of thoseAlthough 18 (17%) respondents reported daily use of either the flocculent-disinfectant or the sodium hypochlorite solution on the questionnaire, only 14 reported chlorinating the water stored in their home at the time of the interview; 11 of these 14  had free chlorine present in their stored water.p = 0.6).On bivariate analysis, drinking water from turbid sources at least 4 months per year was reported by 96 (92%) respondents. Socioeconomic status was not associated with reported consistent use . Socioeconomic status was not significantly associated with reported consistent use after adjustment for these other factors. These associations were essentially unchanged regardless of whether reported consistent use was defined by reported volume of flocculent-disinfectant used or by confirmation of presence of free chlorine in the household water at the time of the interview.On multivariate analysis, after adjusting for economic status and awareness of the previous CDC/KEMRI study, respondents who reported consistent use were less likely than reported sporadic users to express the belief that their drinking water made their family sick  were included in the survey. Five-hundred-thirty-one (37%) compounds reported ever using the sodium hypochlorite solution compared with 105 (7%) who reported ever using the flocculent-disinfectant. Two-hundred-twenty-four (15%) reported use of the sodium hypochlorite in the past 7 days while 14 (1%) reported using the flocculent-disinfectant in that time period. Overall, 549 (38%) compounds reported ever using some form of household-level water treatment and 231 (16%) reported household-level water treatment in the past 7 days. Village-specific rates for ever using the flocculent-disinfectant varied from 0.7% to 16%. Rates for use of flocculent-disinfectant the past 7 days ranged from 0% (6 villages) to 13%. Reports of ever using the sodium hypochlorite solution ranged from 21% to 59%, while rates of use in the past 7 days ranged from 7% to 27%.et al. have demonstrated that even the experience of decreased diarrheal disease burden among residents of rural Guatemala was not adequate to motivate consistent use [These studies demonstrate a complex array of issues contributing to use of household-level water treatment products in western Kenya. While initial use of the flocculent-disinfectant was strongly associated with having turbid drinking water, this association did not persist in the study of reported consistent use. Although cost is often cited anecdotally as a reason for lack of use of household-level water treatment products, in our study economic status was not associated with reported consistent use among early users. Improvements in health do not seem to definitively influence use either: Luby tent use .versus 78%). The relatively high use of sodium hypochlorite despite the turbid water burden may be a reflection of familiarity with the sodium hypochlorite solution. Lower cost or greater ease of use for sodium hypochlorite may also have been determinants of use despite the advantages of flocculent-disinfectant for those using tubid water. Our data suggest that consumers often tried both locally available products, but reported using sodium hypochlorite more consistently than flocculent-disinfectant, for both the past year and the past week. Seventy-five percent of those who used the flocculent-disinfectant since 2003 also used sodium hypochlorite solution in that time period. In both the community as a whole and among those who used flocculent-disinfectant at baseline, the prevalence of sodium hypochlorite use eventually surpassed flocculent-disinfectant use. Thus, although dependence on turbid water correlated with trying flocculent-disinfectant, other factors appear to influence the decision to treat household water consistently and what product to use for this treatment. Since the time of the study, flocculent-disinfectant has expanded to national distribution networks in Kenya; this expansion may increase use by addressing the issues of availability that we found in our study.Dependence on a turbid water source emerged as the strongest motivator for flocculent-disinfectant use in this setting. The association with turbidity persisted after adjusting for socioeconomic status, spending on personal care items, and beliefs about the relationship between water and health. This result supports the hypothesis that the ability of flocculent-disinfectant to visibly clear turbid water is a compelling impetus to initial use. However, the allure of clearer water was not associated with reported consistent use based on the data from the follow-up survey. In this cohort with prior experience with flocculent-disinfectant and a high dependence on turbid water, sporadic use of sodium hypochlorite solution was comparable to use of the flocculent-disinfectant (71% Economic factors clearly influenced usage patterns. The choice of sodium hypochlorite over flocculent-disinfectant may largely be a function of the difference in retail cost as sodium hypochlorite solution cost less than 1 US cent per 20 L of water treated while flocculent-disinfectant cost 12 US cents per 20 L treated. Use did in fact decline remarkably in the follow-up survey with 25% of initial users reporting they never used the flocculent-disinfectant product again; however, the lack of a statistically significant relationship between reported consistent use and socioeconomic status in the follow-up survey suggests that something besides finances also affects usage patterns. The manufacturer is undertaking price-reduction studies in rural Kenya for the flocculent-disinfectant; these may provide a sense of how much affordability ultimately impacts use.dukas, markets and chemist shops. These locales are part of the indigenous consumer culture, and availability there made sodium hypochlorite much more accessible than flocculent-disinfectant, which had minimal penetration into these venues. Further penetration into the conventional retail sector may contribute to increased use of the flocculent-disinfectant through more consistent availability.Lack of availability emerged as an important determinant of flocculent-disinfectant use based on data from the cohort of prior users. Based on qualitative data from interviews, problems with flocculent-disinfectant distribution caused gaps in availability that in turn pre-empted use. Availability of flocculent-disinfectant in the local market decreased dramatically after the change in credit policy at SWAP. Rural community groups who served as vendors during the initial phase of sales did not have adequate cash resources to purchase flocculent-disinfectant in bulk. Without income generation from the wholesale purchases, these groups could not sustain the retail market. Those who reported buying sodium hypochlorite reported purchases from multiple sources including et al. found that although awareness of household-level water treatment products was high across wealth quintiles, use dropped precipitously in the lowest quintile [The documented prevalence of nearly 40% for ever using household-level water treatment products in this rural Kenyan setting demonstrates their potential as a way for even severely economically disadvantaged persons to benefit from safe water. The challenge lies in getting households to adopt this proven intervention. Behavior change communication can help; teaching safe water handling in elementary schools and clinics has demonstrated increased household use of water treatment products in pilot studies ,16. Thesquintile . In the Inspiring sustained use will require consistent availability, affordability in the local context, and a more comprehensive understanding of the factors that motivate those who consistently treat their water. This understanding will require further research and data-driven implementation strategies that address the behavioral and economic issues along with the public health issues. Such strategies could be informed by more in-depth behavioral research to further explain the behaviors and choices documented in our studies and specifically assess the relationships between use and social marketing activities. The World Health Organization’s International Network to Promote Household Water Treatment and Safe Storage, a collaboration of UN agencies, bilateral development agencies, international non-governmental organizations, research institutions, international professional associations, the private sector, and industry associations provides an integrated forum for identifying research needs on household-level water treatment and informing policies and programs .The study was limited by several factors. Low prevalence of usage in the community made it difficult to determine robust statistical associations for factors affecting flocculent-disinfectant use. Small sample size also prevented comparisons between those who used various combinations of water treatments, and analysis of seasonality of use; however, the sample sizes were sufficient to identify major factors associated with use.Courtesy bias likely resulted in some over-reporting of use, based on the results of the utilization study in which 16% of those reporting chlorination did not test positive for residual free chlorine. In addition, cross-sectional studies do not permit an objective assessment of consistent use.4.Household-level water treatment offers an immediate method for providing safe water to millions of people who will not have access to improved water delivery systems in the foreseeable future. These benefits cannot be realized without a better understanding of factors motivating use of the products. To increase usage of household-level water treatment in western Kenya, treatment products must first be consistently available at prices at risk-populations can afford. Availability in the traditional retail sector and through non-traditional vendors will maximize consumer access. NGOs play an important role in generating a consumer impulse for household-level water treatment products through community education and social marketing, but consistent use may require ongoing cost subsidies if the products are to reach those who need them most. The target population without access to adequate water infrastructure is generally the population with minimal financial resources. Visible clearing of turbid water and concern about waterborne diseases drive usage to some extent, but more complex factors appear to ultimately determine selection and consistent use of household-level water treatment products. If household-level treatment products are to fulfill their potential for improved health through safe water, multi-disciplinary implementation programs will need to address both the key barriers of access and affordability and the more nuanced challenge of positive behavior change.


Massively parallel pyrosequencing is a high-throughput technology that can sequence hundreds of thousands of DNA/RNA fragments in a single experiment. Combining it with immunoprecipitation-based biochemical assays, such as cross-linking immunoprecipitation (CLIP), provides a genome-wide method to detect the sites at which proteins bind DNA or RNA. In a CLIP-pyrosequencing experiment, the resolutions of the detected protein binding regions are partially determined by the length of the detected RNA fragments (CLIP amplicons) after trimming by RNase digestion. The lengths of these fragments usually range from 50-70 nucleotides. Many genomic regions are marked by multiple RNA fragments. In this paper, we report an empirical approach to refine the localization of protein binding regions by using the distribution pattern of the detected RNA fragments and the sequence specificity of RNase digestion. We present two regions to which multiple amplicons map as examples to demonstrate this approach. Mutations disrupting function of either cis-acting RNA elements or trans-acting RNA binding proteins (RBP) form the basis for many heritable human diseases [RNA-protein interactions influence virtually every step of post-transcriptional gene expression including pre-mRNA capping, splicing, polyadenylation and mRNA export, stability, localization and translation . These rdiseases -5. Despiin situ RNA binding specificity are required to elucidate the functions of RNA binding proteins in an efficient and comprehensive manner.The analysis of RNA binding proteins is further complicated by the observation that many are multifunctional . Elucidain vivo may be the immunoprecipitation (IP)-microarray assay [The most widely used technique for studying the interactions of RNA binding proteins with their RNA targets ay assay ,5. RNA bay assay . The croay assay ,8. CLIP ay assay ,8.in vivo, we have coupled the CLIP purification strategy from with high-throughput pyrosequencing of CLIP amplicons; we call this pyro-CLIP technology (pCLIP). The 454 sequencing platform  is an integrated system of emulsion-based PCR amplification of hundreds of thousands of DNA/RNA fragments linked to high throughput parallel pyrosequencing in picoliter-sized wells [In order to conduct global profiling of the RNA sites bound by RNA binding proteins ed wells -11. PyroPyro-CLIP experiments generate hundreds of thousands of RNA fragments (CLIP amplicons) to which an RBP binds. Substantial bioinformatics analysis is required to accurately identify the protein binding regions genome-wide. When the experimentally-detected RNA fragments (CLIP amplicons) are aligned to the human genome, many regions are observed to be encompassed by multiple fragments. In this study, we developed an empirical approach to refine the detection of protein binding regions by using the distribution pattern of the detected RNA fragments and the sequence specificity of RNase digestion along the amplicon-enriched genomic region.cis-acting element recognized by the studied RNA binding protein. The number of detected amplicons in each genomic region depends on various factors including the abundances of RNA transcript. Fig. Pyro-CLIP experiments result in hundreds of thousands of amplicons (RNA sequences with 50 to 70-bp in length). Preliminary data suggested that in a pyro-CLIP experiment, dozens of amplicons were usually detected within one genomic region. Hypothetically, all the detected amplicons should encompass at least one In the preliminary data, we found that the distribution of detected amplicons in each genomic region is similar to the pattern shown in Fig. The overall procedure includes three key steps: (1) pre-processing of CLIP amplicons, (2) estimation of sequence specificity of RNase digestion, and (3) prediction of RNA binding regions.blastn program [In the pyro-CLIP experiment, two short RNA linker sequences were ligated on both 5′- and 3′-end of each amplicon. These linker sequences were used as nested primer binding sites for reverse transcription and amplification. After removing both 5′- and 3′-end linker sequences, the remaining amplicons were mapped back into human genome using  program . In the blastn program [In practice, the number of genomic regions that match with the query amplicon varies depending on the selection of parameters while using the  program . We remoIn the pyro-CLIP assay, RNase digestion was used to trim the cross-linked RNA fragments to 50-70 nucleotides. Instead of cutting RNA fragments at random locations, RNase digestion has sequence specificity. The RNases used in the experiment were RNase I and RNase T1 .After mapping the detected amplicons back to the genome, we observed a large number of RNA fragments that shared identical 5′- and 3′-end genomic locations as in Fig 6=4096 potential 6-bp motifs based on all the detected amplicons and their mapped genomic locations.Each detected amplicon was generated by two RNase digestion sites Fig. . After mThe distribution of 6 nt sequences in the human transcriptome is not random, so in order to estimate sequence specificity of RNase digestion, the number of cutting events on each 6-bp motif should be corrected based on the frequency of the site. For instance, all the motifs that contain a CpG di-nucleotide appear in a much lower frequency than the ones without. Therefore, a lower number of detected cutting events on these motifs do not necessarily imply a low sequence specificity of RNase digestion. Based on this principle, we estimated the likelihood of RNase digestion on each 6-bp motif by normalizing the number of detected cutting events to the frequency of occurrence of the motif in the human transcriptome:kL depicts the likelihood of RNase digestion on the k-th 6-bp motif; if denotes the number of cutting events detected on the i-th 6-bp motif; and 0if represents its frequency of occurrences in the human transcriptome .where The size of detected amplicons ranges from 50 to 70 nucleotides. If there were no sequence specificity to the RNase digestion, the overlapped pattern of multiple amplicons would provide very good resolution of the detection of protein binding Fig. . HoweverOur strategy is based on several assumptions:cis-acting element to which the protein binds;1) All the detected amplicons should encompass the 2) The probability of each being selected as an RNase digestion site is proportional to the estimated likelihood of RNase digestion on its 6-bp motif, calculated in Eq. 1;3) Each genomic region where multiple amplicons are detected is located within one RNA transcript;For each genomic region in which multiple amplicons are detected, we simulate the distribution of amplicons that would occur based upon RNase sequence specificity, and then simulate the subset that would be selected if a protein bound to a specific site. This simulation is repeated as the binding site is moved along the RNA. We then compare the distribution of experimentally-detected amplicons Fig.  and of s1) Randomly generate a large number of RNA fragments based on the estimated likelihood of RNase digestion (Eq.1) along the genomic region in which multiple amplicons were detected plus a 70-bp extension towards both 5′- and 3′-side Fig. .i), randomly select M simulated amplicons from among the RNA fragments generated in the previous step that encompass the current site (i). M is the number of experimentally-detected amplicons  For each possible protein binding site , in both experimentally-determined group and simulated group; for each group, F(i) is a vector whose number of elements is equal to the length of genomic region being searched.3) For each genomic locus Pearson correlation coefficient of the resultant F(i) vector between experimentally-detected amplicons and simulated amplicons assuming that the protein binds on the i-th genomic locus, C(i).4) Calculate the 5) Continue steps 2 to 4 for the entire region. The region that results in highest correlation coefficient will be regarded as the region with the highest binding probability.In order to evaluate the robustness of the proposed approach to various noises resulted from multiple biological and computational steps, we created a simulated sets of virtual amplicons based on the sequences of a randomly-selected RNA region, and a pre-defined protein binding site. While generating these testing data, we deliberately included noises from various sources, including:1) Antibody non-specificity;2) Inaccurate estimation of the likelihood of RNase digestion on different 6-bp motifs;3) Noises associating with BLAST search.We tested the performance of the proposed approach under different levels of measurement noise based on the capability of the model to predict the pre-defined protein binding site.ip*logNpi), where ip is the probability that 6-bp motif i is selected as cutting site ip=if/ Σif. By definition, the resultant entropy should be a number from 0 to 1. A larger entropy implies less sequence specificity of the RNase digestion. The entropy for the frequencies of enzyme digestion on 6-bp motif is 0.42. This suggests that RNase digestion has significant preference for certain motifs. This finding is different from the reported cleavage specificity by the manufacturer, where RNase A cleaves at 3′ of single strand C's and U's, and RNase T1 cleaves at 3′ of single strand G's .In the preliminary dataset (unpublished data), a total of 29,071 RNA fragments ranging from 50-70 bp in length were used to estimate the sequence specificity of RNase digestion. We calculated the number of occurrences of all the potential 6–bp motifs at both ends of the RNA fragments, totaling 58,142 6-bp elements. We observed significant preference of RNase digestion on some 6-bp motifs. Only 967 of the 4,096 potential 6-bp motifs (24%) were observed as digestion sites. Strikingly, 26% of RNase digestion occurs at AATAAA sites. Fig. In vertebrate genomes, some sequences appear less frequently than others. For example, the number of occurrences of motifs that contain a CpG dinucleotide is significantly reduced. Therefore, we calculated the frequencies of all the 6-bp motifs in human transcriptome, and further estimated the likelihood of RNase digestion on each potential 6-bp motif based on Eq. 1. With this adjustment, the entropy level of cutting likelihood becomes 0.56. Top 1% 6-bp motifs with highest cutting likelihood are listed in Table The 29,071 RNA fragments cover 52 non-overlapping genomic regions that contain at least 10 fragments. Most enriched regions contain one or two protein binding sites. In this report, we use two examples to demonstrate the potential application of the proposed approach.1) Genomic regions that contain one protein binding sitePPEF2 .One enriched region on chr4: 77,026,287-77,026,348 contains 20 identical RNA fragments Fig. . This isWe generated a distribution of RNA fragments based on the likelihood of RNase digestion along the local chromosomal region  Genomic regions that contain two protein binding sitesThe proposed approach can also be used to refine the detection of protein binding regions that contain two or more binding sites. Instead of searching for the position of one binding site, as shown in Fig. i, j) represents the similarity of the distribution pattern between the experimentally-detected fragments , is shown in Fig. nts Fig.  and simutes Fig. . The numtes Fig. , where iblastn mapping errors.To evaluate the robustness of the proposed approach to various sources of experimental noises from both biological and computational steps, we created a simulated set of virtual amplicons by assuming known protein binding sites and deliberately introducing three different types of noise: antibody non-specificity, inaccurate estimation of sequence specificity of RNase digestion, and a) Antibody non-specificityBiological noise caused by antibody non-specificity may potentially undermine the ability of the proposed approach to accurately identify protein binding regions. Within the genomic region that is enriched by the detected RNA fragments, antibody non-specificity may cause false detection of RNA fragments that are not really bound by the proteins. These fragments may or may not encompass the real protein binding loci.N virtual fragments (50bp to 70bp) that were generated from two sources:We tested the ability of the proposed approach to detect the pre-defined protein binding site while introducing different levels of antibody non-specificity. Within a certain genomic region, we composed a set of n fragments that appeared randomly within the region, with no association with the pre-defined protein binding site and no correlation with 6-bp motif composition of local sequence. Hypothetically, these n fragments result from antibody non-specificity.1. N-n fragments that were simulated based on the 6-bp motif composition of local sequence and pre-defined protein binding site. These N-n fragments simulate the desired RNA fragments that associate with the studied protein.2. n/N). It is clear, however, that the proposed approach retains reasonably good robustness against antibody non-specificity while the proportion of detected RNA fragments contains less than 30% noise, with the correlation coefficient > 80%.Fig. b) Inaccurate estimation of sequence specificity of RNase digestion.The inaccurate estimation of the sequence specificity of RNase digestion may potentially decrease the accuracy of the model prediction. In this step, we deliberately added measurement noise to the estimated likelihood of RNase digestion on each 6-bp motif and tested the ability of the proposed approach to recover the pre-defined protein binding site. The correlation coefficient of the sequence frequency (number of RNA fragments that encompass each genomic locus) between RNA fragments simulated with and without added noise are shown in Fig. c) Noise from BLASTblastn is not always accurate. This is in part due to the sequencing error in the experimental stage, or due to the blastn parameters used. The consequences of BLAST noise on the model prediction are similar to the prediction inaccuracy caused by the antibody non-specificity, i.e. RNA fragments are inaccurately assigned to the studied genomic region. These RNA fragments usually have no association with the real protein binding sites, and have no correlation with the 6-bp motif composition of the studied genomic region. The effect of such noise on the model prediction is evaluated in the Fig. Mapping the detected fragment back to human transcriptome using de novo binding regions of DNA/RNA binding proteins in a genome-wide scale.In this study, we propose a computational approach to refine the detection of protein binding regions from pyro-sequencing-derived RNA fragments. As an ultra-high-throughput technology, pyro-sequencing allows sequencing of more than 100,000 amplicons in a single experiment [Using pyro-CLIP experiment as an example, we present a computational framework to increase the detection resolution within an RNA-fragment-enriched region by using (1) the distribution patterns of the detected amplicons Fig.  and 6B, Multiple RNA fragments can be detected in each enriched region. Intuitively, protein binding region can be localized by finding the common genomic regions that are encompassed by all the local fragments. This is based on the assumption that only those RNA fragments that contain the protein binding site can be immunoprecipated by the antibody, and therefore can be detected by the system. Localization is compromised by the sequence selectivity of RNase digestion. Therefore, the distribution pattern of the RNA fragments contains less information than the one that would result from random sequence cutting. Incorporating the sequence preference of the RNase digestion into the model can partially recover the information. In conclusion, the proposed approach demonstrated great potential in refining the detection of protein binding region using pyrosequencing-derived RNA fragments.The authors declare that they have no competing interests.YL and JRS contributed to the design of the study. JRS and XW performed the CLIP and pyrosequencing assays. XW and YL designed and performed the bioinformatics analysis and drafted the manuscript. CS, LL, SDM, HJE, and JRS participated in coordination, discussions related to result interpretation and revision of the manuscript. All the authors read and approved the final manuscript.


Tubules and sheets of endoplasmic reticulum perform different functions and undergo inter-conversion during different stages of the cell cycle. Tubules are stabilized by curvature inducing resident proteins, but little is known about the mechanisms of endoplasmic reticulum sheet stabilization. Tethering of endoplasmic reticulum membranes to the cytoskeleton or to each other has been proposed as a plausible way of sheet stabilization.Here, using fluorescence microscopy we show that the previously proposed mechanisms, such as membrane tethering via GFP-dimerization or coiled coil protein aggregation - do not explain the formation of the calnexin-induced organized smooth endoplasmic reticulum membrane stacks. We also show that the LINC complex proteins known to serve a tethering function in the nuclear envelope are excluded from endoplasmic reticulum stacks. Finally, using cryo-electron microscopy of vitreous sections methodology that preserves cellular architecture in a hydrated, native-like state, we show that the sheet stacks are highly regular and may contain ordered arrays of macromolecular complexes. Some of these complexes decorate the cytosolic surface of the membranes, whereas others appear to span the width of the cytosolic or luminal space between the stacked sheets.Our results provide evidence in favour of the hypothesis of endoplasmic reticulum sheet stabilization by intermembrane tethering. A key function of most cellular membranes is to form organelles enclosing biochemically distinct subcompartments in the cell essential for a multitude of cellular processes. The endoplasmic reticulum (ER) comprises the most abundant and highly versatile component of the endomembrane system. The specialized sub-compartments of the ER include: (i) the nuclear envelope (NE), composed of two adjacent membrane sheets surrounding the nucleus, and (ii) the peripheral ER, including membrane sheets and a complex network of tubules ,2. ER haThe overall architecture of the ER is highly evolutionarily conserved from yeast to mammals, with luminal intermembrane distances ranging between 50 and 100 nm . Inter-cIn contrast, the mechanism of ER sheet stabilization has been elusive and the identities of the proteins involved are unknown. Several protein complexes have been proposed to stabilize and sustain the extended flat double sheet morphology of the nuclear envelope, such as the SUN proteins that span the whole width of the NE lumen, connecting the nucleus to the cytoskeleton via Nesprin family proteins . The perWhile working on interactions of ER chaperones with neurotransmitter transporters, we found that overexpression of calnexin, an ER-resident lectin chaperone with a single transmembrane-spanning domain, induces formation of stacked OSER membranes, detected by fluorescence and cryo-electron microscopy . We haveHere, we revisit the mechanisms of ER sheet stabilization and stacking and present a detailed investigation into the organization of the stacked smooth ER sheets. Our results indicate that the OSER membrane stacks are highly regular structures that may be maintained through the ordered tethering of membranes by a large native complex, rather than through interactions between heterologously overexpressed proteins.DMEM and standard cell-culture reagents were from GIBCO . SUN1 and SUN2 antibodies were a generous gift from Sue Shackleton ; Nesprin-1 antibody was from Abcam . Anti-rabbit antibody conjugated with Texas Red was from Invitrogen . Climp63-GFP construct was generously provided by Hans-Peter Hauri . Calnexin constructs were described previously .2. Transfections were performed using Lipofectamine-2000 (Invitrogen), according to supplier's instructions. A total of 2-4 μg of plasmid DNA were used in an individual transfection reaction per 3 cm plate; 10 μg of plasmid DNA were used to transfect cells growing in 10 cm dishes.HEK293 cells were cultured at 37°C, supplemented with 5% COFor confocal microscopy cells were grown on poly-L-glutamine-coated (Sigma) glass cover-slips until 50-80% confluence, followed by transfection and/or fixation. Cells were fixed with 4% paraformaldehyde (Sigma) in phosphate-buffered saline (PBS), permeabilized for 30 min at room temperature using PBS with 1% BSA and 0.01% Triton-X100 and stained for 1 h with primary and secondary antibodies  where appropriate. Coverslips with stained cells were washed four times in PBS and mounted onto glass slides in Vectashield medium (Vector Laboratories) for microscopy. Images were acquired using Zeiss LSM 510 confocal microscope with a 63× objective lens.The procedure of cell preparation for cryo-electron microscopy of vitreous sections (CEMOVIS) was described previously . BrieflyImages were scanned and digitised using an MRC KZA scanner at pixel size of 2 or 2.3 Å, dependent on magnification during cryo-EM experiments. All image analysis - selection of the regions of interest, distance measurements, Fourier transformation, identification of strong reflections - was performed using Ximdisp . For meaInspection of the images confirmed that appearance of strong reflections in the selected regions of interest does not correlate with the direction of the knife marks (data not shown). The artefacts caused by the diamond knife - knife marks and crevasses  - in most cases result in appearance of very low resolution reflections (250-350 Å), outside the range where we observed ordered features (40-100 Å). Image analysis was performed on images devoid of crevasses.We have previously shown that calnexin-YFP or calnexin-CFP overexpression causes OSER expansion, based on immunofluorescence and low-magnification CEMOVIS images ; we alsoWe therefore tested whether calnexin tagged with a monomeric fluorescent protein would be capable of inducing multilamellar ER stacks. If fluorescent protein dimerization were a prerequisite for OSER formation, one would expect that calnexin tagged with a monomeric fluorescent protein should not induce multilamellar bodies. As shown in Figure Another hypothesis of ER sheet stabilization and stacking involved Climp63, a microtubule-binding ER protein . Climp63We co-expressed calnexin-CFP with Climp63-GFP in HEK293 cells. No colocalization of the two proteins in the OSER could be observed Figure ; Climp63Some of the OSER compartments were previously suggested to be derived from NE membranes ,12, makiTo test this hypothesis, we used immuno-fluorescence to visualize the endogenous SUN proteins in cells overexpressing calnexin. Antibody staining of SUN1 and SUN2 revealed the presence of SUN proteins in the NE, partially colocalizing with calnexin-CFP Figure  and 1D. We have also tested whether endogenous Nesprin-1 enters the multilamellar bodies. Proteins of the Nesprin family include several giant isoforms (M.W. up to 1 MDa) with large cytosolic regions. The classical function of Nesprins is to link the nuclear envelope to actin cytoskeleton ,24. CytoTo obtain a more detailed view of the ER membrane stack organization, we performed a CEMOVIS analysis of HEK293 cells over-expressing YFP-tagged calnexin. CEMOVIS is a method that preserves the native structure of the cell and allows imaging of the cellular architecture at the molecular level . We haveHere, we analysed a total of 55 good-quality vitreous section micrographs. The high magnification CEMOVIS imaging revealed the absence of a delimiting membrane Figure . The samThe long distances between cytosolic and luminal faces of the membranes clearly indicated that overexpressed proteins on their own would not be able to stabilize the OSER. Calnexin, even tagged with a fluorescent protein (the length of the YFP barrel is ~4 nm) on its cytosolic C-terminus (90 amino acid residues), would not be able to reach out and connect two ER membranes. Likewise, the length of the luminal domain of calnexin, based on its X-ray structure , is insuThe ectopically expressed membrane proteins inducing and/or populating the OSER membranes have previously been shown to be mobile, rapidly diffusing in the bilayer plane ,15. FRETThe high regularity of the ER membrane stacks suggested that tethering complexes could exist. A mechanism similar to that by which coiled-coil proteins organize Golgi membrane stacks  could acThe images obtained by CEMOVIS are very noisy, allowing only the gross features, such as membranes or large protein complexes , to be observed directly. Visualization of smaller objects directly is, in most instances, not possible. However, if the molecules are arranged in an orderly fashion, this can be detected in Fourier space and, if the order is sufficiently good, the structures of the arrayed molecules can be determined to near atomic resolution in favourable cases, e.g., from 2D crystals of membrane proteins . We therThe image analysis routine included digitization of the micrographs and selection of well-preserved regions of interest  and the cutting direction (indicated by an open arrow). We found no correlation between the cutting direction and the observed ordered densities in the best images, suggesting that these features are not created during sample preparation (data not shown).To exclude the possibility of an artefact caused by sample preparation or imaging, 3 adjacent areas of the same image were analysed as described above Figure . Only thAnalysis of the selected regions of interest revealed the presence of periodic densities, indicative of the ordered arrangement of molecules between the OSER membranes. Unfortunately, several factors forbid analysis of the vitreous sections of OSER membranes as 2D crystals: (a) the vitreous sections of OSER membranes represent only slices across the tentative 2D crystalline areas; (b) as a soft biological material, the OSER membranes are prone to distortions, bending and folding; (c) even if the specimen is crystalline, minute changes in the specimen thickness may lead to data corruption due to inclusion of disordered regions or extraneous ordered regions in the final projection. Together, these factors prohibit indexing of the observed diffraction patterns. Therefore, for illustration purposes, using only the strong reflections we masked the Fourier transforms imposing a simple 1-dimensional lattice and calculated filtered images of the regions of interest by back-transformation, visualizing the density features between the stacked ER membranes Figure .Based on our selection criteria, in a total of 55 micrographs, we found 17 reasonably well-ordered regions on the cytosolic side Figure  and 4 reThe observation of densely packed, periodic structures connecting two ER membranes suggests that these structures may act as molecular tethers. An absence of correlation between the parameters of these tethers, namely the spacing, the angle and the distance between the membranes, is indicative of high flexibility of these tethering complexes, which in turn is in line with the previously suggested dynamic nature of the proteinaceous matrix that shapes the ER .To the best of our knowledge, this is the first report detailing ER membrane sheet organization in a native environment, i.e. unfixed, hydrated cells. To date, the reports of attempts to visualize the ultrastructure of the ER membranes included freeze fracture electron microscopy of the crystalloid ER (tubular OSER) in the UT-1 cells, which showed macromolecular complexes of approximately 10 nm in diameter , and a fWe used CEMOVIS methodology to preserve the native ultrastructure and to image the intracellular membranes of the HEK293 cells by cryo-EM at unprecedented level of detail. A sub-compartment of ER, OSER, was used as a model system, which allowed us to sample a sufficient amount of stacked ER sheet micrographs for analysis. Using projection images of the OSER membranes we have identified ordered cytosolic and luminal macromolecular arrays supporting ER membrane stacking. The imaging of the OSER membranes may not allow us to measure directly the interaction between OSER membranes and thus we may not unequivocally rule out the possibility that the ordering of the molecules is secondary to ER stack formation. However, we propose that the OSER membranes may be stabilized by these arrays.in trans interaction between overexpessed proteins. It is unlikely that the discrepancy between our results and those published previously [trans-interaction between the ER membranes leading to membrane tethering. It is worth noting that a broad range of ER-localized proteins have been known to induce OSER formation, including: cytochrome b5 [We have shown that induction of ER sheet stacks by YFP-tagged calnexin overexpression is not driven by fluorescent protein dimerization. Monomeric fluorescent protein-tagged calnexin induces OSER formation; the stacked membranes are too far apart for direct eviously  resultedeviously  and calneviously ) are knoeviously , and areeviously . It is phrome b5 , HMG-CoAhrome b5 , inositohrome b5 , cytochrhrome b5 , microsohrome b5 , torsinAhrome b5  and otheSeveral intracellular compartments, both in prokaryotes and eukaryotes, have been studied by CEMOVIS previously with a comparable level of detail -41. OrdeA number of questions remain open, the most important being the identity of the proteins involved in OSER stabilization. We have ruled out the obvious candidates, represented by the coiled-coil domain-containing Climp63, and the LINC complex proteins that have been proposed to perform a similar role in the NE. This shows that, although the OSER membranes may originate from the NE at least in some cases ,12, the Our results support a hypothesis according to which active maintenance of organized smooth ER  may be achieved in part by molecular tethering complexes. The potential significance of our findings is underscored by the recently shown cell cycle dependent 'tug of war' between sheet and tubular ER morphologies . It is oVMK conceived and designed the study, performed fluorescence and electron microscopy and drafted the manuscript; BZ performed cryo-sectioning and electron microscopy, participated in the design of the study and helped draft the manuscript.


We explored the utility of population- and pedigree-based analyses using the Framingham Heart Study genome-wide 50 k single-nucleotide polymorphism marker data provided for Genetic Analysis Workshop 16. Our aims were: 1) to compare identity-by-descent sharing estimates from variable amounts of data; 2) to apply each of these estimates to a case-control association study designed to control for relatedness among samples; and 3) to contrast these results to those obtained using model-based and model-free linkage analysis methods. The study of quantitative traits has led to the development of tools of varying complexity designed to identify chromosomal regions associated with disease. This has been coupled in recent years by the increasing availability of data sets that include hundreds of thousands of markers. We investigated the utility of using more data and more sophisticated analyses by applying analytical methods of variable complexity to a single data set and comparing their results. We also estimated the same statistics using variable amounts of marker data to investigate the influence of the amount of marker data on the estimates. Generally speaking, we asked whether we really benefit from these new tools and large data sets, or do they simply increase the complexity of our research? More specifically, we: 1) compared estimates of identity-by-descent (IBD) sharing from increasing amounts of marker data; 2) evaluated how well different IBD estimates corrected a case-control association study for relatedness; and 3) compared the results of our case-control study and various linkage analyses to contrast any signals of association between genotype and phenotype.We focused exclusively on the Genetic Analysis Workshop (GAW) 16 50 k marker data, and primarily analyzed only chromosome 7. We matched the position of each chromosome 7 marker to the sex-averaged Kosambi map sequence position on the Rutgers map [We filtered markers with >3% missing data and with minor allele frequency <0.05. We used chi-square tests to test the null hypothesis of Hardy-Weinberg equilibrium, and removed markers yielding the largest 1% of the test statistics, leaving 2132 markers on chromosome 7. A "thinned" marker panel was obtained by selecting approximately every tenth marker from this "dense" filtered marker set, preferentially selecting markers with higher rare allele frequencies among founders because they are more suitable for linkage analysis. The final thinned data set included 214 markers on chromosome 7 (and 3465 genome-wide markers) with a marker density of ~1 per cM.To ensure compatibility with the linkage analysis programs in the MORGAN package , we mergFor linkage-based analyses, we focused on high-density lipoprotein level (HDL) and chromosome 7 due to previous evidence of linkage within the Framingham Heart study (FHS) -8. We usWe performed Bayesian oligogenic segregation analysis using the software package Loki 2.4.7  to identk-coefficients, where ki is the probability that i alleles are shared IBD, using the thinned chromosome 7 and genome-wide panels of markers, as well as subsets of 214 and 1000 genome-wide markers. We estimated k-coefficients for all possible pairs of independent people (n = 1827), using all founders in the pedigrees and other unrelated individuals, and for all pairs of individuals within each pedigree. Kinship coefficients, Φ, were subsequently computed as Φ = 0.25k1 + 0.5k2 and pairs of individuals with Φ > 0.2 were noted.Without reference to the pedigree structures, we estimated We selected four pairs of individuals showing high apparent relatedness as estimated from the thinned chromosome 7 markers while differing with respect to pedigree numbers. For each pair, the dense chromosome 7 markers were used to detect IBD segments using the model of Thompson . We usedth percentiles of the trait distribution in the full data set. The correction for relatedness essentially eliminates inflated test statistics resulting from inclusion of related individuals. We corrected the naïve chi-square statistic p-values using three types of kinship coefficients: pedigree-based prior, pedigree-based posterior, and population-estimated kinship coefficients. The pedigree-based prior was computed based on pedigree structure alone, while the pedigree-based posterior was based on the gl_auto results (described below) that used both pedigree structure and marker data. The dense chromosome 7 marker panel was used for the case-control study, while the thinned chromosome 7 marker panel was used for estimation of kinship coefficients. The population-estimated kinship coefficient was a maximum-likelihood estimate based on the thinned chromosome 7 marker data.We used a new "case-control" study design that corrects for relatedness (both known and estimated as cryptic kinship) within the sample , choosinTwo MORGAN  programsw-score [w-score analyses were performed only on the size 4-9 pedigrees, while VC analysis was performed on this subset as well as on all pedigrees for comparison. We summarized the results using randomized p-values for the conditional test [p-values for the VC analysis through trait simulation and the inheritance vectors described above [th out of 500,000 iterations were used to compute Bayes' factors for the presence of a QTL within each 2-cM bin.Genetic linkage can be detected with pedigree data using inheritance vector realizations. We used the inheritance vectors obtained from gl_auto for two linkage analysis methods: 1) standard variance-components (VC) analysis using SOLAR, and 2) a novel conditional inheritance vector test using the w-score , which inal test  and empied above , where ek1 or k2 values for chromosome 7. This indicates there is non-negligible relatedness among independent individuals across pedigrees.We found several pairs of independent individuals that share non-zero average IBD Figure , but thek1 decreased as the number of markers used increased for two of the four pairs of individuals. The IBD segments analysis estimated the full nine-state IBD probabilities along the chromosome, with the average of these values reported in Table Detailed analysis of four such pairs of independent individuals with unique pedigree numbers are summarized in Table Figure p-value = 0.0087). All linkage analyses detected one or more peaks in the region between 20 and 40 cM. A single Loki run is represented in Figure Linkage analysis results are summarized in Figure p-values summarize test significance as well as uncertainty of the test results. For example, although we find significant evidence for linkage near 38 cM using VC analysis and trait resimulation, the conditional test p-values in the same region are uncertain, as indicated by the range of p-values estimated at that position. To resolve these uncertainties, we would need to use markers at greater density or with greater polymorphism levels to infer the inheritance vectors in this region.Randomized k1 > 0.8 than 214 markers from chromosome 7.As a rule, more marker data increases the stability and apparent accuracy of IBD estimates Figure , as the k-statistics estimated from 214 vs. 1000 genome-wide markers, little difference is observed between estimates from 1000 vs. 3465 genome-wide markers. This suggests that we can thin our marker panels to avoid the effects linkage disequilibrium without strongly altering our inferred k-statistics. This also allows for the generation of multiple, equivalent, thinned data sets from a single dense data set, to be used for replication purposes.However, a relatively modest number of markers were needed to achieve this stability. Although we see a dramatic difference between the Even with thousands of markers, relationships between some pairs of independent people were detected by several approaches. Some of these pairs shared pedigree numbers but could not be connected in the pedigree file. This suggests that our methods were able to detect real relatives even when they were not labelled as such. However, some pairs had unique pedigree numbers, suggesting some cryptic relatedness among the FHS participants and raising the possibility that adjustment for such relationships might be necessary in some analyses.p-values in the case-control study. Fortunately, our corrections using pedigree- and marker-estimated relatedness worked well. Because many case-control studies do not have access to pedigree data, this suggests that our method may be applied to genome-wide association studies without the need for additional pedigree information. The pedigree-posterior estimate of relatedness overcorrected our test statistic, although as the analysis used only the thinned chromosome 7 markers, this is not surprising given the results in Figure Relatedness, known or cryptic, clearly inflated uncorrected w-score and MCMC-based oligogenic linkage analyses. The w-score also identified a possible linkage signal near 95 cM, although the confidence in actual p-values varied across the chromosome. Analysis of the size 4-9 pedigrees emphasized the peak near 20 cM at the expense of the peak near 40 cM, while analyses of all pedigrees identified a modest signal near 180 cM. This signal was detected in a previous GAW [The number and strength of linkage signals varied across methods and by the amount of data used. Linkage analyses, but not the case-control analyses, provided evidence for HDL loci on chromosome 7. The bimodality of the linkage signal between 20 and 40 cM was more clearly defined with the more computationally intensive and trait-model-based w-score provided both localization and confidence of information in linkage analysis. Although encompassing a wide range of approaches, these methods show clear promise for future work.Our novel methods were useful in a variety of situations. Inheritance vectors generated by gl_auto were used in the VC analyses and in empirical significance testing. Analysis of IBD segments identified wide swathes of shared chromosomal regions between pairs of independent people with patterns not visible in a single summary statistic. The method to correct case-control studies for relatedness was practical and effective, and the The use of additional data and analytical methods of increasing complexity appears to have paid dividends. However, there are clearly limits. More markers provide better IBD sharing estimates, but a marker density greater than between 1 and 3 cM would likely give only a slight improvement. Correcting case-control studies for relatedness is effective, relatively simple, and can be done using marker data alone. Linkage analyses of greater complexity identified more, albeit weak, linkage signals than simpler analyses. It would appear that all association and linkage methods are capable of detecting strong and clear signals. Because not all studies are fortunate enough to have strong signals, sophisticated analytical tools and large, but not too large, data sets may deliver additional results along with their complexity.BMI: Body mass index; FHS: Framingham Heart Study; GAW: Genetic Analysis; Workshop; HDL: High-density lipoprotein; IBD: Identity by decent; MCMC: Markov-chain Monte Carlo; QTL: Quantitative trait locus; SNP: Single-nucleotide polymorphism; VC: Variance components.The authors declare that they have no competing interests.All authors participated in study design and approved the final manuscript. EAT, FB, and YC participated in the IBD analyses, and YC performed the case-control study. EEM, YD, CC, MS, and EMW participated in the linkage analyses. EEM drafted the manuscript.


Cognitive model of panic disorder have proposed that panic attacks result from the catastrophic misinterpretation of certain bodily sensations. Cognitive-Behavioral Therapy (CBT) for panic disorder aims to change these catastrophic cognitions. CBT intervention successfully caused reduction of catastrophic cognitions and symptomatic improvement in the majority of cases. However there are some patients who fail to modify their catastrophic cognitions or rather experience an increase in them during CBT treatment. It is clinically and theoretically important to understand about cognitive sensitization of panic disorder during CBT sessions. The purpose of the present study is 1) to clarify the baseline characteristics of panic patients who would experience sensitization of their catastrophic cognitions through the CBT treatment, and 2) to examine the course of symptomatic changes for them.Of ninety-five outpatients with panic disorder started the group CBT program for treatment of panic disorder, seventy-nine completer were classified as "cognitively sensitized (CS)" or "cognitive responding (CR)" or "no-responder" according to the difference of the Agoraphobic Cognitions Questionnaire score across treatment. We compared the CS and CR patients in terms of their baseline clinical characteristics. Then we assessed the symptomatic and functional changes for both groups.At the start of the CBT program, despite of the same degree of panic disorder severity, CS scored significantly lower on ACQ score than CR. CS also showed significantly lower score on anticipatory anxiety compared to CR. At the end of treatment CS showed significant improvement in severity of panic disorder, although the degree of improvement was smaller than that for CR. Then CS would progressively reduce their agoraphobic fear and avoidance, and would improve their functional impairment up to three month of follow-up.Panic patients who would experience sensitization of their catastrophic cognitions through the CBT treatment could nonetheless gradually improve. They showed a relatively low level of catastrophic cognition and anticipatory anxiety before starting the CBT program. We might conclude that temporary sensitization of catastrophic cognition may be necessary before improvement especially among those with initially low catastrophic body sensation fears and that we need not be concerned too much with temporary increase in catastrophic cognition in the process of CBT for panic disorder. A number of studies have supported the effectiveness of cognitive behavioral therapy (CBT) for panic disorder -3. SeverCognitive model of panic disorder have proCBT for panic disorder aims to change these catastrophic cognitions. Taylor The purpose of present study is 1) to clarify the baseline characteristics of panic patient who would experience sensitization of their catastrophic cognitions through the CBT treatment, and 2) to examine the course of symptomatic changes for them.Subjects were 137 consecutive patients who sought treatment for panic disorder at Nagoya City University Hospital Department of Psychiatry between June 2001 and March 2006. Of 137 patients, 95 subjects attended the group cognitive-behavioral therapy program.At the start of the CBT program, all the patients met the following entry criteria:(a) principal Axis I diagnosis of panic disorder with or without agoraphobia according to the DSM-IV criteria, as assessed by the Structured Clinical Interview for DSM-IV(SCID) ; (b) absUse of antidepressants to control their anxiety and/or depression was permitted throughout the CBT period. Because these drugs do not interfere with CBT treatments . In caseAt the start of the CBT program, 56 patients used antidepressants, 19 patients used benzodiazepines regularly or occasionally.The Ethics Committee of Nagoya City University Graduate School of Medical Sciences approved the study protocol and all subjects provided written informed consent after full explanation of the purposes and procedures of the study.CBT program used in this study was based on the CBT program developed by the Clinical Research Unit for Anxiety and Depression at the University of New South Wales, Sydney, Australia . This trOne group consisted of three to four patients. Two trained therapists  conducted two-hour highly structured sessions once a week for ten weeks, using a detailed manual.The senior psychiatrist, who had more than 15 years' experience as a clinician and had observed the PD-CBT program at St. Vincent's Hospital, trained the other therapists. With the aim of training, the junior psychiatrists or psychologists attended a series of CBT sessions as co-therapist, which facilitated mainly by the senior therapist. Then trained junior psychiatrist or psychologist began to lead a group CBT sessions assisted by another junior therapist. The senior psychiatrist supervised the other therapists at a CBT clinical conference held once a week.Before enrolment into the CBT program, all participants received a semi-structured interview judging the anxiety and mood disorders section of the SCID and the Panic Disorder Severity Scale (PDSS). This interview was conducted by one of the clinicians carrying out the CBT program. Then at the start of the program, all patients completed five self-report questionnaires. To assess the intensity of catastrophic cognitions, patients were asked to fill out the Agoraphobic Cognitions Questionnaire (ACQ). To evaluate the clinical status and personality characteristics, patients completed the Work, Home and Leisure Activity Scales (WHLS), the Fear Questionnaire-agoraphobic subscale (FQ-ag), the Mobility Inventory for agoraphobia-alone subscale , and the NEO-Five Factor Inventory (NEO-FFI). The same sets of instruments except for the personality inventory were repeated at the end of the program. Three months after the end of the program, we mailed follow-up questionnaires including ACQ, FQ-ag, MI-alone, and WHLS. Details of these instruments are described below. Since we followed up by post mail, PDSS which is based on a semi-structured interview was not administered at 3-month follow-up.The ACQ is a 14-item self-report instrument to assess "fear of fear" or cognitions concerning catastrophic consequences of experiencing anxiety. Each item is rated on a five-point scale ranging from 1(thought never occurs) to 5, according to the frequency with which this thought occurred when the client was anxious. Good reliability and validity have been shown for both the original and the Japanese versions of this questionnaire,21.The PDSS is a seven-item interview-based scale of panic disorder severity in which the clinician rates the severity of seven features of panic disorder on a scale rating from 0 (none) to 4 (extreme). The features that are rated include frequency of panic attack, distress during panic attack, anticipatory anxiety, agoraphobic fear/avoidance, interoceptive fear/avoidance, work impairment/distress, and social impairment/distress. Adequate inter-rater reliability and validity have been reported for both the original and the Japanese versions ,23.The Work, Home and Leisure Activity Scales was designed to assess role functioning in the areas of work, home management, social leisure, and private leisure activities. Each item is rated on a nine-point scale ranging from 0  to 8 (very severely impaired). Satisfactory reliability and construct validity have been reported .The Fear Questionnaire-agoraphobia subscale is a self-report instrument for measuring the severity of agoraphobic avoidance of five typical situations for agoraphobia. Each situation is rated on a nine-point scale ranging from 0 (would not avoid it) to 8  to show how much he/she would avoid each of the listed situations because of fear or other unpleasant feelings. Test-retest reliability and factor validity have been confirmed .The Mobility Inventory for agoraphobia is a self-report instrument for measuring the severity of agoraphobic avoidance. Patients are asked to rate on a five-point scale ranging from 1(never avoid) to 5 to show how they feel about 31 places or situations that they may avoid because of anxiety or phobia, when they are alone. This instrument has been shown good reliability and construct validity ,27.The NEO-FFI is a 60-item self-reported inventory aimed to assess five personality factors of neuroticism, extraversion, conscientiousness, openness, and agreeableness .σ = standard deviation of normal population, γ = test-retest reliability coefficient). Since there was no available data revealing the stability of ACQ for a Japanese sample, we calculated the SEM for ACQ by using the data from Chambless et al.[EM for ACQ total score (SEM = 2.41), we set the cut-off point at 3 as minimum change score. Each subject was classified as a "cognitive responding (CR)," or a "cognitively sensitized (CS)" based on their differences of ACQ total score across the treatment. The subject whose ACQ score at post-treatment was 3-point or more lower than pre-treatment ACQ score was classified as CR, and those whose post-treatment ACQ score was 3-point or more higher than pre-treatment ACQ score was classified as CS. Others who change the ACQ ranged from +2 to -2 across the treatment were considered as non-responder in cognition.To estimate the change of catastrophic cognition, we calculated the differences in ACQ total score across the treatment for each subject. Then we considered the standard error of measurement  for continuous variables and chi-squared test of independence for categorical variables. If the data distribution did not follow the normality assumption, Kruskal-Wallis Test was utilized.t-test, as long as the variables were normally distributed within each group and the variation of scores in the two groups were not reliably different. If the data distribution did not follow the normality assumption, Mann-Whitney U test was utilized.We then focused on the differences between the CS and CR patients. First we compared these two groups in terms of their baseline clinical characteristics. The group mean values were compared using the independent Student d.For the assessment of changes in symptom severity and functional impairment across treatment in both groups, Student's t-test for dependent samples was used. If the data distribution did not follow the normality assumption, Wilcoxon rank-sum test was utilized. The within-group treatment effect size (ES) was calculated by Cohen's Further ANOVA for repeated measures with PDSS total score as the dependant variable was performed to evaluate whether the degree of improvement differed between the two groups.To examine the course of symptomatic changes for the two groups, we conducted a 2(group: CS vs. CR) × 3(time: baseline vs. post-treatment vs. three-month follow-up) repeated measures ANOVA with FQ-ag score, MI-alone score and WHLS as the dependant variable, followed by the post-hoc Dunnett's test when appropriate. All data undergoing ANOVA were tested for assumption of sphericity with Mauchly's test. In cases the sphericity did not hold, we adjusted the degree of freedom for the F-test using the Greenhouse-Geisser epsilon.Statistical analyses were performed using the SPSS program version 15.0. All the statistical tests were two-tailed, and results were considered significant when p < 0.05.Of ninety-five patients who started the CBT program, sixteen patients withdrew from the program before its completion, because of symptom improvement, lack of improvement, inconvenience of attending the program, concurrent physical illness or worsening of depression. The seventy-nine completers (26 men and 53 women) with a mean age of 36.1(SD = 11.3) were classified as CS(N = 11) or CR(N = 52) or no-responder(N = 16) according to the difference of ACQ score across the treatment. Table Table By definition, as can be seen in Table For both groups, PDSS total scores improved significantly; CS patients' mean score moved from 11.1(SD = 4.1) at baseline to 9.1(SD = 4.4) at endpoint. CR patients' mean score moved from 13.2(SD = 4.7) at baseline to 5.9(SD = 4.3) at endpoint. Repeated measures ANOVA for PDSS total score showed a significant main effect of time  = 32.239, p < 0.001), and Group × Time interaction  = 10.80, p = 0.002). The degree of improvement differed between two groups.With regard to symptomatic and functional changes across treatment, CR patients showed significant improvement for all variables. In contrast, CS patients significantly improved in impairment of social functioning and WHLS, and showed non-significant trend to improve in MI-alone.Results of repeated measures ANOVA which examined the courses of symptomatic changes from baseline to three month follow-up for the two groups are presented in Table As can be seen in Table With regard to the first objective of the present study, we were able to delineate the baseline characteristics of panic patient who would experience sensitization of their catastrophic cognitions through the CBT treatment as follows. Despite the same degree of panic disorder severity, CS patients showed relatively low level of catastrophic cognitions than CR patients preceding CBT treatment. Furthermore, CS patients reported lower level of anticipatory anxiety and interoceptive fear/avoidance before treatment. These findings are generally consistent with prior research conducted by Beck & Shipherd who founThe second purpose of this study was to examine the course of symptomatic change of CS patients. We found that at the end of treatment CS patients showed significant improvement in severity of panic disorder, although the degree of improvement was smaller than that for CR patients. The improvement for CS patients at the end of treatment was mainly due to restoration of social functioning. This improvement for CS patients appears to continue at least for three months following the end of treatment. Consistent with previous findings ,11,14,30It is interesting to note that panic patients who would experience sensitization of their catastrophic cognitions through the CBT treatment could nonetheless gradually improve. It has already been pointed out that exposure to feared situations or bodily sensations can initially increase anxiety. Otto et al. emphasized that these experience help patients to learn that they can be "OK" despite the presence of anxiety . We may EM as the cut-off point. Second, the sample size was rather small to generalize our results and some of our negative findings might be due to lack of power. However, we can be sure of what positive findings we were able to identify. Third, the study did not include a control group and one cannot be sure if the significant reduction in panic symptomatology observed in the present cohort might be due to passage of time rather than to CBT treatment per se. Fourth, we did not confirm the change of symptomatorogy for depression with objective measure during the treatment and follow-up phase. Therefore we could not perfectly exclude the possibility that the results of this study affected any symptom of depression. Fifth, we did not confirm whether the patient underwent other treatments in the post treatment follow-up phase. Therefore strictly we couldn't rule out the possibility the improvement of patients due to some other treatment.Several limitations of current study should be mentioned. First, the procedure of categorization that classified patients into "cognitively sensitized" or "cognitive responding" may appear rather arbitrary. A different categorization strategy may have been possible but in the absence of agreed-upon procedure, we set the Set al.[Casey et al. concludeIn conclusion, we found that CS patients had a lower level of catastrophic cognition and anticipatory anxiety than CR patients at baseline before starting the CBT program.Through and at three months after the CBT program, CR patients demonstrated robust and continued improvement in symptomatology and functioning, while CS patients lagged behind CR patients, because they scored worse at the end of acute phase treatment than CR patients but tended to catch up with them at three-month follow-up. When clinically paraphrased we might conclude that temporary sensitization of catastrophic cognition may be necessary before improvement especially among those with initially low catastrophic body sensation fears and that we need not be concerned too much with temporary increase in catastrophic cognition in the process of CBT for panic disorder.TAF has received research funds and speaking fees from Asahi Kasei, Astellas, Dai-Nippon, Eisai, Eli Lilly, GlaxoSmithKline, Janssen, Kyowa Hakko, Meiji, Organon, Pfizer, Tsumura, Yoshitomi, and Zelia. All the other authors declare that they have no competing interests to declare.YNoda was the primary investigator; YNakano, KL, SO, YK, TF, NW, JC, YNoguchi, MK, MS and TAF performed the clinical investigation ; TAF participated on the design of the study and supervised the overall conduct of the study. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:


Drug resistance in parasitic nematodes is an increasing problem worldwide, with resistance reported to all three commonly used classes of anthelmintics. Most studies to date have sought to correlate the resistant phenotype with genotypic changes in putative target molecules. Although this approach has identified mutations in several relevant genes, resistance might result from a complex interaction of different factors. Here we propose an alternative mechanism underlying the development of drug resistance based on functional differences in microRNA activity in resistant parasites. microRNAs play an important role in resistance to chemotherapeutic agents in many tumour cells and here we discuss whether they might also be involved in anthelmintic resistance in parasitic nematodes. Onchocerca volvulusTeladorsagia circumcincta and Haemonchus contortus, resistance has been reported to all three classes of drug and, indeed, triple-resistant parasites exist in parts of the world, including Scotland Parasitic nematodes of humans and animals remain a major problem in many areas of the world. Control of these parasites relies almost exclusively on the use of chemotherapeutic agents, of which three classes are in common use. These are the tetrahydropyrimidines/imidazothiazoles , the benzimidazoles (e.g. fenbendazole) and the macrocyclic lactones . Whereas most drugs retain efficacy against human parasitic nematodes, there are indications that resistance to ivermectin might be developing in the filarial nematode Caenorhabditis elegansben-1, in which mutations conferred resistance to benzimidazole (BZ) compounds in C. elegansH. contortus, mutations in the β-tubulin isotype 1 gene (Hc-iso-1) are also associated with BZ resistance. Transformation of BZ-resistant C. elegans with the wild-type Hc-iso-1 allele restores sensitivity to BZ compounds, whereas transformation with the mutant Hc-iso-1 gene had no such effect C. elegans have indicated that resistance to ivermectin might be polygenic and that mutations at any one contributing locus may be insufficient for a fully resistant phenotype Understanding the mode of action of anthelmintics and the mechanisms of resistance is important in terms of attempting to monitor and modulate the resistant phenotype. Much of our knowledge of mechanisms of drug action and the likely genes involved in resistance comes from studies on C. elegans  of miRBase (http://www.mirbase.org/) identifies 940 miRNAs in humans and 175 in C. elegans. In addition, up to one third of all human mRNAs might be regulated by miRNAs let-7 and lin-4 have been identified in the B. malayi genome miRNAs are one of a family of endogenous small non-coding RNAs, including small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs), that have been identified in a diverse range of organisms mdr-1 gene), an important mechanism of drug efflux. The 3′-UTR of the mdr-1 gene contains a binding site for miR-451, which negatively regulates mdr-1 expression  near the binding site of miR-24 in the 3′-UTR of the human dihyrofolate reductase (DHFR) gene prevents repression by miR-24, resulting in overexpression of DHFR and resistance to methotrexate It is possible that a link between miRNAs and drug resistance is more likely to occur in cancer cells, as the cancerous state can itself be due to miRNA mis-expression. However, the study of miRNAs in cancer has clearly established that miRNA-mediated alterations in levels of drug targets, drug transporters, metabolic enzymes or cell apoptosis proteins can lead to drug resistance. Altered gene expression associated with drug resistance in other systems indicates that examination of miRNA activity and 3′-UTR interactions in parasitic nematodes is warranted to improve our understanding of drug resistance mechanisms.C. elegansC. elegans, miR-1 negatively regulates the expression of two nicotinic acetylcholine receptor (nAChR) subunits, unc-29 and unc-63mir-1 mutants the expression of both UNC-29 and UNC-63 subunits is increased, and this corresponds with decreased muscle sensitivity to acetylcholine and levamisole  Hc-GGR3 3′-UTR of resistant worms. It was proposed that this polymorphism might be associated with resistance, although functional analysis is required to test this. Given the high degree of conservation of some miRNA sequences across species, including the miR-1 sequence, C. elegans presents a suitable model system in which to functionally test potential miRNA-3′-UTR interactions, which cannot readily be examined directly in parasitic species.Interestingly, in a very different approach using immunoprecipitation to isolate miRNA-target complexes in H. contortus and O. volvulus, treatment with ivermectin results in a reduction in allele frequency and overexpression of P-glycoprotein and other ABC transporter genes H. contortus that were selected for BZ resistance show an elevated frequency of the same P-glycoprotein allele C. elegans also found that exposure to increasing concentrations of ivermectin resulted in increased expression of several P-glycoprotein and ABC transporter genes As well as changes in expression levels of potential drug targets, alterations in the expression of drug transporters have been reported in parasitic nematodes following anthelmintic treatment. This might be analogous to the changes in transporter levels in tumour cells exposed to drugs, as discussed previously. In in silico approaches C. elegans as a heterologous expression system.Herein the possibility that changes in gene expression levels associated with drug resistance in parasitic nematodes are mediated by miRNAs has been discussed. This might be a mechanism of altering drug efficacy; given the link between miRNAs and drug responses in cancer cells, this area is ripe for detailed investigation. With the number of genome sequencing projects underway, miRNAs present in parasitic nematodes can be identified using both Currently, the potential correlation between antihelmintic resistance and changes in particular gene sequence or expression level has been limited to the study of a small number of candidate genes. Examining and comparing miRNA levels, as well as mRNA expression in drug resistant and susceptible isolates, through the use of microarrays will produce a global picture of changes that might correlate with drug resistance. In addition, miRNA inhibitors and mimics  are avai


J Chem Inf Model 2007, 47: 626-634) of the TMACC descriptor suggests it is competitive with the current state of the art.The topological maximum cross correlation (TMACC) descriptors are alignment-independent 2D descriptors for the derivation of QSARs. TMACC descriptors are generated using atomic properties determined by molecular topology. Previous validation  and dihydrofolate reductase (DHFR). In the case of the ACE inhibitors, the TMACC interpretation shows features specific to C-domain inhibition, which have not been explicitly identified in previous QSAR studies.http://comp.chem.nottingham.ac.uk.The TMACC interpretation can provide new insight into the structure-activity relationships studied. Freely available, open source software for generating the TMACC descriptors can be downloaded from The online version of this article (doi:10.1186/1758-2946-1-22) contains supplementary material, which is available to authorized users. Quantitative structure-activity relationship (QSAR) models correlate molecular chemical structure to biological activity. The underlying principle for QSAR modelling is the similar property principle: molecules with similar chemical structures will exhibit similar biological properties . This pr2D QSAR models are generated using descriptors derived from the two-dimensional graph representation of a molecule. In contrast, 3D QSAR models correlate activity with descriptors based on spatially localised features. Although 3D descriptors may allow more detailed descriptions of the molecular binding interactions between ligands and receptors, 3D methods are more time-consuming, due to the requirement of precise conformational detail on the molecule and exact alignment . In manyClassic QSAR methods, developed by Hansch , provideOver the last decade, advances in computational technology combined with contemporary methodologies have led to a vast array of new descriptors . TopologPrevious validation of the TMACC descriptors was promising, with leave-one-out (LOO) cross-validated correlation coefficients comparable to those achieved by the state-of-the-art 2D QSAR method, hologram QSAR . An exteThe present study aims to evaluate the ability to identify known structure-activity relationships using the TMACC descriptors. To exemplify the TMACC descriptors, we investigate two datasets, which were previously used in a comprehensive comparison of modern QSAR approaches . Models 50) values for each molecule. TMACC descriptors were generated using the topological data of each molecule. All nonpolar hydrogen atoms were removed and their atomic value added to the heavy atom to which they were bonded. Polar hydrogen atoms were considered explicitly. Physicochemical properties were then assigned to each atom. Four parameters were used to represent these properties: Gasteiger partial charges . Bo. Bo32]. 379Val and 380Val in the S1' sub-site of the C-domain of sACE provides hydrophobic interactions between the sub-site and the P1' residue of inhibitor molecules, such as the P1' methyl group of captopril and enalaprilat pyrimidine inhibitors assigned by the TMACC interpretation showed the C8 atom of quinazoline inhibitors to be identified as positive for activity, whilst the N8 of pyrido pyrimidines was most frequently negative for activity. This interpretation reflects experimental evidence, which indicates the presence of a C8 (quinazoline) is more potent for DHFR inhibition than an N8  .115Val in human DHFR [22Leu may be formed by these groups [The presence of a 5-methyl group increases inhibitor potency by forming hydrophobic interactions with man DHFR . Methyl e groups . The bene groups , 51. ThiThe TMACC interpretation identified 5-methyl groups as positive for activity, consistent with experimental data, which suggests this feature is important for the inhibition of human DHFR. The interpretation also identified methyl groups in the 9 and 10 positions as positive for activity. This is consistent with the known hydrophobic interactions formed by these features within the DHFR active site . The TMAAnalyses of the TMACC QSARs modelled for the ACE and DHFR data sets have shown that the TMACC interpretation can identify distinctive features of a structure-activity relationship. The TMACC interpretation provided a clear and precise representation of the activity of specific groups. Amalgamation of the atomic activity values determined for such groups within a data set, showed strong correlation with experimental evidence, which shows the TMACC interpretation can produce models which accurately depict the features of a structure-activity relationship.Overall, the TMACC interpretation modelled the ACE inhibitor structure-activity relationship highlighted important features for C-domain selective ACE inhibition. The TMACC interpretation provided a consistent representation of the structure-activity relationship present in the ACE data set. However, the insight into the structure-activity relationship of ACE inhibitors produced by the TMACC interpretation was limited by the size of the data set. To obtain a more detailed analysis of components important or detrimental to the ACE inhibitor structure-activity relationship, it would be necessary to investigate a data set which represents a comprehensive range of functional groups and structural components. Investigation of the activity of features important for C-terminal domain selective inhibition in comparison to features important for N-terminal domain selective inhibition would provide further insight into the interpretive ability of the TMACC descriptors.An inherent weakness, due to the 2D nature of the TMACC descriptor, is insensitivity to chirality. However, the use of chirality descriptors derived from topological data may provide a solution to this limitation and may also improve the predictive ability of the QSAR models . Investi