In the electroencephalogram (EEG), early anticipatory processes are accompanied by a slow negative potential, the initial contingent negative variation (iCNV), occurring between 500 and 1500 ms after cue onset over prefrontal cortical regions in tasks with cue-target intervals of about 3 s or longer. However, the temporal sequence of the distributed cortical activity contributing to iCNV generation remains unclear. During iCNV generation, selectively enhanced low-beta activity has been reported. Here we studied the temporal order of activation foci in cortical regions assumed to underlie iCNV generation using source reconstruction of low-beta (13–18 Hz) activity. During the iCNV, elicited by a cued simple reaction-time task, low-beta power peaked first (750 ms after cue onset) in anterior frontal and limbic regions and last (140 ms later) in posterior areas. This activity occurred 3300 ms before target onset and provides evidence for the temporally ordered involvement of both cognitive-control and motor-preparation processes already at early stages during the preparation for speeded action. The anticipation of a response signal after presenting a cue evokes a contingent negative variation (CNV) in the human electroencephalogram (EEG)early CNV (iCNV) component has not been clearly shown yet.In addition to these heterogeneous findings, several studies have investigated the temporo-spatial pattern of brain activity underlying the CNV globally, that is, without differentiating early and late components During the iCNV, selectively enhanced oscillatory beta power was found in the EEG EEG beta rhythms have been found to represent activity in pyramidal cells subserving information transfer between distant brain structures The main goal of this study was the identification of areas engaged in preparatory processes during the typical iCNV interval and the temporo-spatial pattern of their activity as revealed by event-related beta oscillations. The activity pattern was examined by analyzing the dynamics of the cortical projections of the time-varying cross-spectral density of the low-beta band.The study was approved by the local ethics committee of the Hospital of the University of Tuebingen. All subjects gave written informed consent prior to entering the study.Twenty-eight healthy subjects  participated in a forewarned reaction-time task. The paradigm consisted of an auditory cue , followed 4200 ms later by a target tone ; the intertrial interval was 12 s. Stimuli were presented via in-ear phones. Subjects were instructed to keep their eyes closed, to hold their right hand on a computer mouse placed on the table in front of them, and to respond as quickly as possible to S2 with a click on the left mouse button. The data reported here resulted from one of three conditions of an experiment. The total duration of one condition was 20 min.The EEG was recorded at 56 positions of the 10/10 system. Activity at both mastoids was recorded for optional re-referencing; two ground electrodes were placed at the earlobes. The vertical and horizontal eye movements were recorded with separate electrodes and were used later in addition to the 10/10 EEG electrodes for blind source separation with the second-order blind identification algorithm (SOBI) The values of the perturbations were averaged for frequencies between 13–18 Hz and for the time window starting 480 ms and ending 1660 ms after S1 onset. The averaged values were tested against the averaged value of a baseline interval (1502-314 ms before S1) for each channel.http://www.uzh.ch/keyinst/loreta.htm) was used to compute the cortical three-dimensional distribution of current density. The eLORETA method is a discrete, three-dimensionally distributed, linear, weighted minimum norm inverse solution. The eLORETA method has no localization bias even in the presence of structured noise and therefore is an improvement over the previously developed tomographies LORETA The exact low resolution brain electromagnetic tomography (eLORETA) software . Subsequently, current source density of low-beta (13–18 Hz) oscillations was estimated in eLORETA for 6239 cortical voxels as a function of time. The mean of the baseline interval (380-32 ms before S1) was statistically tested against the mean of the iCNV interval (500–1500 ms) with a paired t-test without additional normalization and logarithmization. A permutation method using 5000 randomizations established the critical value for a significance level of 1%. Clusters that showed t-scores above 5.3 were used for further analysis.At the frontal electrodes, a negative wave occurred during the time interval of the iCNV, whereas a positive wave occurred at posterior electrode positions. Event-related spectral perturbations around the 13-Hz band were observed over anterior as well as posterior regions see .2 = .117]. Simple contrasts showed that beta power was significantly higher at the left-central frontal cluster compared to the right frontal , the left-central parietal , and the right parietal  clusters. As illustrated in The Bonferroni-corrected p-values of the paired t-tests showed significant perturbations in the iCNV interval for all EEG channels. The highest values for event-related spectral perturbations in the low-beta band were found in a left-central frontal cluster. This cluster included the electrodes AF3, AFz, F5, F3, F1, and Fz. The mean of this cluster  was tested against the mean of a right-frontal cluster , a left-central posterior cluster  and a right-posterior cluster . The Greenhouse–Geisser-corrected ANOVA for repeated measures showed a main effect of clusters  and including left middle frontal cortex and anterior cingulate gyrus. Simple contrasts showed that in this cluster, the 95% threshold was reached significantly earlier (p<.05) than in seven other, more posterior cortical clusters  between time to threshold and MNI y-coordinate (anterior–posterior axis) of the 10 selected clusters.During the iCNV interval, we found a broad distribution of significant event-related spectral perturbation for beta power during the interval of the early component of the CNV. Similar to Molnar et al. A broadly distributed pattern of activity was also reported by previous studies using paradigms that evoked iCNVs and our regions overlapped with regions related to the CNV generation in previous fMRI studies The temporal order of this neural activity, however, could not be elucidated because of the insufficient time resolution of fMRI. Our major aim was to uncover the temporal order in the interplay between different parts of the network that are active 550 to 1450 ms after cue onset. Our results revealed a temporal order of cluster-specific maxima in beta amplitudes in anterior–posterior direction: First, beta power peaked significantly earlier in anterior than in posterior areas. Second, there was a significant correlation between clusters y-coordinates (anterior–posterior axis) and the onset latencies of the beta peaks in the different clusters. Therefore, brain activity during the iCNV-related time interval (500–1200 ms after cue onset) appears to be characterized by a specific temporo-spatial pattern, evolving from anterior to posterior regions.The early activity in the left and mid-ACC provides additional evidence for the crucial role of the ACC for the cue-induced expectation of upcoming events. The specific role of the ACC during the iCNV time interval might, at least in part, lie in the motivationally guided mobilization of preparatory processes via the adjustment of cortical arousal in order to optimize upcoming information processing. The ACC may realize this mobilization via pyramidal cell connections with the amygdala and the locus coeruleus These findings suggest that these areas may be involved in activating the appropriate task set by modulating computational activity in more posterior areas. It is tempting to conjecture that the parallel early activation peaks in ACC, insula and PFC reflect the initiation of two parallel streams of control, a basal energetical and a higher-order computational one, that are both essential for optimal task performance. Coull et al. In conclusion, the increased low-beta activity may reflect activity in long axons of pyramidal bundles


Decreasing a number of hospital admissions is important for improving outcomes for people with schizophrenia. The Information Technology Aided Relapse Prevention Programme in Schizophrenia (ITAREPS) programme enables early pharmacological intervention in psychosis by identification of prodromal symptoms of relapse using home telemonitoring via a phone-to-PC SMS platform.This study was a 1-year extension of a previously published mirror-design follow-up evaluation of programme clinical effectiveness. In total, 73 patients with psychotic illness  collaborating with 56 family members participated in the clinical evaluation.There was a statistically significant 77% decrease in the number of hospitalisations during the mean 396.8 ± 249.4 days of participation in ITAREPS, compared with the same time period before participation in ITAREPS , as well as significantly reduced number of hospitalisation days when in the ITAREPS .The ITAREPS programme represents an effective tool in the long-term treatment of patients with psychotic disorders. Studies published so far suggest that crisis interventions such as increasing the dose of antipsychotic medication, based on early detection within a relapse prevention programme, reduce readmission rates in schizophrenia. Previous findings from ITAREPS mirror-image design follow-up showed a statistically significant 60% decrease in the number of hospitalisations during the mean 283.3 ± 111.9 days of participation in ITAREPS compared with the same time period before entering the programme .The ITAREPS enables early pharmacological intervention in psychosis by identification of prodromes of relapse using home telemonitoring via a phone-to-PC SMS platform. ITAREPS demonstrated sustained effectiveness for 1 year after the conclusion of the previous mirror-image design follow-up, decreasing the total number of hospitalisations by 77% and the number of hospitalisation days by 58% respectively, during the time spent in ITAREPS compared with a period of equal length prior to ITAREPS enrolment.The need for providing more efficient healthcare services, coupled with major advancements in information and high-tech communication technology, has led to novel approaches to medical treatment during the last decade. E-health technologies combine the use of electronic communication and information technology in the healthcare for clinical, education and administrative purposes.The Information Technology Aided Relapse Prevention Programme in Schizophrenia (ITAREPS), developed by the Prague Psychiatric Centre in the Czech Republic, presents a mobile phone-based e-health solution for remote patient monitoring and disease management in schizophrenia and psychotic disorders in general.The main incentive behind ITAREPS development was emerging evidence that acute psychotic state may result in certain pathophysiological processes in the central nervous system (CNS) responsible for deterioration of the overall clinical condition . IncreasThese findings emphasise the need for a decrease in the number of relapses as one of the main goals of long-term treatment of psychotic illness.The Information Technology Aided Relapse Prevention Programme in Schizophrenia-mediated telemonitoring identifies early warning symptoms of an illness. It enables early pharmacological intervention and helps to prevent relapses and recurrent hospitalisations. Previous findings from a mirror-image design  follow-up evaluation of clinical effectiveness of the ITAREPS programme in 45 patients with psychotic illness  showed aThe main aim of this clinical evaluation was to confirm sustained effectiveness of the programme, one additional year after the conclusion of the previous mirror-image design follow-up.Each participant, a patient and her/his family member enrolled in the ITAREPS programme, completes a 10-item Early Warning Signs Questionnaire . Reporting on psychometric properties and structure of EWSQ has been published elsewhere ,9. ReminIf a total EWSQ score exceeds a given score threshold, an automatically generated ALERT is declared and a treating psychiatrist is notified by an e-mail message. According to the Early Intervention Algorithm (EIA), a part of ITAREPS, the presence of early warning signs warrants an immediate increase in baseline maintenance dose of antipsychotic by 20% within the next 24 h. However, data about nature of early intervention were not captured.http://www.itareps.com) offers an authorised physician, a user-friendly process to enter patient data. It also provides summaries and continuous reporting on current clinical status of each patient participating in the ITAREPS.An ITAREPS web-based interface -10 criteria for diagnosis of schizophrenia, schizoaffective disorder or acute polymorphic psychotic disorder with or without symptoms of schizophrenia . Of the The following baseline patient data were obtained and entered by psychiatrists: demographic data; diagnosis; illness history; Clinical Global Impression Severity Scale (CGI-S) and current medication. Compliance was evaluated by psychiatrists using a questionnaire with a 5-point scale [one = always and five = never taking antipsychotic medication ].As this follow-up used only clinical information without specific patient identifiers and the procedures required no deviation from standard clinical practice, informed consent was not obtained from participants. The protocol of ITAREPS programme was approved by the Ethics Committee of the Prague Psychiatric Centre.The number of hospitalisations was assessed individually for each patient during the period of her/his participation in ITAREPS programme compared with equal period of time prior to ITAREPS enrolment.Z = 4.86, p < 0.00001).In total, there were 14 hospitalisations during a mean of 396.8 ± 249.4 days follow-up compared with 60 hospitalisations during the same length of time before entering the programme. The results showed a statistically significant reduction of 77% in the number of hospitalisations during the ITAREPS programme participation .There was a statistically significant reduction of 58% in the number of hospitalisation days during the programme participation compared with the mirror period before the ITAREPS entry  during the follow-up. The participants′ high co-operation was set up as at least 70% of the expected SMS with the EWSQ rating were delivered to the system.There was 100% decrease in the number of hospitalisations (from 25 to zero) in patients within highly co-operative patient/family member pairs .This finding remains unchanged from the previous follow-up results where 100% decrease in the number of hospitalisations (from 13 to zero) was observed in patients from highly co-operative patient/family member pairs , length of participation in the ITAREPS programme, number of ALERT messages, involvement of a family member or separately assessed co-operation of a patient or a family member respectively. A controlled randomised study should be warranted to find out what effect these variables have on effectiveness of ITAREPS.However, the group of patients without hospitalisation showed a significantly higher proportion of highly co-operative patient/family member pairs compared with subjects with hospitalisation during ITAREPS programme participation .The effectiveness of the ITAREPS programme, in conjunction with its low set-up and operating costs, makes ITAREPS an attractive option in the long-term treatment and management of patients with schizophrenia and psychotic disorders in general. Albeit user-friendly and easy-to-understand system with minimal eligibility requirements for patient implying its utilizability in common clinical settings, the programme applicability in severely ill, less compliant subjects warrants further investigation.The results of the clinical follow-up should be interpreted with caution because of methodological limitations of the mirror-image design . In addition, although a specific pharmacological intervention was recommended according to EIA, the exact nature of the intervention during the ALERT states could not be documented in this clinical evaluation.Therefore, a prospective, double-blind, randomised trial with the ITAREPS programme is warranted to confirm these promising findings.


The incorporation of disease-associated covariates into studies aiming to identify susceptibility genes for complex human traits is a challenging problem. Accounting for such covariates in genetic linkage and association analyses may help reduce the genetic heterogeneity inherent in these complex phenotypes. For Genetic Analysis Workshop 15 (GAW15) Problem 3 simulated data, our goal was to compare the power of several two-stage study designs to identify rheumatoid arthritis-related genes on chromosome 9 (disease severity), 11 (IgM), and 18 (anti-cyclic citrinullated protein), with knowledge of the answers. Five study designs incorporating an initial linkage step, followed by a case-selection scheme and case-control association analysis by logistic regression, were considered. The linkage step was either qualitative-trait linkage analysis as implemented in MERLIN-nonparametric linkage (NPL), or quantitative-trait locus analysis as implemented in MERLIN-REGRESS. A set of cases representing either one case from each available family, one case per linked family (NPL ≥ 0), or one case from each family identified by ordered-subset analysis was chosen for comparison with the full set of 2000 simulated controls. As expected, the performance of these study designs depended on the disease model used to generate the data, especially the simulated allele frequency difference between cases and controls. The quantitative trait loci analysis performed well in identifying these loci, and the power to identify disease-associated alleles was increased by using ordered-subset analysis as a case selection tool. There are many possible mechanisms by which environmental or clinical covariates may either influence the risk of complex human diseases directly, or partially account for genetic heterogeneity. For example, they may act as independent environmental risk factors, or increase the disease risk in concert with genetic susceptibility via gene × environment interaction, or define a more homogeneous subgroup of patients in which the main effect of a particular susceptibility gene is more apparent. The purpose of our analysis of the Genetic Analysis Workshop 15 (GAW15) simulated data was to evaluate the power of several two-stage study designs consisting of separate linkage and association analysis steps. These study designs incorporated disease-associated continuous covariates in several different ways. Power comparisons were focused on two distinct factors: 1) thresholds used for the linkage analysis step, which determined the subset of markers included in a subsequent case-control association analysis, and 2) criteria for selecting cases (one per family) to include in this association analysis.With knowledge of the answers, we analyzed the simulated GAW15 microsatellite and SNP data on chromosomes 9, 11, and 18 in an attempt to detect the loci responsible for three disease-associated covariates: disease severity, IgM, and anti-cyclic citrinullated protein (CCP) values, respectively. To investigate Type I error, we also analyzed the relationship of anti-CCP values and genotypes on chromosome 15, which does not harbor any disease-associated loci ("null chromosome"). We analyzed covariate and genotype data from all 1500 nuclear families, and genotype data from all 2000 unrelated controls. We used the MERLIN package  and the p-value threshold 0.05, 10-cM region centered on the linkage peak) and loose . Each design consisted of two stages. If the first-stage linkage analysis of 1500 families using the microsatellite marker map met the linkage threshold, it was followed by a second-stage association analysis of the SNPs in the linkage region in unrelated cases (one per family) and 2000 controls, using logistic regression with an additive allele coding. The case selection strategies are summarized in Table p-value from the logistic regression survived the Bonferroni correction for the number of analyzed markers.We examined five distinct study designs Table , each ofTable p-value thresholds affect the type I error rate of the Stage 2 association analysis.It was previously shown that linkage and association test statistics are statistically independent under the null hypothesis of i) no linkage and no association; ii) linkage and no association; iii) association and no linkage . ConsistOur study demonstrates that the incorporation of disease-related covariates into a combined linkage and association analysis can help identify genes that contribute directly or indirectly to the risk of RA. Specifically, results for chromosome 18 show that the efficiency of a case-control association analysis can be greatly increased when linkage and covariate information are used to select the cases. For the simulation models used to generate the GAW15 data, the OSA method worked particularly well in this regard because it uses both the family-specific identify-by-descent (IBD) sharing information, and the relationship between covariate distribution and IBD sharing across families to enrich the case sample for the disease allele of interest. For the data sets simulated here, the "linked best" strategy (Design B in our study) was able to achieve the exact same power as Design A with a 34% reduction in the number of analyzed cases, even though it ignored covariate information  difference between cases and controls and almost complete LD with the causal allele  resulted in improved power for the association analysis (from near 0 to 20–30%). However, because OSA used family-specific NPL scores for the binary affection status as input, regardless of disease severity, the overall power of Design E remained low (16% at best). A family-based association analysis of disease severity with the QTDT (quantitative transmission-disequilibrium test) package  or a logp-value threshold, improved power for chromosome 18, and to a lesser extent chromosome 11, since it identified a subgroup of cases with reduced allelic heterogeneity, even though the linkage evidence in this subgroup continued to be low. In real data sets, a SNP map of the density simulated here is unlikely to include SNPs in high enough LD with susceptibility or quantitative trait loci to detect strong association signals, and the two-stage approach presented here continues to be of practical importance.The GAW15 data provided very weak linkage signals for the three loci considered here, presumably due to substantial within-family heterogeneity with respect to the simulated disease loci Table . This maThe author(s) declare that they have no competing interests.


Chromatin immunoprecipitation combined with massively parallel sequencing methods (ChIP-seq) is becoming the standard approach to study interactions of transcription factors (TF) with genomic sequences. At the example of public STAT1 ChIP-seq data sets, we present novel approaches for the interpretation of ChIP-seq data.in vivo. While a large majority of genomic sites with high ChIP-seq signal is associated with a nucleotide sequence ressembling a STAT1 binding site, only a very small subset of the over 5 million potential STAT1 binding sites in the human genome is covered by ChIP-seq data. Furthermore a surprisingly large fraction of the ChIP-seq signal (5%) is absorbed by a small family of repetitive sequences (MER41). We compare recently developed approaches to determine STAT1 binding sites from ChIP-seq data. Assessing the content of the established consensus sequence for STAT1 binding sites, we find that the usage of “negative control” ChIP-seq data fails to provide substantial advantages. We derive a single refined probabilistic model of STAT1 binding sequences from these ChIP-seq data. Contrary to previous claims, we find no evidence that STAT1 binds to multiple distinct motifs upon interferon-gamma stimulation The observation of the binding of activated STAT1 protein to a specific repetitive element bolsters similar reports concerning p53 and other TFs, and strengthens the notion of an involvement of repeats in gene regulation. Incidentally MER41 are specific to primates, consequently, regulatory mechanisms in the IFN-STAT pathway might fundamentally differ between primates and rodents. On a methodological aspect, the presence of large numbers of nearly identical binding sites in repetitive sequences may lead to wrong conclusions about intrinsic binding preferences of TF as illustrated by the spacing analysis STAT1 tandem motifs. Therefore, ChIP-seq data should be analyzed independently within repetitive and non-repetitive sequences. The precise spatial and temporal regulation of gene expression remains poorly understood despite an increasing number of species with nearly complete genome sequences available. Proteins which regulate the expression of genes by binding to specific DNA sequences in the vicinity of their targets have been termed transcription factors (TFs). The nucleotide sequences of observed binding sites generally display a considerable variation, which may cause difficulties in the description of binding preferences using sequence motifs. Recent benchmarking studies confirmed that transcription factor binding sites (TFBS) prediction based on statistical motif discovery approaches is unreliable and thus remains a major bottleneck in the study of transcriptional regulatory regions Methods for interpreting ChIP-seq data are currently under intensive development. A common aspect of emerging solutions includes the possibility to recognize and process separately sequence reads from the + and the − strand of pulled down fragments STAT1 is a member of an intensively studied family of TFs with implications in the regulation of immune responses. In resting cells STAT1 is mainly located in the cytosol. Upon stimulation with the cytokine interferon gamma (IFN-γ), STAT1 is trans-located to the nucleus to bind to target DNA sequences. Early studies on relatively few binding sequences defined a consensus sequence TCCNNNGAA of the IFN-γ-activated site GAS . SubsequRepetitive sequence elements constitute almost half of the human genome, however their potential functions are still poorly defined. A number of recent studies either predict TFBS within repeat sequences The present re-analysis of ChIP-seq data extends the analysis of STAT1 binding for the first time to repetitive sequences. We furthermore show that ChIP-Seq data can be used to analyze the relation of in vivo interactions and extended sequence features such as spacing within regulatory modules of binding sites. A large number of virtually identical binding sites within repeats potentially induces a strong bias in a corresponding binding site model and could lead to wrong conclusions regarding preferential associations and constraints in distances to other TFBS. We present therefore approaches to analyze ChIP-seq data which also take into account the emerging roles of repeats in the regulation of gene expression and evolutionary aspects. All results shown in this article are based on public ChIP-Seq data described in ChIP-peak see , definesNext, we aim to compare the results of our peak detection approach to previously described STAT1 sites (or center positions of ‘binding regions’) derived from the identical ChIP-seq data set Position-specific scoring approaches using weight matrices (PWM) or Hidden Markow Models (HMM) feature superior performances as descriptors of binding preferences We use the PWM  derived in vivo STAT1 site with an algorithm reporting multiple motifs (MEME). In this case, the results obtained with the complete and repeat-filtered sets are strikingly different. With the complete set, we found three motifs with E-values in the order of 10−1000 or lower  and phylogenetic conservation are combined.This study aims at the identification of a reference set of robustly induced STAT1 binding sites upon IFN-γ stimulation. Many of the recent approaches in the analysis of ChIP-seq data use methods with similar underlying principles to determine genomic loci with elevated ChIP-seq signal (peak detection). Main differences consist in the determination of the threshold of signal intensities which separate signal from background noise. The noise level in ChIP-seq is still poorly characterized. Therefore the STAT1 binding motif obtained in the present study does not take into account putative low affinity sites. For the detection of low affinity binding sites, specialized approaches may be used The limited correlation of the predicted affinity (PWM score) and the observed STAT1 ChIP-seq signal as presented here suggests that genomic features additional to the nucleotide sequence determine the genomic binding of STAT1. Such mechanisms are likely generalized to all transcription factors and obvious candidates include cooperative effects between multiple DNA binding proteins. Epigenetic modifications may reduce the accessibility of the DNA by chromatin compaction The present study extends the analysis of STAT1 binding for the first time to repetitive elements. In distinction to previous reports on binding sites of other transcription factors within repeats ChIP-seq signals are at present not reliably predictable by features of genomic sequences. Extended analysis of ChIP-seq data in HeLa cells including additional DNA binding proteins The binding of STAT1 within repetitive sequences of the LTR class might relate to the description of functional GAS sites in retroviruses Our analysis confirms the previously described enhanced resolution of the ChIP-seq approach. However, ‘negative control’ data exploited by some of the current peak calling algorithms don't provide substantial advantages. We extend initial characterizations of STAT1 ChIP-seq data sets to binding sites within repetitive sequence elements. The selective induction of ChIP-seq signal at specific repeats upon cellular stimulation corroborates specific binding by STAT1. These observations bolster previous reports on binding sites of other TFs within repeats. Repetitive elements may however bring in biases deflecting the analysis of binding sequences. We recommend therefore an independent analysis of sequences derived from ChIP-seq data within repetitive and within non-repetitive genomic sequences, in order to avoid incorrect conclusions on general properties of binding sites.http://www.bcgsc.ca/downloads/chiptf/, providing 2 files with the genomic coordinates of 12.9 million, and 15.1 million mapped sequence tags of unstimulated and stimulated HeLa cells, respectively.The ChIP-seq data underlying this study are described in http://ccg.vital-it.ch/chipseq/, http://sourceforge.net/projects/chip-seq/. An SGA file is a tab-delimited text file with five obligatory fields per line: chromosome, feature name, position, strand, and a number representing the count of sequenced tags mapping to this position. The position field corresponds to the chromosomal position of the 5′ end of the mapped sequence tag, that is the beginning of the matching region for tags on the positive (+) strand, or the end position of tags on the negative (−) strand. SGA files are sorted by chromosome, position and strand, allowing for rapid, sequential processing by downstream analysis tools.The source files were converted in SGA (Simple Genome Annotation) format, the working format of our ChIP-Seq tools available at The average length of the pulled-down fragments was estimated with the aid of the ChIP-cor program of the ChIP-seq tools. The ChIP-cor program generates a histogram indicating how many times a − strand tag is found at a particular distance of a + strand tag. In ChIP-seq experiments, + and − strand tags tend to occur in equivalent numbers in clusters around the transcription factor binding site. The relative displacement of + and − tags visualized by ChIP-cor serves as estimation of the typical fragment size, found to be 140 bp in these ChIP-seq experiments. An additional ChIP-center program generates “centered” SGA files by adding or subtracting a user-defined distance from the positions of the + and − tags, respectively, in the input SGA files. The strand field is changed to 0 to reflect the unoriented nature of the center positions defined in the output SGA file. Based on the estimated fragment length, we used a centering distance of 70 bp for SGA files of both stimulated and unstimulated ChIP-seq experiments.The centered SGA files were used as input to the peak detection program ChIP-peak. The following summarizes the concepts of the ChIP-peak program. Each line of the sorted input SGA file is considered as candidate peak, for which the total number of mapped tags in a window of chromosomal positions is computed using neighboring lines in the SGA file. This window is centered on the position, with a user-specified width. In order to be retained as a peak, a candidate position must have at least a threshold number of total tags. Moreover, it must be the position with the highest number of counts within a so-called vicinity range, an additional user-specified parameter of the width of a position-centered window. If an input SGA line is retained as a peak, its position may be optionally redefined as the center of gravity of the tag counts in the surrounding window. The weight of sporadically occurring positions with suspiciously high tag counts can be decreased by a user-specified count cut-off value. Counts in the input file exceeding this value are replaced by the cut-off value. In this work, we used a window width of 200 bp, and a vicinity range of 400 bp, a (stringent) threshold of 50 counts, and a permissive count cut-off value of 999999. A posteriori peak refinement was turned off.The refinement of the STAT1 binding motif description was carried out using the MArkow MOdeling Tool MAMOT  startingib is the probability of base b at binding site position i, and wib is the corresponding weight in the scoring matrix. The function int rounds the argument to the nearest integer. The choice of the scaling factor 3/log(2) is arbitrary. Note that 3 score units correspond to a factor of 2 in terms of base frequencies. Putative binding sites can be scored by aligning their nucleotide sequence to the PWM and sum the matching scores over all positions.The initial model considers alternative spacer lengths of 2, 3 and 4 bases between the consensus half-site motifs TTC and GAA in the initial model. The resulting trained HMM assigned very low probabilities (below 1%) to the spacing classes 2 and 4, which allows to ignore these classes and to represent the STAT1 binding specificity by a standard position weight matrix (PWM) with spacer length 3. The probability matrix extracted from the trained HMM , and the natural logarithm was calculated of the ratio ‘stimulated’ over ‘unstimulated’ (log ratio).http://ccg.vital-it.ch/chipseq/) and applying count density normalization.PhastCons scores on human genome coordinates derived from the 17-way vertebrate genomes alignment were obtained from the UCSC genome browser File S1List of 4446 STAT1 binding sites in sga format (uploaded to ChIP-seq web server).(0.14 MB TXT)Click here for additional data file.File S2List of 4446 STAT1 binding sites in bed format (uploaded to UCSC Genome Browser).(0.15 MB TXT)Click here for additional data file.


Baculovirus-expressed HIV-1 Pr55gag Virus-Like Particles (HIV-VLPs) induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with a production of Th1- and Th2-specific cytokines.The analysis of genomic transcriptional profile of MDDCs, obtained from normal healthy donors and activated by HIV-VLPs, show the modulation of genes involved in the morphological and functional changes characterizing the MDDCs activation and maturation. Similar data are obtained using peripheral blood mononuclear cells (PBMCs), without further selection, showing the feasibility of a direct and “simplified” experimental procedure.ex vivo generated MDDCs, can be observed also in CD14-expressing freshly derived PBMCs, with the possible identification of genetic predictors of individual response to immunogens.The results here described show that the maturation pattern induced by HIV-VLPs in  Dendritic cells (DCs) are professional antigen-presenting cells (APC) able to initiate immune responses ,2. Immatin vitro from peripheral blood monocytes, generating monocyte-derived DCs (MDDCs)  , washed and then fixed with 2% paraformaldehyde for analysis with a FACScalibur flow cytometer (BD Pharmingen). Data analysis was carried out with FlowJo software . The cell fraction that responded by upregulation of activation markers on the cell surface was calculated by overlaying the histograms of treated and untreated cells and Overton subtraction of the curves.DCs were harvested, washed twice in PBS and lysed in 350ul RLT buffer with fresh addition of 2-Mercaptoethanol per each well of the 6-well plate. Total RNA was isolated using RNeasy minikits (Qiagen), according to the manufacturer's protocol, and RNA quality and quantity was estimated by Agilent Bioanlayzer  and NonoDrop. Amplified antisense RNA (aRNA) was obtained from total RNA (0.5-3 µg) via two round of in vitro transcription, according the protocol previously described . 6ug of ) and retrieved after median centered, filtering of intensity (>300) and spot elimination . Data were further analyzed using Cluster and TreeView software [et al. [Hybridized arrays were scanned at 10-µm resolution on a GenePix 4000 scanner (Axon Instruments) at variable PMT voltage to obtain maximal signal intensities with less than 1% probe saturation. Resulting jpeg and data files were deposited at microarray data base (mAdb) . Amplified antisense RNA (aRNA) was obtained from total RNA extracts  and hybrex vivo purified MDDCs and in vivo “unselected” PBMCs.Supervised cluster analysis obtained on either MDDCs or PBMCs samples stimulated with HIV-VLPs shows the segregation of the untreated from the HIV-VLP-treated samples, at a significance threshold of univariate test <0.005 Fig. A and B. The differential gene expression in HIV-VLP-treated DCs, compared to untreated samples, was considered statistically significant only when supported by a p<0.005, and treatment–induced changes in gene profiling were analyzed using Student's t test. Considering only genes showing at least a 1.5-fold modulation (increase or reduction) in the transcriptional levels, it has been possible to identify unique genes in the profile induced by HIV-VLPs  populations. This observation confirms our previous report, showing that HIV-VLPs induce a specific transcriptional profile pattern in MDDCs, distinct from either PBS or LPS treatment .In particular, merging the genes modulated in the analyses performed on MDDCs and PBMCs, a significant set (>100) of common genes with a differential (up or down regulation) transcription has been identified. Moreover, a specific subset of these genes (>25) shows a differential transcription >2 in both analyses, suggesting the recognition of a strong transcriptional “signature” pattern common to both cell target populations Fig. .The PBMCs transcriptional profile was analyzed on the basis of the cellular pathways modulated by the HIV-VLP-treatment. In respect to the main focus of this study, only the pathways involved in immune activation have been evaluated in more detail.ex vivo derived MDDCs [The HIV-VLPs induction of PBMCs results in the activation of genes associated with antigen presentation functions. A set of cytoskeletal genes that may potentially mediate shape change and migratory behavior of activated APCs is also observed. The increase in the expression of immune cytokines, chemokines, and receptors contribute to the recruitment of monocytes, DCs, and macrophages to the site of infection. Moreover, they modulate both innate and adaptive immune response, including the polarization of Th cells, and the down-regulation of the inflammatory response, which may significantly interfere with the immune response. The induction of signaling genes and transcription factors may be involved in preparing the DC to be receptive to regulatory signals in the lymphatics and lymph nodes. All these categories of genes are extremely similar to what observed by us upon HIV-VLP induction of ed MDDCs .It is extremely interesting that among the common upregulated genes, identified in the analyses performed on MDDCs and PBMCs, some of them are intimately related to the immune activation . In particular, CCL3, CCL4 and CCL20 are chemokines actively participating in the host response to invading pathogens by regulating the trafficking and activation stage of inflammatory cells, including the Toll-like receptor signaling pathway. They all exert similar effects on monocytes and are potent chemoattractants for lymphocytes and dendritic cells. More specifically, CCL3 selectively attracting CD8+ Tcells, CCL4 selectively attracting CD4+ Tcells and CCL20 promoting the adhesion of memory CD4+ Tcells -39.Collectively, the transcriptional profile induced by HIV-VLPs in freshly drawn PBMCs reflects a significant cellular and immunological reprogramming of cells directly involved in the host immune response.ex vivo generated MDDCs, can be observed also in CD14-expressing freshly derived PBMCs. Moreover, the genomic transcriptional profile induced by HIV-VLPs in PBMCs shows the activation of unique genes and cellular pathways, reflecting a distinctive cellular and immunological reprogramming of circulating cells deputed to trigger the host immune response.The results here described show that the maturation pattern induced by HIV-VLPs in ex vivo generated MDDCs identifies a cluster of genes whose expression is similarly modulated, indicating that a specific transcriptional signature is found between ex vivo generated MDDCs and “unselected” PBMCs.A supervised Eisen's clustering analysis confirms the specificity of the observation, given that the PBMCs samples, derived from the HIV-VLP treatment, cluster together indicating that the pattern of specifically modulated genes is consistent all across the analyzed samples. A comparison with the pattern induced by HIV-VLP treatment in Among the pathways and specific genes activated in PBMCs treated with HIV-VLPs, those directly involved in the biological functions as antigen presenting cells (APCs) have been analyzed in detail. The functional maturation and activation of CD14-positive cell population present in PBMCs by HIV-VLPs has been shown and, in particular, the activation of genes involved in cellular control  as well as in functional activity  has been observed.These results are extremely interesting, indicating the sensibility and the specificity of the gene transcriptional profile analysis for identifying in PBMCs, regardless the heterogeneity of cell populations unrelated to the host immune response, a cluster of genes whose transcription is specifically modulated in response to an antigen, such as the HIV-VLPs. The cell populations likely involved in this process are the blood DCs , as well as the additional professional APCs such as Macrophages and B-cells. Their specific individual involvement in the observed reponse will be further evaluated.ex vivo generated MDDCs [ex vivo selection of DCs. If the reported data will be confirmed on a larger scale, the possibility of screening the donor susceptibility to an antigen treatment using PBMCs would greatly simplify the identification of “responsive” vaccinees and the understanding of eventual failures in individuals enrolled in clinical trials.These results confirm the data obtained by HIV-VLP treatment of selected ed MDDCs . In thisMicroarray approach allows quantitative and simultaneous analysis of gene expression of a large amount of genes and the systematic studies of expression patterns are extremely useful for identify molecular events and key pathways involved in cellular functions induced by specific stimuli. In particular, data on the global pattern of gene expression underlying the modifications induced by HIV-VLPs in PBMCs, at early stages of antigen administration, may be extremely helpful for the identification of exclusive activation markers to trace the biological effects of modifications/optimizations of the HIV-VLP vaccination strategy.The authors declare that they have no competing interests.LB carried out experiments on DCs and PBMCs; wrote the article; AM carried out gene expression experiments on PBMCs; EA carried out gene expression experiments on DCs; EW supervised gene expression experiments on PBMCs; MLT participated to production of VLPs; GKL participated in designing experiments on DCs and PBMCs; FMM participated in designing gene expression experiments; FMB supervised the overall project.


This microarray platform changes the conventional view of transcript analysis since it allows the evaluation of the expression level of a transcript by querying each exon component. The Exon 1.0 ST platform does however raise some issues regarding the approaches to be used in identifying genome-wide alternative splicing events (ASEs). In this study an exon-level data analysis workflow is dissected in order to detect limit and strength of each step, thus modifying the overall workflow and thereby optimizing the detection of ASEs.A new microarray platform  do not affect the efficacy of statistical tools in detecting ASEs. However, data pre-filtering is mandatory if the detected number of false ASEs are to be reduced. MiDAS and Rank Product methods efficiently detect true ASEs but they suffer from the lack of multiple test error correction. The intersection of MiDAS and Rank Product results efficiently moderates the detection of false ASEs.To optimize the detection of ASEs we propose the following workflow: i) data pre-filtering, ii) statistical selection of ASEs using both MiDAS and Rank Product, iii) intersection of results derived from the two statistical analyses in order to moderate family-wise errors (FWER). Whilst the conventional Affymetrix GeneChips feature a probe set consisting of 11–20 probes selected from the 3' end of the mRNA sequence, the new all-exon arrays have 4 probes selected from each putative exonic region. To generate the target, Exon 1.0 ST arrays use T7 linked random hexamers for cDNA synthesis, instead of those of all previous Affymetrix expression arrays, which employed an oligo-dT linked T7 and thus required an intact poly-A tail. Importantly, this new WT Sense Target Labeling Assay generates DNA targets and therefore results in DNA/DNA duplex formation during hybridization, as opposed to DNA/RNA hetero-duplexes in conventional arrays. It has been shown that there is close agreement between the conventional Affymetrix 3'IVT arrays and the new Exon 1.0 ST arrays [The GeneChipThere has however been no way to date of defining the efficacy of this workflow or of different statistical methods in the detection of alternative splicing events. The ideal instruments to evaluate the effect of data pre-processing and the efficacy of different statistical methods on differential expressions are benchmark spike-in experiments , where aExon skipping events were generated using the experimental data, kindly provided by Abdueva . The AbdTo build the exon skipping benchmark experiment 4 out of the 5 groups of spike-in genes (20 out of 25 genes) were used. We focused on those because they were all part of the Exon 1.0 ST core annotation subset. The overall idea of the generation of synthetic exon skipping events is based on the availability of exon-level signals for spike-in genes. Therefore, it is possible to create new genes characterized by skipping events combining, for the same gene, exon-level expressions derived from different spike-in concentrations. An example is given in figure 2Intensity (log2I) measured upon a spike-in of 128 pM unless the exon skipped, which has the log2I measured upon a spike-in of 32 pM  within each synthetic gene , in order to retain only those genes where the skipping event represents the smallest intensity signal or Splice Index  = -2 for the comparison of the 512 versus the 128-32 subset and by log2(32/128) = -2 for the comparison 128 versus 32-2 subset. It is instead log2(2/32) = -4 for the comparison 32 versus 2-0 subset.To identify exon-skipping events a comparison between two different conditions, i.e. unspliced versus spliced, is needed. Detection of exon-skipping events for the subset 128-32 was done comparing it to the unspliced set spiked-in at 512 pM , for the subset 32-2 comparing it to the unspliced set spiked-in at 128 pM  and for the subset 2-0 comparing it to the unspliced set spiked-in at 32 pM . These comparisons embed a certain level of differential expression at gene level. The expected gene-level differential expression is given by logRMA and PLIER algorithms were used to combine the intensities belonging to the probes of each probe set to form one expression measure for each gene/exon-level probe set (summarization). The effect of these summarization methods on detection of alternative splicing events was investigated using MiDAS. A Receiver Operating Characteristic (ROC) curve was used to evaluate the effect of intensity summaries on alternative splicing detection . Specifically, using the exon-level probe set annotation information provided by Affymetrix, we removed all probe sets where all the probes in the probe set perfectly match more than one sequence in the putatively transcribed array design content as well as those where the probes either perfectly match or partially match more than one sequence in the putatively transcribed array design content. This filter could have an important effect on the correct association of the gene expression signal. However, it affects a very limited number of exon-level probe sets and therefore it does not produce an important reduction of the size of non-informative data Table . True Po annotation package . EGs are used to query ENSEMBL database and all ENSEMBL transcripts associated to any of them are retrieved. Subsequently, the filter procedure retains only those EGs associated to more than one ENSEMBL transcript. The EGs, retained by this filtering procedure, are mapped again to their gene-level probe sets.This filter uses the Affymetrix annotation that links each gene-level probe set to a specific GeneBank (GB) accession number (ACC), which represents the target sequence used to design the probes associated to a gene-level probe set. Then, Entrez Gene Ids (EGs) are retrieved querying with these ACCs a specific organism oriented Bioconductor Multiple mRNAs filter strongly reduces the number of core exons because it retains only exons of genes which are linked to multiple transcripts in the ENSEMBL database and for this reason it results to be more effective than the other filters as shown both in Table In EXON 1.0 ST GeneChips, to determine if a given probe signal is detected above background (DABG), its intensity is compared to a distribution of background probes with the same G/C content. A p-value  is computed representing the probability that the signal intensity is part of the null distribution. Specifically the DABG p-value filter, used in this work, is designed to retain only probe sets characterized by a DABG p-value ≤ 0.05 in all the arrays. Although this filter reduces the data set under analysis  and is faster since permutations are calculated only once and not gene by gene.We evaluated the efficacy of the detection of Alternative Splicing Events (ASEs), using a linear model based algorithm (MiDAS) and a permutation based algorithm (RP). MiDAS was applied on SI transformed data as RP was applied instead using SI, RPI  is the probability of making one or more false discoveries (FP) among all the hypotheses when performing multiple pairwise tests. Since FWER controlling procedures are often too conservative in high dimensional screening studies , they arThe semi-synthetic data set presented here represents a suitable instrument for testing the efficacy of new statistics for exon-level analysis. Furthermore, it allowed us to test the efficacy of a basic workflow Fig.  for ASEs. Rank Product (RP) is a non-parametric statistics that detects items that are consistently highly ranked in a number of lists and the significance of the detection is assessed by a non-parametric permutation test [, modifying the available implementation (Bioconductor [SI) or intensities (RPI) for all exon data set without considering their association to a specific gene and the significance of the detection is assessed using 500 permutations of those lists. Gene-level implementation of RP, i.e. running RP only on the subset of exons belonging to a specific gene, is computationally demanding and it is characterized by a very poor sensitivity (data not shown).Exon-skipping events were generated using experimental data kindly provided by Abdueva . MiDAS pion test . RP was onductor  RankProdThe modified RP method as well as all the filtering procedures are embedded in Bioconductor oneChannelGUI  package.Project name: Dissecting an alternative splicing analysis workflow;;Project home page: Operating system(s): Platform independent, require at least 8 Gb RAM;, Bioconductor 2.2 .Programming language: R 2.7.0 Licence: The Artistic License, Version 2.0;CDB developed the benchmark data set and carried out the analyses described in this work. FC participated in the benchmark data set preparation. RAC supervised the study.


Adeno-associated virus (AAV) is well known for suppression of oncogenesis in rodents, but its inhibitory effects on human carcinoma are less well understood. We report the differential ability of AAV to inhibit the tumorigenicity of two human cervical carcinoma cell lines. The wild-type AAV-2 DNA carried by a pSV2Neo vector was transfected into HeLa cells, which contain 50 copies of human papillomavirus type 18 (HPV-18), and SiHa cells, which contain 1-2 copies of HPV-16. About 1-3 copies of AAV genome were introduced per cell. AAV transfection moderately reduced the growth rate and anchorage-independent activity of the cells. In nude mice, the size of tumours arising from SiHa cells was reduced by 87%, in contrast to no reduction in tumour size arising from HeLa cells. This suggests that the differential suppression exerted by AAV may be due to differences in HPV copy number. To define the region that is responsible for the oncosuppression, mutation analyses were conducted. The results of nude mice assays showed that both the replication gene and inverted terminal repeats of AAV were important for the inhibition. This study may provide a model system for further studies on the underlying mechanism of AAV oncosuppressive activity.


Activation of hepatic stellate cells (HSCs) plays an important role in the development of cirrhosis through the increased production of collagen and the enhanced contractile response to vasoactive mediators such as endothelin-1 (ET-1). The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that is highly expressed in liver, kidneys, adrenals, and intestine. FXR is also expressed in HSCs and activation of FXR in HSCs is associated with significant decreases in collagen production. However, little is known about the roles of FXR in the regulation of contraction of HSCs. We report in this study that treatment of quiescent HSCs with GW4064, a synthetic FXR agonist, significantly inhibited the HSC transdifferentiation, which was associated with an inhibition of the upregulation of ET-1 expression. These GW4064-treated cells also showed reduced contractile response to ET-1 in comparison to HSCs without GW4064 treatment. We have further shown that GW4064 treatment inhibited the ET-1-mediated contraction in fully activated HSCs. To elucidate the potential mechanism we showed that GW4064 inhibited ET-1-mediated activation of Rho/ROCK pathway in activated HSCs. Our studies unveiled a new mechanism that might contribute to the anti-cirrhotic effects of FXR ligands. Hepatic fibrosis or cirrhosis is a chronic scarring process of the liver that is associated with increased and altered deposition of extracellular matrix (ECM). During the chronic liver injury, hepatic stellate cells (HSCs) undergo a process of transdifferentiation from a resting, fat-storing phenotype to a myofibroblast-like phenotype characterized by expression of fibroblastic cell markers such as α-smooth muscle actin (α-SMA). Activated HSCs produce increased amounts of ECM components that contribute significantly to the fibrotic changes in cirrhosis 4-induced liver injury The farnesoid X receptor (FXR) belongs to the superfamily of ligand-activated transcriptional factors. FXR has the typical nuclear receptor structure and can be activated by structurally different ligands, including several primary and secondary bile acid (BA) species conjugated to either taurine or glycine in vitro when cultured on plastic in serum-containing media, as evidenced by an induction of α-SMA at mRNA , ruling out the possibility that FXR ligands affect HSC contraction via regulating the expression levels of the components in the RhoA/Rho-kinase signaling.We then studied whether ET-1-induced activation of RhoA was altered in HSCs following treatment with GW4064. RhoA activities were determined by pull-down of GTP-RhoA. As shown in To test if the reduced ET-1-stimulated RhoA activation in GW4064-treated HSCs affects Rho-kinase activity, we studied the phosphorylation of the Rho-kinase substrate moesin at Thr-558 with a site- and phosphor-specific antibody. As shown in One of the major consequences of activation of RhoA/Rho kinase pathway is the phosphorylation of myosin light chains (MLC), which is critically involved in the contraction of HSCs. To further elucidate the mechanism by which GW4064 inhibits HSC contraction, we studied if GW4064 affects the ET-1-induced phosphorylation of MLC. As shown in We have confirmed and expanded previous reports that activation of FXR in HSCs inhibits their transdifferentiation. We have also shown for the first time that FXR ligands exert inhibitory effect on their contractile response to ET-1, a potent vasoconstrictor.It has been well established that activation of HSCs plays a key role in the development of cirrhosis A number of nuclear receptors have been reported to show inhibitory effects on HSC activation. These include retinoid X receptor, PPARs, pregnane-X-receptor, and FXR As an initial step to address this question we studied the effect of GW4064, a synthetic FXR ligand, on the expression of ET-1 and its corresponding receptors on stellate cells. As shown in Despite the inhibitory effect on ET-1 expression, GW4064 showed no effect on the expression of either ETA or ETB receptor in stellate cells. A study by Chi and colleagues showed that retinoic acid exerted an inhibitory effect on ETB expression in stellate cells but showed no effect on either ET-1 or ETA expression The freshly isolated stellate cells that were continuously treated with GW4064 for 7 days showed reduced contractile response to ET-1. This is unlikely due to changes in the expression levels of ET-1 receptors as shown in our RT-PCR studies. It is likely due to the fact that GW4064-treated cells are less activated and are equipped with less active contractile mechanism. It has been shown that α-SMA is involved in the stellate cell contraction After demonstrating a reduced contractile response in GW4064-treated, partially activated stellate cells we further observed a similar inhibitory effect of GW4064 in fully activated stellate cells. To understand the underlying mechanism we focused on examining the effect of GW4064 on RhoA/Rho kinase signaling as this pathway plays a key role in stellate cell contraction We then examined if GW6064 affects RhoA activation. Western analysis clearly showed that the ET-1-mediated activation of RhoA, measured as pull down of active GTP-RhoA, was significantly inhibited by GW4064 treatment. So far, the mechanism responsible for this inhibition is unclear. Preliminary studies showed that there were no significant changes in the mRNA expression levels of the receptors and several GTPase-activating proteins (p190 A-RhoGAP and p190 B-RhoGAP) and guanine nucleotide exchange factors  following treatment with GW4064 (data not shown). It remains to be determined if GW4064 treatment can induce any changes in the expression of these molecules at protein level. Nonetheless, the impaired activation of RhoA clearly resulted in a reduced activity of Rho-kinase, as shown by reduced phosphorylation of moesin, a member of the family of ezrin-radixin-moesin (ERM) proteins, which is phosphorylated at Thr-558 by Rho-kinase.The inhibition of RhoA/Rho kinase signaling is clearly implicated in the GW4064-mediated inhibition of stellate cell contraction as the phosphorylation of the downstream MLC is significantly decreased. Many studies have shown that, similar to smooth muscle cells, contraction of stellate cells is powered by myosin II through its action on the actin cytoskeleton, a process that is activated by phosphorylation of its myosin regulatory light chain In summary, in addition to its known anti-fibrotic effect, our study suggests a novel function of FXR in regulating intrahepatic vascular resistance through its inhibitory effect on stellate cell contraction. Although more studies are required to better understand the underlying mechanism, inhibition of RhoA/Rho kinase is likely to play a role in the FXR-mediated negative regulation of stellate cell contraction.All experimental manipulations and protocols in this study were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. The animals were housed and maintained in accordance with standards established in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (permit number: 0706848).in situ proteinase/collagenase perfusion followed by density gradient centrifugation as described Retired male Sprague-Dawley rats were used for primary HSC isolation. HSCs were isolated via Total RNA was extracted from cells with TRIzol reagent (Invitrogen) and the first-strand cDNA was synthesized by use of SuperScript III reverse transcriptase (Invitrogen). Real-time PCR analysis of rat genes was performed by use of SYBR Green-based assays with the ABI 7300 Real-Time PCR System  Immunodetection of actin2 was performed following our published protocol Protein extraction and Western blot analysis were performed as described previously Cells were treated with and without ET-1 (10 nM) for 10 min for all experiments in which RhoA was assayed. To the diluted buffer  in water, 10 µg/mL of aprotinin and 10 µg/mL leupeptin from Roche Molecular Biochemicals  were added. Cells were rinsed twice with PBS, and lysis buffer was added. Rho reagent slurry (Upstate) was added, samples were incubated, and beads were pelleted via brief centrifugation. Beads were resuspended in Laemmli-reducing buffer from Invitrogen  and boiled for 5 minutes. Supernatant and agarose pellets were mixed, and samples were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Invitrogen) and blotted to a nitrocellulose membrane. Anti-Rho antibody and mouse monoclonal IgG1 (Upstate) were applied for overnight, and appropriate secondary antibody was used for 1 hour. Chemoiluminescent from Pierce  was applied to the membrane for 5 minutes and developed on radiographic film.5 cells/mL) in DMEM supplemented with 10% fetal bovine serum and antibiotics, and plated on the collagen gels (2 mL cell suspension/well). After incubation overnight to allow cell attachment, serum-free conditions were introduced for 4 hours and the cells were washed three times with serum-free DMEM. Gels were detached from the plates. HSCs were treated with GW4064 for 18 hour. ET-1 (10 nM) was then added to the culture media just before the lattices were detached. Buffer without ET-1 was used as a control. The gels were photographed every hour for 6 hours. Surface area of the collagen gels was measured using digital image analysis software. All experiments were performed as triplicate.Contractility of HSCs was evaluated using collagen gel lattices on 24-well culture plates as described t test. Comparisons between three or more groups were made with analysis of variance followed by Tukey-Kramer post hoc analysis. In all cases, P<0.05 was considered statistically significant.All data are expressed as means ± S.E.M. unless otherwise stated. Comparisons between two groups were made with unpaired Student's


Astrocytic tumours often become resistant to a variety of chemotherapeutic agents in advanced stages and frequently possess mutations in the p53 tumour-suppressor gene. Previous studies using established cell lines to investigate the relation between mutated p53 genes and altered resistance to anti-cancer agents brought inconsistent results. In this report, we examined the status of the p53 gene in 56 astrocytic tumour specimens by single-strand conformation polymorphism and their in vitro chemosensitivity to 30 different kinds of anti-cancer agents. The chemosensitivity was determined by drug-induced cell death using flow cytometry. We found that the mutated p53 gene correlated with increased resistance to DNA-damaging agents but the sensitivity to anti-microtubule agents was independent of the mutation, suggesting a clinical significance of the status of p53 gene in astrocytic tumours and a rational application of anti-microtubule agents to the patients with p53-mutated astrocytic tumours.


SRC 531 mutation in colorectal cancers from North-European and Asiatic patients, raising the hypothesis that the genetic activation of src in colon cancer might be restricted to patients belonging to specific ethnic groups. We investigated a large series of colorectal cancers from Italian patients (155 cases) with a high prevalence of liver metastasis (43%). Using a PCR-RFLP assay, the occurrence of a SRC 531 mutation was ruled out in all the investigated specimens of primary tumours and/or metastases. Our results demonstrate that SRC Gln531AMB plays no role in the development or in the progression of colorectal cancer among Italian patients. © 2001 Cancer Research Campaign http://www.bjcancer.comA truncating mutation (C to T transition) at codon 531 of the human protooncogene c-src, possibly accounting for the activation of c-src tyrosine kinase, has been recently identified in a subset of advanced colorectal cancer from North-American patients. However, two subsequent studies have failed to confirm the occurrence of


A curvilinear dose response of myeloid leukaemia induction in the CBA male mouse was obtained after single whole-body X-irradiation at about 3 months of age. This strain has a very low spontaneous incidence of the disease and no cases were found in the unirradiated controls. The incidence was independent of dose rate over the range used (4.2-552 rad/min). Diagnosis required histopathological examination of various body tissues, the gross anatomical changes being easily confused with other haemopoietic disorders, but a few cases were recognized in life from blood samples. Curve-fitting to various models based on theories of radiocarcinogenic mechanism is described.


The interaction between moderate-dose radiation and cisplatin or carboplatin was studied in a cisplatin-sensitive (GLC4) and -resistant (GLC4-CDDP) human small-cell lung cancer cell line. Cellular toxicity was analysed under oxic conditions with the microculture tetrazolium assay. For the platinum and radiation toxicity with the clinically relevant dose ranges applied, this assay was used to obtain information on cell survival after the treatments. Apart from effects on cell survival effects on DNA were also investigated. Configurational DNA changes could be induced by platinum drugs and thereby these drugs might change the frequency of DNA double-strand breaks (dsbs). DNA fragmentation assayed with the clamped homogeneous electric field (CHEF) technique was used as a measure for dsbs in DNA. The radiosensitising effect of the platinum drugs was expressed as enhancement ratio (ER) calculated directly from survival levels of the initial slope of the curve. The highest ER for cisplatin in GLC4 was 1.39 and in GLC4-CDDP 1.38. These were all at 75% cell survival. Carboplatin showed increased enhancement with prolonged incubation up to 1.21 in GLC4 and was equally effective as cisplatin in GLC4-CDDP. According to isobologram analysis, prolonged incubation with both platinum drugs showed at least additivity with radiation for both cell lines at clinically achievable doses. GLC4-CDDP showed cross-resistance to radiation. The radiosensitising capacity of both lung cancer cell lines was not dependent on their platinum sensitivity. The formation of dsbs in DNA directly after radiation was not influenced by pretreatment of either drug in the sensitive or in the resistant cell line. Drug treatment resulted in decreased DNA extractability in control as well as in irradiated cells. Modest enhancement ratio for radiosensitisation by platinum drugs cannot be explained on the level of dsb formation in DNA in both cell lines. Interaction of radiation with the clinically less toxic carboplatin can be improved by prolonged low-dose carboplatin exposure before irradiation and is as potent as cisplatin in the resistant lung cancer cell line. This suggests an advantage in combining radiation and carboplatin in lung cancer patients.


In mammals, genetically-directed cell labeling technologies have not yet been applied to the morphologic analysis of neurons with very large and complex arbors, an application that requires extremely sparse labeling and that is only rendered practical by limiting the labeled population to one or a few predetermined neuronal subtypes.IRES-CreER knock-in mice were constructed to permit labeling selectively in cholinergic or catecholaminergic neurons [choline acetyltransferase (ChAT)-IRES-CreER or tyrosine hydroxylase (TH)-IRES-CreER], predominantly in projection neurons [neurofilament light chain (NFL)-IRES-CreER], or broadly in neurons and some glia [vesicle-associated membrane protein2 (VAMP2)-IRES-CreER]. When crossed to the Z/AP reporter and exposed to 4-hydroxytamoxifen in the early postnatal period, the number of neurons expressing the human placental alkaline phosphatase reporter can be reproducibly lowered to fewer than 50 per brain. Sparse Cre-mediated recombination in ChAT-IRES-CreER;Z/AP mice shows the full axonal and dendritic arbors of individual forebrain cholinergic neurons, the first time that the complete morphologies of these very large neurons have been revealed in any species.In the present study we have addressed this application by using CreER technology to non-invasively label very small numbers of neurons so that their morphologies can be fully visualized. Four lines of IRES-creER lines should prove useful for studying a wide variety of questions in neuronal development and disease.Sparse genetically-directed, cell type-specific neuronal labeling with  A central organizing principle in all nervous systems is the division of neurons into different classes based on their distinctive dendritic and/or axonal morphologies. This principle was first appreciated over a century ago following the systematic analysis of individual neuronal morphologies in Golgi stained preparations A second general strategy for visualizing neuronal morphology relies on the selective expression of genes coding for enzymes or fluorescent proteins. These genes can be introduced acutely into target neurons by viral infection, electroporation, or particle bombardment In the mouse, genetically directed neuronal labeling has typically been achieved by the selective expression of a fluorescent protein or enzyme under the direct control of a cell-type-specific promoter or under the indirect control of a pharmacologically-regulated fusion between Cre recombinase and a mutated estrogen receptor ligand-binding domain The mouse studies reported thus far have not applied genetic labeling technologies to the morphologic analysis of neurons with large and complex arbors, an application that requires extremely sparse labeling and that is only rendered practical by limiting the labeled population to one or a few predetermined neuronal subtypes. In the present study we have addressed this application by developing and characterizing a series of mouse lines that express CreER in distinct neuronal subsets and we have used these lines in conjunction with a plasma membrane-anchored alkaline phosphatase reporter to visualize the morphologies of large CNS neurons.IRES) and a CreER coding region were inserted into the 3′ untranslated region (UTR) of three genes that are expressed in defined subsets of neurons and one gene that is expressed in neurons and a subset of glia  cells. We chose this approach rather than conventional or bacterial artificial chromosome (BAC) transgenesis because we presumed that a knock-in allele would have the greatest likelihood of precisely recapitulating the expression pattern of the endogenous gene. Although constructing gene-targeted mouse lines is more labor intensive than constructing BAC transgenic mice, it has the virtue that only one knock-in mouse line needs to be characterized for each construct. In contrast, in a transgenic approach, multiple lines are typically characterized for each construct to assess the effects of line-to-line variation in transgene structure, copy number, and integration site, as this variation can affect the expression level or lead to variegated expression. A potential disadvantage of the knock-in strategy is that the insertion of foreign sequences within the 3′ UTR could compromise the function of the target gene, for example by altering mRNA transport, stability, or translation efficiency. In the present instance, no phenotypic effects were apparent for any of the IRES-CreER alleles in the heterozygous state or for the ChAT-IRES-CreER, NFL-IRES-CreER, or TH-IRES-CreER alleles in the homozygous state. However, homozygosity for the VAMP2-IRES-CreER allele appears to be lethal.For each of the four target genes, IRES-CreER sequences into the 3′ UTR places the exogenous DNA segment at a greater distance from the start site of transcription than would insertion of CreER sequences in the 5′ UTR, a design feature that, together with Flp-mediated excision of the Frt-flanked Phospho-glycerate kinase (PGK) promotor-neomycin resistance (neo) selectable marker (‘FNF’), should minimize perturbations in gene expression in the targeted allele (IRES-CreER cassette are: 57 kb (ChAT), 3.5 kb (NFL), 6.8 kb (TH), and 2.0 kb (VAMP2). As shown in IRES-CreER-FNF cassette also includes a bacterial tetracycline resistance gene (TetR) 3′ of the CreER sequences, a selectable marker that facilitated the identification of bacterial colonies harboring the plasmids into which this cassette had been inserted.The strategy of inserting d allele . For theIRES-CreER expression, each targeted line was crossed to the Z/AP reporter line Z/AP locus ubiquitously expresses a beta-galactosidase-neo fusion protein (beta-geo). Cre-mediated recombination excises the beta-geo coding sequence and simultaneously activates expression of human placental alkaline phosphatase (AP), a heat-stable enzyme that localizes to the plasma membrane via a GPI anchor Z/AP reporter in neurons leads to the efficient accumulation of AP on individual axons and dendrites, which can be visualized by histochemical staining To characterize the cell type specificity of IRES-CreER;Z/AP lines were treated with a single dose of 4-hydroxytamoxifen (4HT) during the first two postnatal weeks, and >1 month later tissues were harvested and the distribution of AP revealed by histochemical staining. The specificities of the NFL-IRES-CreER and ChAT-IRES-CreER lines were analyzed in flatmounts of adult retina. As seen in NFL-IRES-CreER;Z/AP mice is confined to retinal ganglion cells, each of which projects an axon to the optic disc, and less frequently to horizontal cells  amacrine cells, recognizable by their characteristic pinwheel morphology; these are the only cells in the retina that express choline acetyltransferase VAMP2-IRES-CreER;Z/AP mice is observed in a wide variety of neurons as well as in glia  in patterns that closely match those obtained by classical labeling methods such as diI and neurobiotin filling , that is enriched for projection neurons (NFL-IRES-CreER), or that is broadly targeted to neurons and some glia (VAMP2-IRES-CreER). We note that in the context of typical CreER-based tissue- and time-specific conditional knockout experiments, inefficient Cre-mediated recombination of target genes is generally considered undesirable In this paper we have used the CreER technology to non-invasively label very small numbers of neurons so that their morphologies can be fully visualized. The four lines of 4–108 neurons, depending on CNS location. In the system described here, this inefficiency arises from a combination of four factors: (1) the relatively inefficient translation of CreER coding sequences mediated by the IRES element Z/AP locus that renders it ∼1,000 fold less recombinogenic than other constructs in which a pair of loxP sites directs excision of an intervening segment of several kilobases ; (3) the dose and route of administration of 4HT; and (4) a progressive decrease in Cre-mediated recombination when 4HT is administered at later postnatal ages.For genetically-directed cell labeling to be useful for the analysis of neuronal morphology, Cre-mediated recombination following 4HT exposure should label only ∼1 in 10ChAT, NFL, and TH genes is low or undetectable in proliferating progenitors, Cre-mediated recombination in the corresponding IRES-CreER knock-in lines should also be low in proliferating progenitors regardless of the time of 4HT injection. By contrast, in the case of VAMP2-IRES-CreER;Z/AP mice, 4HT injection at P2 produced clones of several hundred granule cells in the cerebellum (data not shown), implying expression of CreER from the VAMP2 locus in proliferating granule cell progenitors.Sparse labeling also requires targeting of postmitotic cells to avoid generating clonally related clusters of neurons. For those CNS regions in which neuronal proliferation occurs prenatally  this is readily achieved by 4HT injection at any postnatal age; for those regions in which neuronal proliferation continues postnatally (e.g. retina and cerebellum) 4HT injection can be delayed until ∼P8–P10. Furthermore, since expression of the Two properties of AP histochemistry are particularly useful for the morphologic analysis of large neurons: (1) efficient diffusion of the low-molecular weight histochemical substrates into fixed tissue and (2) the stability of the AP reaction product, which survives immersion in the tissue clearing agent benzylbenzoate∶benzyl alcohol (BBBA). These attributes of substrate and product permit processing and imaging of a small number of relatively thick sections (200–400 um thickness), thus minimizing the technically challenging alignment of neuronal processes between sections. We also note that the NBT/BCIP precipitate does not bleach or fade with visible light exposure and, if the tissue is stored in ethanol, the precipitate can be preserved for years with no apparent loss or diffusion. These attributes facilitate data collection over many imaging sessions. Although not utilized in the present study, AP activity can also be visualized by transmission electron microscopy via the production of a lead-phosphate precipitate, thus facilitating a correlation between light microscopic and ultrastructural analyses Cholinergic neurons with cell bodies located in the basal forebrain modulate cellular activity throughout the cerebral cortex, with wide-ranging physiologic effects. For example, cholinergic input modifies the response properties of neurons in primary sensory cortices, enhances the response to behaviorally significant stimuli in association areas, facilitates cortical control of motor output, and regulates vascular tone Interest in the forebrain cholinergic system has been greatly stimulated by the observation that postmortem Alzheimer's disease brains exhibit a decrease in the number of cell bodies of cholinergic neurons in the basal forebrain and a parallel loss of enzymes involved in cholinergic transmission (e.g. ChAT) in the cerebral cortex ChAT-IRES-CreER;Z/AP mice permits the visualization of the full axonal and dendritic arbors of individual forebrain cholinergic neurons. To the best of our knowledge, this is the first time that the complete morphology of these cells has been revealed in any species. Earlier studies in which individual basal forebrain cholinergic neurons were labeled by injection of neurobiotin revealed their dendritic arbors but only incompletely filled their much larger axonal arbors 2 in the mouse. The large size of these cells is consistent with the simple idea that their loss in a variety of degenerative CNS diseases might reflect a correspondingly high metabolic burden and/or a high burden of toxic insults from amyloid deposition or reactive chemical species. Ideas along these lines have gained wide currency in the context of motor neuron disease and peripheral sensory neuropathies, with the enhanced susceptibility to disease in these contexts being related to the presence of an extremely long axon Sparse Cre-mediated recombination in The most widely used methods for defining the morphologies of individual mammalian neurons involve fluorescent dye or neurobiotin filling by intracellular injection. In the simplest version of these methods, each neuron is identified solely on the basis of properties that are revealed after micro-electrode penetration: morphology, physiology, or both. However, this approach is extremely tedious if the goal is to target a particular neuronal subset, and it is impractical if that subset is rare.A more efficient derivative of this method takes advantage of nondestructive approaches that reveal particular neuronal subsets in living tissue to guide intracellular tracer injection. For example, a particular subset of cell bodies can be visualized by retrograde labeling with fluorescent dextran, a method that has been widely used to analyze the morphologies of retinal ganglion cells that project to defined retinorecipient areas in the brain The sparse labeling approach described here can be easily extended to Cre-control of other proteins. An especially interesting extension involves producing fluorescent fusion proteins that localize to subcellular structures such as synapses, mitochondria, or the cytoskeleton. This type of labeling has recently been reported following biolistic transformation of neurons in retina explants ChAT, NFL, TH, and VAMP2 genes were isolated from an ES cell genomic library in bacteriophage lambda (a gift of Dr. Se-Jin Lee). For each knock-in allele, a cassette consisting of an IRES-CreER, the pBR322 tetracycline resistance gene (TetR), and a Frt-flanked phosphoglycerate kinase promotor-neomycin resistance gene (FNF) was inserted into the 3′ untranslated region within a genomic segment that had been subcloned into a plasmid carrying a Herpes Simplex Virus thymidine kinase gene. The TetR gene was included within the IRES-CreER/FNF cassette to facilitate identification of the final plasmid construct. The restriction site in the 3′ UTR into which the IRES-CreER/TetR/FNF cassette was inserted, together with the sequence of ten nucleotides on each side of the insertion sites are as follows. ChAT: Sac I, GGGCTGGGAGCTCCCTCTGA; NFL: Pci I, AGTCAATACATGTATAATTC; TH: Acc65 I, TGCATAGGGTACCACCCACA; VAMP2: BamH I, CTCCCTTGGATCCGTGTGTG. Following electroporation into ES cells and positive and negative selection , colonies were screened by Southern blot hybridization with ∼500 bp probes generated by PCR as shown in Genomic DNA segments encompassing the 3′ most exons of the mouse PGK-neo cassette was excised by crossing to mice expressing Flp in the germline. Lines were maintained on a mixed Sv129×C57BL/6 background. VAMP2-IRES-CreER homozygotes are inviable, but the other three IRES-CreER lines can be maintained as homozygous stocks. All four lines have been deposited at the Jackson Laboratories and are freely available. Mice were handled and housed in accordance with the Johns Hopkins University Animal Care and Use Protocols and IACUC guidelines.Following germline transmission of the targeted allele, the TetR primer (primer C), a sense strand 3′ UTR primer 5′ of the IRES-CreER/TetR insertion site (primer A) and a 3′ UTR antisense primer 3′ of the IRES-CreER/TetR insertion site (primer B) produces PCR products of different size for the untargeted allele and the knock-in allele, thus distinguishing homozygous WT, heterozygote, and homozygous knock-in genotypes. PCR was performed with Advantage 2 Polymerase  using 35 cycles of 30 seconds denaturation at 94°C, 30 second annealing at 60°C, and 1 minute elongation at 72°C. Rapid testing for the presence of the Z/AP allele was performed by X-gal staining of a small piece of tail.As shown in These procedures were performed essentially as described in ref. 14. In brief, 4HT was dissolved in ethanol, mixed with sunflower seed oil , centrifuged under vacuum to remove the ethanol, and delivered as a single intraperitoneal injection between P0 and P10 at a dose of 0.05–0.4 mg per mouse. After at least one month, the mice were anesthetized, and subjected to transcardiac perfusion with 4% paraformaldehyde in PBS. The brain was set in a block of 3% standard or low-melting point agarose in PBS and a complete series of 300 um coronal sections were cut with a vibratome. Brain sections and intact retinas in PBS were heated in a water bath at 65°C for 75 minutes to inactivate endogenous AP and, for those embedded in low melting point agarose, to melt the agarose surrounding the tissue; heat-inactivated tissues were then reacted in NBT/BCIP overnight at room temperature with gentle agitation. After washing and post-fixation, tissues were dehydrated with a graded ethanol series, and clarified in 2∶1 benzylbenzoate∶benzyl alcohol (BBBA). For long-term storage, tissue samples were returned to ethanol. AP-stained tissues can be stored in ethanol for several years with no change in appearance.Images were captured on a Zeiss Stemi SV11 dissecting microscope equipped with OpenLab software and a Zeiss Axio Imager Z1 microscope equipped with a motorized stage and AxioVision software. Typically, 1 um or 2 um Z-steps were used to capture grey-scale bright-field or DIC images. As BBBA leads to tissue shrinkage of circa 30%, a complete Z-stack at 2 um intervals of what was initially a 300 um section can be captured with ∼100 images; the size of the resulting image stack is ∼180 Mbytes. For each vibratome section, a two-dimensional projection of AP-labeled neurites was obtained by manually tracing over the electronic images using an electronic stylus  and Illustrator CS2 software.