To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues.Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most pronounced differences between tissues were seen for the frequencies of alternative 3' splice site and alternative 5' splice site usage, which were about 50 to 100% higher in the liver than in any other human tissue studied. Quantifying differences in splice junction usage, the brain, pancreas, liver and the peripheral nervous system had the most distinctive patterns of AS. Analysis of available microarray expression data showed that the liver had the most divergent pattern of expression of serine-arginine protein and heterogeneous ribonucleoprotein genes compared to the other human tissues studied, possibly contributing to the unusually high frequency of alternative splice site usage seen in liver. Sequence motifs enriched in alternative exons in genes expressed in the brain, testis and liver suggest specific splicing factors that may be important in AS regulation in these tissues.cis-regulatory elements and trans-acting factors likely to have important roles in tissue-specific AS in human cells.This study distinguishes the human brain, testis and liver as having unusually high levels of AS, highlights differences in the types of AS occurring commonly in different tissues, and identifies candidate  The differentiation of a small number of cells in the developing embryo into the hundreds of cell and tissue types present in a human adult is associated with a multitude of changes in gene expression. In addition to many differences between tissues in transcriptional and translational regulation of genes, alternative pre-mRNA splicing (AS) is also frequently used to regulate gene expression and to generate tissue-specific mRNA and protein isoforms -5. Betwesrc N1 exon [CD44 gene [Drosophila melanogaster sex determination [trans-acting splicing-regulatory factors, implicating members of the ubiquitously expressed serine/arginine-rich protein (SR protein) and heterogeneous nuclear ribonucleoprotein (hnRNP) families, and tissue-specific factors including members of the CELF [cis-regulatory elements in exons or introns that play key regulatory roles have also been identified, using a variety of methods including site-directed mutagenesis, systematic evolution of ligands by exponential enrichment (SELEX) and computational approaches [Insights into the regulation of AS have come predominantly from the molecular dissection of individual genes (reviewed in ,12). Pro N1 exon , cancer-D44 gene  and the mination . Biochemthe CELF  and NOVAthe CELF  familiesproaches -22. In aproaches -26 and aproaches . These nFrom another direction, the accumulation of large databases of cDNA and expressed sequence tag (EST) sequences has enabled large-scale computational studies, which have assessed the scope of AS in the mammalian transcriptome ,8,10,28.cis-regulatory elements and trans-acting factors involved in tissue-specific AS.Here, we describe an initial effort to answer these questions using a large-scale computational analysis of ESTs derived from about two dozen human tissues, which were aligned to the assembled human genome sequence to infer patterns of AS occurring in thousands of human genes. Our results distinguish specific tissues as having high levels and distinctive patterns of AS, identify pronounced differences between the proportions of alternative 5' splice site and alternative 3' splice site usage between tissues, and predict candidate Alternative splicing events are commonly distinguished in terms of whether mRNA isoforms differ by inclusion or exclusion of an exon, in which case the exon involved is referred to as a 'skipped exon' (SE) or 'cassette exon', or whether isoforms differ in the usage of a 5' splice site or 3' splice site, giving rise to alternative 5' splice site exons (A5Es) or alternative 3' splice site exons (A3Es), respectively . For each such gene, a random sample of 20 of these ESTs was chosen (without replacement) to represent the splicing of the given gene in the given human tissue. For the gene and tissue combinations included in this analysis, the median number of EST sequences per gene was not dramatically different between tissues, ranging from 25 to 35 . The sampled ESTs for each gene were then compared to each other to identify AS events occurring within the given tissue . The random sampling was repeated 20 times and the mean fraction of AS genes observed in these 20 trials was used to assess the fraction of AS genes for each tissue Figure . DiffereThe human brain had the highest fraction of AS genes in this analysis Figure , with moAlternatively spliced genes in this analysis exhibited on average between one and two distinct AS exons. Analyzing the different types of AS events separately, we found that the human brain and testis had the highest levels of skipped exons, with more than 20% of genes containing SEs Figure . The higFXR1) gene [An example of a conserved exon-skipping event observed in human and mouse brain tissue is shown in Figure R1) gene ,36. In tR1) gene -39.Analyzing the proportions of AS events involving the usage of A5Es and A3Es revealed a very different pattern Figure . NotablyALDOB, IDH1), protein and amino acid metabolism (for example, BHMT, CBP2, TDO2, PAH, GATM), detoxification or breakdown of drugs and toxins (for example, GSTA3, CYP3A4, CYP2C8).From the set of genes with at least 20 human liver-derived ESTs, this analysis identified a total of 114 genes with alternative 5' splice site and/or 3' splice site usage in the liver. Those genes in this set that were named, annotated and for which the consensus sequences of the alternative splice sites were conserved in the orthologous mouse gene  are listed in Table BHMT and CYP2C8 - are shown in detail in Figure BHMT, the exons involved are highly conserved between the human and mouse orthologs  between the 20-dimensional vectors of splicing-factor expression values between all pairs of 26 human tissues. The DNA microarray studies analyzed 10 tissues in addition to the 16 previously studied . The unusual splicing-factor expression in the human liver was seen consistently in data from two independent DNA microarray studies using different probe sets  . The enrTo quantify the differences in splicing patterns between mRNAs or ESTs derived from a gene locus, a new measure called the splice junction difference ratio (SJD) was developed. For any pair of mRNAs/ESTs that align to overlapping portions of the same genomic locus, the SJD is defined as the proportion of splice junctions present in both transcripts that differ between them, including only those splice junctions that occur in regions of overlap between the transcripts Figure . The SJDi, j), with transcript i coming from set 1 (for example, heart-derived ESTs), and transcript j coming from set 2 (for example, lung-derived ESTs), and dividing this number by the total number of splice junctions in all pairs of transcripts compared, again considering only those splice junctions that occur in regions of overlap between the transcript pairs considered. Note that this definition has the desirable property that pairs of transcripts that have larger numbers of overlapping splice junctions contribute more to the total than transcript pairs that overlap less. As an example of the splice junction difference between two sets of transcripts, consider the set S1, consisting of transcripts (1,2) from Figure S2, consisting of transcripts (3,4) from Figure S1,S2) = d(S1,S2) / t(S1,S2) = [d(1,3) + d(1,4) + d(2,3) + d(2,4)]/ [t(1,3) +t(1,4) + t(2,3) + t(2,4)] = [3 + 4 + 2 + 3]/ [3 + 4 + 4 + 5] = 12/16 = 0.75, reflecting a high level of dissimilarity between the isoforms in these sets, whereas the SJD falls to 0.57 for the more similar sets S1 = transcripts (1,2) versus S3 = transcripts (2,3). Note that in cases where multiple similar/identical transcripts occur in a given set, the SJD measure effectively weights the isoforms by their abundance, reflecting an average dissimilarity when comparing randomly chosen pairs of transcripts from the two tissues. For example, the SJD computed for the set S4 = (1,2,2,2,2), that is, one transcript aligning as transcript 1 in Figure S5 = (2,2,2,2,3) is 23/95 = 0.24, substantially lower than the SJD value for sets S1 versus S3 above, reflecting the higher fraction of identically spliced transcripts between sets S4 and S5.The SJD value can be generalized to compare splicing patterns between two sets of transcripts from a gene - for example, to compare the splicing patterns of the sets of ESTs derived from two different tissues. In this case, the SJD is defined by counting the number of splice junctions that differ between all pairs of transcripts  values for all such genes, divided by the sum of t(SA,SB) values for all of these genes, where SA and SB refer to the set of ESTs for a gene derived from tissues A and B, respectively, and d(SA,SB) and t(SA,SB) are defined in terms of comparison of all pairs of ESTs from the two sets as described above. This analysis uses all available ESTs for each gene in each tissue (rather than samples of a fixed size). A large SJD value between a pair of tissues indicates that mRNA isoforms of genes expressed in the two tissues tend to be more dissimilar in their splicing patterns than is the case for two tissues with a smaller inter-tissue SJD value. This definition puts greater weight on those genes for which more ESTs are available.To make a global comparison of patterns of splicing between two different human tissues, a tissue-level SJD value was computed by comparing the splicing patterns of ESTs from all genes for which at least one EST was available from cDNA libraries representing both tissues. The 'inter-tissue' SJD value is then defined as the ratio of the sum of The SJD values were then used to globally assess tissue-level differences in alternative splicing. A set of 25 human tissues for which at least 20,000 genomically aligned ESTs were available was compiled for this comparison  and the SJD values were then computed between all pairs of tissues in this set Figure . A clustThe systematic analysis of transcripts generated from the human genome is just beginning, but promises to deepen our understanding of how changes in the program of gene expression contribute to development and differentiation. Here, we have observed pronounced differences between human tissues in the set of alternative mRNA isoforms that they express. Because our approach normalizes the EST coverage per gene in each tissue, there is higher confidence that these differences accurately reflect differences in splicing patterns between tissues. As human tissues are generally made up of a mixture of cell types, each of which may have its own unique pattern of gene expression and splicing, it will be important in the future to develop methods for systematic analysis of transcripts in different human cell types.cis-regulatory elements in exons or introns and trans-acting splicing factors. The current study has integrated these three elements, inferring alternative transcripts from EST-genomic alignments, identifying candidate regulatory sequence motifs enriched in alternative exons from different tissues, and analyzing patterns of splicing-factor expression in different tissues. Our results emphasize differences in the frequencies of exon skipping versus alternative splice site usage in different tissues and highlight the liver, brain and testis as having particularly high levels of AS, supporting the idea that tissue-regulated AS plays important roles in the differentiation of these tissues. The high levels of alternative splice site usage in the liver may relate to the unusual patterns of splicing-factor expression observed in the adult liver, suggesting aspects of developmental regulation of AS at the tissue level. Obtaining a more comprehensive picture of AS will require the integration of additional types of data upstream and downstream of these core interactions. Upstream, splicing factors themselves may be differentially regulated in different tissues or in response to different stimuli at the level of transcription, splicing, or translation, and are frequently regulated by post-translational modifications such as phosphorylation, so systematic measurements of splicing factor levels and activities will be required. Downstream, AS may affect the stability of alternative transcripts (for example, in cases of messages subject to nonsense-mediated mRNA decay), and frequently alters functional properties of the encoded proteins, so systematic measurements of AS transcript and protein isoforms and functional assays will also be needed to fully understand the regulatory consequences of AS events. Ultimately, it will be important to place regulatory events involving AS into the context of regulatory networks involving control at the levels of transcription, translation and post-translational modifications.Understanding the mechanisms and regulatory consequences of AS will require experimental and computational analyses at many levels. At its core, AS involves the generation of alternative transcripts mediated by interactions between Chromosome assemblies of the human genome (hg13) were obtained from public databases . TranscrThe GENOA genome annotation script  was usedIn total, GENOA aligned about 85,900 human cDNAs and about 890,300 ESTs to the human genome. The relatively low fraction of aligned ESTs about 18%), and average aligned length of about 550 bases , reflect the stringent alignment-quality criteria that were imposed so as to be as confident as possible in the inferred splicing patterns. The aligned sequences yielded about 17,800 gene regions with more than one transcript aligned that exhibited a multi-exon structure. Of these, about 60% exhibited evidence of alternative splicing of internal exons. Our analysis did not examine differences in 3'-terminal and 5'-terminal exons, inclusion of which is frequently dictated by alternative polyadenylation or alternative transcription start sites and therefore does not represent 'pure' AS ,74. The 8%, and aTo quantify the difference in splicing patterns between mRNAs or ESTs derived from a gene locus, the splice junction difference ratio (SJD) was calculated. For any pair of mRNAs/ESTs that have been aligned to overlapping portions of a genomic locus, the SJD is defined as the fraction of the splice junctions that occur in overlapping portions of the two transcripts that differ in one or both splice sites. A sample calculation is given in Figure k-mers) were identified by comparing the number of occurrences of k-mers (for k in the range of 4 to 6 bases) in a test set of alternative exons versus a control set. In this analysis, monomeric tandem repeats (for example, poly(A) sequences) were excluded. The enrichment score of candidate k-mers in the test set versus the control set was evaluated by computing χ2 (chi-squared) values with a Yates correction term [et al. [k-mers over-represented in the sampled subset versus the remainder of the control set were computed as above. The estimated p-value for observing the given enrichment score  associated with an over-represented sequence motif of length k was defined as the fraction of subsets that contained any k-mer with enrichment score  higher than the tested motif. Correcting for multiple testing is not required as the p-value was defined relative to the most enriched k-mer for each sampled set. For sets of skipped exons from human brain- and testis-derived EST sequences, the test sets comprised 1,265 and 517 exons skipped in brain- and testis-derived ESTs, respectively, and the control sets comprised 12,527 and 8,634 exons constitutively included in human brain- and testis-derived ESTs, respectively. Candidate sequence motifs in skipped exons from brain and testis-derived ESTs with associated p-values less than 0.002 were retained. For the set of A5E and A3E events from human liver-derived EST sequences, the test set comprised 44 A3Es and 45 A5Es, and the control set comprised 1,619 A3Es and 1,481 A5Es identified using ESTs from all tissues excluding liver. In this analysis, A3Es and A5Es with extension sequences of less than 25 bases were excluded and sequences longer than 150 bases were truncated to 150 bases, by retaining the exon sequence segment closest to the internal alternative splice junction. Over-represented sequence motifs in A3Es and A5Es from liver-derived EST sequences with associated p-values less than 0.01 were retained.Over-represented sequence motifs (ion term , using a [et al. . We randSR proteins, SR-related proteins, and hnRNPs were derived from published proteomic analyses of the spliceosome -48. ExprAdditional data files containing the following supplementary data, tables and figures are available with the online version of this paper and from the GENOA genome annotation website . The lisThe average and median number of ESTs per gene and tissue, and the total number of genes per tissue using different minimum numbers of ESTsClick here for additional data fileThe average total number of AS genes and AS genes containing SEs, A3Es and A5Es using ESTs derived from normal, non-diseased tissuesClick here for additional data fileThe number of constitutively spliced and AS genes, and AS genes containing SEs, A3Es and A5EsClick here for additional data fileThe average fractions of AS genes and average fractions of AS genes containing SEs, A3Es and A5Es using ESTs derived from normal, non-disease-derived tissuesClick here for additional data fileCategories of cDNA libraries and designated tissues derived from the MGC, IMAGE and CGAPClick here for additional data fileThe average lengths of ESTs that aligned to gene loci expressed in different tissuesClick here for additional data fileHuman splicing factors of SR, SR-related and hnRNP genes, corresponding Ensembl gene numbers and Affymetrix microarray probe identification numbersClick here for additional data fileThe distribution of the average Pearson correlation coefficient values across different tissues for expression levels of random sets of genes obtained from the Affymetrix microarray datatClick here for additional data file


The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRα) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision.Standard-curves were constructed by employing standardized Jurkat cell culture procedures, both for GRα and BCR (breakpoint cluster region), as a normalizing gene. We evaluated standard-curves using five different sets of cell culture passages, RNA extraction, reverse transcription, and qrt-PCR quantification. Intra-assay precision was evaluated using 12 replicates of each gene, for 2 patients, in a single experiment. Inter-assay precision was evaluated on 8 experiments, using duplicate tests of each gene for two patients. Standard-curves were reproducible, with CV (coefficient of variation) of less than 11%, and Pearson correlation coefficients above 0,990 for most comparisons. Intra-assay and inter-assay were 2% and 7%, respectively.This is the first method for quantitation of GRα expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way. Glucocorticoids (GC) are a vital class of steroidal hormones that mediate profound and diverse physiological effects in vertebrates. GC are key hormones in the regulation of glucose homeostasis, but other essential functions are assigned to GC as well, such as regulatory roles in development and other metabolic pathways, stress and immune responses, neurobiology, and programmed cell death. In addition, corticosteroids are among the most widely prescribed class of drugs, primarily for their anti-inflammatory and immunosuppressive roles. They are also used in many chemotherapy regimens for leukemias and other cancers due to their critical capability to induce apoptosis.Cortisol and its synthetic derivatives act upon the glucocorticoid receptor (GR), a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. Prior to ligand binding, GR is primarily localized within the cytoplasm as an oligomeric complex composed of one receptor polypeptide, two molecules of heat shock protein 90 (hsp90), one molecule each of hsp 70, hsp 56 (immunophilin) and hsp23. When the hormone binds to the receptor, the GC-GR complex undergoes conformational changes, followed by dissociation from the hsp complex and dimerization of the GR molecules. The activated GR dimer is translocated into the nucleus and because of its high DNA affinity is able to bind to a specific DNA sequence named glucocorticoid response element (GRE), which is located in the vicinity of the target-regulated gene. The GR-GRE complex interacts with other components of the transcription apparatus to either enhance or repress the expression of the targeted gene -3.There are several molecular mechanisms involved in glucocorticoid resistance or hypersensitivity (reviewed by Yudt, 2002) and GR expression seems to be a key one. These mechanisms are important for the regulation of cell and tissue-specific GC sensitivity, but they can be pathologically modified in clinical conditions such as AIDS, glucocorticoid-resistant asthma, rheumatoid arthritis and familial glucocorticoid resistance, among others -7.Evaluation of GR expression in these conditions presents critical restrictions and has been limited to research protocols, partly due to analytical difficulties. Methods employed so far include ligand-binding assays, northern and western-blots and PCR. These methods could only provide qualitative or semi-quantitative information and a truly quantitative and reproducible evaluation of GR expression was still needed. In this study, we describe a quantitative real-time PCR (qrt-PCR) for GR alpha isoform (GRα) expression that is suitable for patient monitoring and can be easily reproduced.GRα and BCR (breakpoint cluster region) standard-curves were very stable using five different standard preparations, with maximum coefficient of variation (CV) of 10.3% observed for GRα most concentrated standard-point. GRα standard-curves presented CVs of 10.3%, 7.8%, 9.1%, 5.5% and 3.6% for the cycle-thresholds (Ct) obtained with each standard (6 to 2 logs of Jurkat cells). On both genes, standard-curves CV were greater at the most concentrated standard-points. BCR CV was smaller than those observed for GRα CV in all standard-points and all standard-curves. Standards of 6 to 1 log of Jurkat cells presented, respectively, CV of their Ct of 4.5%, 2.7%, 3.1%, 2.7%, 1.9% and 0.9%. BCR standard-curves had greater analytical sensitivity than GRα, with 10 EC/mL versus 100 EC/mL.Linear regression analysis of pairs of standard-curves demonstrated strong correlation for both genes. The smallest Pearson correlation coefficient on GRα curves was 0.982, but most of them were higher than 0.990. Two pairs of BCR curves showed Pearson correlation coefficient of 1.000 , it assess only GR ligand-binding properties in the cytoplasm, providing no information regarding GR in the nucleus and requires the use of radioactive materials. Western blot assays were developed to assess GR protein levels. Despite numerous efforts to make well-characterized and specific antibodies, to date there are no highly specific human monoclonal antibodies targeted to GR isoforms, preventing the development of true quantitative methods, such as ELISA ,10. QuanThree groups recently described qrt-PCR techniques for GRα evaluation. DeRijk et al. developed Taqman evaluation of GRα and GRβ, however they did not describe the use of standard-curves or gene normalization . Since tBoullu-Ciocca et al. evaluated GRα and GRβ expression using real-time PCR in obese and control cases . They usPedersen and Vedeckis evaluated two cell lines using real-time PCR for seven different isoforms of GR (GRα, GRβ, and exon 1 splicing variants: 1A1, 1A2, 1A3, 1B, 1C), total GR (using exons 5–6) and 18S mRNA as normalizing gene . StandarThe aim of this study was to describe and validate the reproducibility for clinical monitoring of absolute quantitation of glucocorticoid receptor alpha isoform using real-time PCR. Our approach used a standardized procedure for Jurkat cell culture to make cDNA standards for both GRα and BCR. Standard-curves were reproducible, with less than 11% CV for all standards used and Pearson correlation coefficient above 0.990 for most comparisons. Intra-assay and inter-assay precision were 2% and 7%, respectively.The BCR gene, derived from normal chromosome 22, was chosen as an appropriate endogenous control. The gene is ubiquitously expressed, has no reported pseudogenes and is a stringent control for the detection of degradation of RNA ,16. We fIn conclusion, we described for the first time a method for quantitation of GRα expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way.2 at 37°C. Culture medium was changed every 72 h, by centrifugation at 500 rpm for 5 min and 10 mL of fresh medium was added. Viable cells were counted in a hemocytometer (Neubauer chamber) using Trypan Blue (Sigma) and re-suspended to obtain a final density of 105 cells per milliliter. This procedure was repeated three times and 24 hours after the third medium exchange, cells were re-suspended to a final density of a 106 cells per milliliter. This strict protocol is necessary to ensure RNA extraction on the log phase of cell growing phase in order to minimize the variation of mRNA expression.We developed standards for real-time quantitation of GRα expression by employing standardized Jurkat (E6-1 clone, ATCC) cell culture. Jurkat cells were grown in RPMI 1640 (Gibco) supplemented with 10% FBS, 1% penicillin-streptomycin (Gibco), 5% COThree normal individuals participated in this study, in accordance with the guidelines proposed in The Declaration of Helsinki and approved by the ethics committee of our institution. Cases 1 and 2 are 30 and 32 year-old females, respectively, and case 3 is a 9 year-old boy. Heparinized blood was collected by venipuncture. After collection of 20 mL blood, mononuclear cells were separated using Histopaque-1077 (Sigma) by density gradient separation after centrifugation at 1750 rpm for 30 minutes. Cells were washed and re-suspended in PBS and 10% DMSO (Sigma) was added before freezing at -80°C. Prior to RNA extraction, DMSO was removed by PBS washing and re-suspension.2, 500 mM each dNTP, 2.5 mM random primers, 1.25 U/mL reverse transcriptase (Multiscribe reverse transcriptase) and DNAse-RNAse-free water as needed. After 10 minutes at 25°C, reaction was carried at 48°C for 30 minutes, followed by enzyme inactivation at 95°C for 5 minutes.Total RNA was isolated from cells using guanidinium thiocyanate-chloroform extraction (Trizol, Gibco), according to the manufacture's recommendations. RNA was dissolved in DNAse-RNAse-free water (Gibco), after which the OD 260/280 was confirmed as >1.7, allowing the estimation of RNA concentration. RNA was stored at -80°C when the RT step was not immediate. cDNA was synthesized from 1 μg of total RNA, using TaqMan Reverse Transcription reagents (N808-0234, Applied Biosystems) with 1 × buffer, 5.5 mM MgClJurkat cell cDNA was serially diluted in 1:10 ratio with DNAse-RNAse-free water in order to obtain 6 logs of standards from one million cells per millimeter to ten cells per milliliter. They were numbered accordingly to their log number (6 to 1).GRα primers and probes were designed with Primer Express v.1.5 (Applied Biosystems) and both GRα and BCR  primers sets target two different exons, in order to prevent genomic amplification. BCR primers were previously used by Branford et al and our group ,17. Prim2, 200 nM of each primer, 100 nM of probe, 0.025 U/μL of AmpliTaq Gold, 5 μL of cDNA and water were incubated in a total volume of 25 μL. Cycle conditions on an ABI 7700 (Applied Biosystems) were: 95°C for 10 minutes (AmpliTaq Gold activation) followed by 45 cycles of 95°C for 15 seconds (denaturation) and 60°C for 90 seconds . No template controls and duplicates of each standard, for both genes, were used in each run. Results were exported to MS-Excel to perform linear regression analysis for each gene, determining a standard-curve for Log of EC  based on the average Ct of duplicates . Briefly, 1 × TaqMan buffer A, 500 μM each dNTP, 4.5 mM MgCltes fig. .The average Ct for each sample was employed in the linear regression equation in order to obtain the EC, for each gene. Then, GRα-EC was divided by BCR-EC in order to establish the GRα-EU (Expression Units).In order to evaluate standard-curve stability with this process, we used five different sets of cell culture passages, trypan blue viable cell counting, RNA extraction, cDNA synthesis and qrt-PCR. These standard-curves sets were named A to E. We calculated the mean, SD and CV for each standard, the CV for standard-curves slopes, as well as Pearson correlation coefficient for each pair of curves.Intra-assay precision was evaluated in a single experiment that included standard-curves for each gene and 12 replicates for each gene, in samples obtained from two normal controls (1 and 2). In order to calculate GRα-EU, pairs of samples (GRα and BCR) were established according to their position on the thermocycler.Inter-assay precision was evaluated over eight different experiments, each one with its own standard-curve and duplicates of each gene, with samples obtained from two normal controls (1 and 3). Duplicates were averaged for calculation of GRα-EU.Standard-curves and GRα-EU were calculated on MS-Excel 2000 for Windows. Linear regression of standard-curves pairs was calculated using SPSS 10.0 (SPSS, Chicago). For comparison of intra and inter-assay results of case 1, we employed Mann-Whitney Rank Sum test on SigmaStat v.2.03 (SPSS, Chicago), which was also used to perform the t-test. Differences of p < 0.05 were considered statistically significant.MRM planned, performed and analyzed real-time PCR reactions and wrote the manuscript; CDCF did sample collection, cell culture optimization, RNA extraction and cDNA synthesis; KCM did statistical calculations and prepared figures and manuscript; NAR provided real-time PCR support; CAL planned the study, revised experimental data and the manuscript.


HAC1 mRNA is the only substrate for the Ire1 nuclease in yeast.Three different genome-scale screens indicate that the  HAC1 mRNA (encoding a UPR-specific transcription factor) is the first step in a nonconventional mRNA splicing pathway; the released exons are then joined by tRNA ligase. Because only the spliced mRNA is translated, splicing is the key regulatory step of the UPR.The unfolded protein response (UPR) allows intracellular feedback regulation that adjusts the protein-folding capacity of the endoplasmic reticulum (ER) according to need. The signal from the ER lumen is transmitted by the ER-transmembrane kinase Ire1, which upon activation displays a site-specific endoribonuclease activity. Endonucleolytic cleavage of the intron from the HAC1 mRNA as a substrate according to three criteria: HAC1 mRNA is selectively cleaved in vitro by Ire1; the HAC1 mRNA sequence contains two predicted Ire1 cleavage sites; and HAC1 mRNA is selectively degraded in tRNA ligase mutant cells.We developed methods to search for additional mRNA substrates of Ire1p in three independent lines of genome-wide analysis. These methods exploited the well characterized enzymology and genetics of the UPR and the yeast genome sequence in conjunction with microarray-based detection. Each method successfully identified Within the limits of detection, no other mRNA satisfies any of these criteria, suggesting that a unique nonconventional mRNA-processing mechanism has evolved solely for carrying out signal transduction between the ER and the nucleus. The approach described here, which combines biochemical and genetic 'fractionation' of mRNA with a novel application of cDNA microarrays, is generally applicable to the study of pathways in which RNA metabolism and alternative splicing have a regulatory role. The unfolded protein response (UPR) regulates the protein-folding and secretory capacity of eukaryotic cells by monitoring conditions within the endoplasmic reticulum (ER) and regulating a downstream gene-expression program (reviewed in -3). In ytrans-autophosphorylation of the cytosolic kinase domain, providing the means by which the signal is transmitted across the ER membrane.The identification and characterization of the UPR signaling components revealed a unique mechanism of signal transduction whose salient features are conserved among all eukaryotes. The UPR is initiated when the amino-terminal portion of the serine/threonine ER-transmembrane kinase Ire1 detects unfolded proteins within the ER lumen ,7. AccumHAC1 mRNA, termed HAC1i mRNA (i for UPR-induced) [HAC1 mRNA splicing reaction mediated by Ire1 and Rlg1 is spliceosome-independent, and utilizes chemistry that closely resembles pre-tRNA splicing [HAC1 mRNA occur independently. In contrast, spliceosome-mediated splicing is a series of two transesterification reactions that have to be strictly ordered: 3' splice-site cleavage cannot occur before 5' splice-site cleavage. Intriguingly, this Ire1-mediated splicing reaction happens on polyribosome-bound HAC1 mRNA in the cytosol [Activated Ire1 acts as a site-specific endoribonuclease, cleaving the mRNA encoding the transcription activator Hac1 at two discrete positions, and removing a 252-nucleotide nonclassical intron ,9. A secinduced) . The HACsplicing . As in p cytosol ,13.HAC1 mRNA, the intron forms a long-range base-pairing interaction with the 5' untranslated region (UTR) that is responsible for preventing its translation [In the unspliced nslation ,13. SpliHAC1 mRNA, whereas the nuclease activity of RNase L shows no sequence specificity. Previously, to demonstrate the sequence specificity of Ire1, we devised an in vitro assay which utilized a purified, recombinant Ire1 protein [HAC1 mRNA faithfully at both 5' and 3' exon-intron junctions and thus recapitulates the substrate specificity of full-length Ire1 in vivo. Other RNAs, including actin mRNA and poly(U) RNA, which has been demonstrated to be an RNase L substrate [The amino-acid sequence of the nuclease region of Ire1 reveals significant similarities to that of RNase L, a mammalian endoribonuclease that is activated by interferon during viral infection ,15. RNas protein . This prubstrate , are notubstrate ,16.bona fide Ire1 orthologs IRE1α and IRE1β [In metazoans, multiple parallel pathways originate from the ER and contribute to the UPR. Two nd IRE1β ,20 are pnd IRE1β -23. Anotnd IRE1β ,25. Actind IRE1β . A thirdnd IRE1β ,28. Accund IRE1β ,29-32, wnd IRE1β .HAC1 mRNA is the only known RNA substrate for yeast Ire1, prompting the question of whether other substrate RNAs exist or whether Ire1 and HAC1 mRNA function as a matched enzyme-substrate pair that interact exclusively with each other. Here we describe three independent genome-scale methods that address this question. Each approach successfully identifies HAC1 mRNA as a substrate of Ire1; none of the approaches identifies any other mRNA as a qualified candidate. Two of these approaches represent novel applications of cDNA array technology and could be adapted to the study of other signal transduction pathways regulated by RNA processing.To date, + RNA fraction from cells and subjected it to in vitro cleavage reactions in the presence or absence of Ire1*. Fragments that lost their poly(A)+ tails due to cleavage by Ire1* were re-isolated, reverse transcribed, fluorescently labeled and hybridized to genomic cDNA microarrays to identify Ire1 substrates.We first devised a molecular screen to identify mRNAs cleaved by the Ire1 nuclease . The GST moiety was removed during the purification by protease cleavage. Upon incubation of Ire1* with in vitro transcribed HAC1 mRNA, we observed efficient and accurate cleavage at both splice junctions as previously described [For these experiments, we expressed and purified a recombinant Ire1* tagged with glutathione-escribed ,11.HAC1 mRNA contained in a total poly(A)+ RNA fraction from yeast would be efficiently cleaved, even in the presence of significant excess of other mRNAs. We incubated total poly(A)+ RNA with Ire1* and then fractionated the products using oligo(dT) cellulose  tail) was recovered exclusively in the oligo(dT)-bound fraction  chromatography. We prepared a mock-treated reference sample using reaction conditions which were identical except that Ire1* was omitted. We reverse-transcribed both samples and fluorescently labeled the resulting cDNA with Cy3  or Cy5 , respectively. The two probes were then simultaneously hybridized to yeast microarrays. We expected that contaminating poly(A)- RNA or uncleaved poly(A)+ RNA would be equally represented in the oligo(dT) unbound fractions of both Ire1*-treated and mock-treated reactions, thus leading to equal representation of both fluorescent probes in the mixture. Indeed, scatter plots generated by quantitating the fluorescence levels of both Cy3 and Cy5 5 Figure  showed t2 Cy3/Cy5 ratio for all mRNAs + RNA fraction. As the Ire1 cleavage sites on other mRNAs could, in principle, be located within the 5' or 3' UTRs, we also hybridized the same probe to genomic microarrays containing yeast intergenic regions in addition to the ORFs. Our results were similar to those observed using ORF-only microarrays, with HAC1 being the only significant outlier (data not shown).We expected those mRNAs that are specifically cleaved by Ire1* to be enriched in the oligo(dT) unbound fraction of the Ire1*-treated reaction compared to the unbound fraction of the mock-treated reaction, resulting in a higher Cy3/Cy5 fluorescence ratio in the microarray hybridization. Depletion of particular mRNAs by Ire1* digestion thus provides an enzymological tool to fractionate substrate from nonsubstrate mRNAs. We therefore plotted a histogram of the logHAC1 mRNA and mutational analysis . In the list of genes identified, only a single gene, - mRNA, resulting in high outlying Cy3/Cy5 ratios; however, none of the genes predicted to bear single HAC1 splice consensus sequences fell more than 3σ from the mean of the distribution . Because previous in vitro analyses have shown that stem-loop structures matching the consensus are sufficient for cleavage, we consider it most likely that the predicted stem-loop structures are either not included in the transcripts or do not fold as predicted in the context of the full mRNA sequences.For the remaining 52 predicted stem-loop structures, the possibility remained open that Ire1 would cleave some substrates at only a single site, perhaps to downregulate particular mRNAs. However, in the light of the data obtained from the microarray analysis described in Figure rlg1-100, previously showed that Ire1-dependent cleavage of HAC1 mRNA occurs normally in the absence of ligation, but that the cleavage products are rapidly degraded. We assume that any other mRNA following this pathway should suffer the same fate, and that we could identify substrates of Ire1 by looking for mRNAs whose steady-state levels drop upon UPR induction in the rlg1-100 mutant, but not in a wild-type cell. The Ire1-dependent selective disappearance of HAC1 mRNA in rlg1-100 cells thus provides us with a tool to assess the spectrum of mRNAs that utilize the Ire1-mediated splicing pathway.In a third genome-scale approach, we exploited the phenotype of a mutant in tRNA ligase that is defective in UPR induction . Charactrlg1-100 cells with either tunicamycin (to induce the UPR by inhibition of N-linked glycosylation in the ER) or with no drug as a control, isolated total mRNA, reverse transcribed and fluorescently labeled the cDNA with Cy5 (for the tunicamycin-treated sample) and Cy3 (for the untreated sample) before simultaneous hybridization to genomic microarrays. mRNAs which are depleted during tunicamycin treatment should have Cy3/Cy5 ratios greater than 1 (log2 Cy3/Cy5 ratios greater than 0). As shown by the data in Figure 2 Cy3/Cy5 ratios followed a tight quasi-normal distribution (mean = 0.11, σ = 0.27) with a single outlier. HAC1, at a log2 Cy3/5 ratio of 1.5, HAC1 falls 5σ from the mean, and is thus successfully identified as a splicing target of the UPR. Once again, no other mRNA clearly satisfied the criteria for identification as an Ire1 substrate. The depletion of HAC1 mRNA upon tunicamycin treatment was specific to rlg1-100 cells. A similar analysis of wild-type cells showed the expected induction of UPR target genes and no depletion of HAC1 mRNA relative to an untreated control (data not shown). Thus, HAC1 mRNA again stands out as the unique substrate for Ire1-dependent cleavage in yeast.We treated HAC1 mRNA as a target of Ire1 nuclease. In vitro cleavage of mRNAs by Ire1*, followed by microarray detection of the fractionated mRNAs, identifies HAC1 mRNA as significantly enriched in the population of mRNAs specifically cleaved by Ire1. A computational search for the experimentally determined Ire1 consensus cleavage sites identifies HAC1 as the unique gene containing two such sequences. Finally, in vivo induction of splicing in a cell containing wild-type tRNA ligase or the mutant rlg1-100, and subsequent microarray detection of 'genetically fractionated' mRNAs, identifies HAC1 as selectively depleted in the absence of the wild-type ligase. No other mRNA met any of these criteria for identification as an Ire1 substrate. We consider it reasonable to conclude that among the set of robustly expressed genes, HAC1 mRNA is the lone substrate of Ire1.The experiments presented in this paper represent three genome-scale approaches for identifying mRNA substrates of the Ire1-dependent mRNA splicing pathway in yeast. Each approach successfully and selectively identifies the HAC1 mRNA. For example, a poorly expressed substrate would exhibit a low signal-to-noise ratio in the microarray readout of the in vitro cleavage assay, or a substrate cleaved close to the 5' end would still hybridize to cDNA arrays with an efficiency comparable to that of the uncleaved mRNA, and hence could have escaped detection. In our computational screen, we might have missed cleavage sites that are significantly divergent from the experimentally defined consensus. Finally, because tRNA ligase would not take part in a cleavage-only reaction on a single-site substrate, we would also not expect our third method to lead to a relative reduction of the abundance of such mRNAs in rlg1-100 mutant cells as compared to the wild-type (though we would have expected such substrates to be identified in the in vitro Ire1* cleavage experiment). Thus rigorously, we cannot conclude that HAC1 mRNA is the only Ire1 substrate. However, because the potential caveats are noncongruent and because HAC1 stands out unambiguously in each of the three methods applied, we consider it highly unlikely that additional substrates exist.In principle, there are many reasons why each of the approaches presented here could have missed identifying an Ire1 substrate other than Previous studies in metazoans have suggested that other RNAs are degraded when Ire1 is activated ,34. WhenHAC1. In contrast, other signaling pathways in the cell are commonly branched, using one enzyme to activate multiple different substrates. The utilization of common principles of protein modification, such as phosphorylation or ubiquitination, readily allows cross-talk among different signaling pathways that use common components. One phosphatase, for example, can dephosphorylate substrates that have been phosphorylated by several different kinases. In contrast, communication between the ER and nucleus through Ire1 appears to be a very private affair. Ire1-dependent splicing offers unique mechanistic advantages, such as the ability to quickly complete the synthesis of partially made Hac1 transcription factor by releasing the translational arrest of stalled polyribosomes. Given that the ER is a topologically distinct compartment of the cell with metabolic considerations quite distinct from those of the cytosol, a communication route that is wired differently and therefore more insulated from other informational 'chatter' in the cell may be particularly beneficial in the secure passage of information from the ER to the nucleus.According to the current evidence, therefore, Ire1 has a single identified target in yeast and thus functions solely for the purpose of post-transcriptional regulation of Δire1 and Δhac1 mutant cells display indistinguishable growth phenotypes and highly correlated gene-expression profiles [HAC1 ortholog XBP1, and the methods developed here may prove useful in searching for such putative, additional Ire1 substrates in metazoans.The linear connectivity (activation of Ire1 → production of Hac1) of the two key players of the yeast UPR is supported by genetic analysis. Both profiles . In highprofiles ,35; in aprofiles ,36. The rlg1-100 cells were treated with 1 μg/ml tunicamycin  for 40 min in the experiments described in Figure + RNA was isolated from total RNA using the PolyA+ Tract mRNA isolation system (Promega) according to the manufacturer's instructions. Poly(A)+ RNA used for in vitro Ire1* nuclease reactions was prepared by two rounds of purification using the PolyA+ Tract system.RNA samples were prepared as described previously . Brieflyin vitro nuclease reactions was analyzed by Northern blot hybridization as described previously [32P]α-dCTP according to the manufacturer's instructions (Amersham). All probes prepared from DNA fragments were generated by PCR amplification of yeast genomic DNA.Total cellular RNA and RNA recovered from eviously  by separIn vitro nuclease reactions were performed by incubating yeast poly(A)+ RNA with recombinant Ire1* (expressed and purified from Escherichia coli) as described previously [+ Tract mRNA Isolation System (Promega) according to the manufacturer's instructions. Control nuclease reactions were performed and processed identically, except that Ire1* was omitted from the reaction mixture.eviously ,11. Foll+ RNA (2 μg) used for the experiment in Figure General protocols for microarray hybridization and for preparation of probe were as described previously . A samplHybridized microarrays were scanned with a GenePix 4000A microarray scanner (Axon Instruments). GenePix Pro was used to analyze and to display the data. All data points with absolute fluorescence intensity less than 250 in either channel were discarded. Cy3 and Cy5 fluorescence intensities were normalized against one another to adjust for differences in labeling efficiency and scanner gain. Quantitative analysis was performed in Microsoft Excel.Saccharomyces Genome Database [Pattern searching was performed using the public-domain software scan_for_matches . SequencDatabase . Patternr1 = {au,ua,gc,cg,ug,gu}RNA base-pairing rules were used; allowed base-pairs are the fields of argument r1.p1 = 4..4 YCNGNNGNG ~p1These parameters instructed the program to use RNA base-pairing rules to find a sequence of four nucleotides (one arm of stem), followed by a sequence matching YCNGNNGNG , followed by the reverse complement of the four nucleotides at the beginning (the second arm of the stem). The output of the computational screen is listed in Additional data file 2.The following additional data are available with the online version of this paper. Additional data file Supplementary Table 1 which lists all the data displayed in Figure 3Click here for additional data fileSupplementary Table 2, which lists the complete output of the computational screenClick here for additional data fileSupplementary Table 3 which lists all data displayed in Figure 5Click here for additional data fileThe captions and keys to the supplementary tablesClick here for additional data file


Transcription factors are frequently involved in the process of cellular transformation, and many malignancies are characterized by a distinct genetic event affecting a specific transcription factor. This probably reflects a tissue specific ability of transcription factors to contribute to the generation of cancer but very little is known about the precise mechanisms that governs these restricted effects.To investigate this selectivity in target gene activation we compared the overall gene expression patterns by micro-array analysis and expression of target genes for the transcription factor EBF in lymphoma and neuroblastoma cells by RT-PCR. The presence of transcription factors in the different model cell lines was further investigated by EMSA analysis.In pre-B cells mb-1 and CD19 are regulate by EBF-1 in collaboration with Pax-5 and E-proteins. We here show that neuroblastoma cells express these three, for B cell development crucial transcription factors, but nevertheless fail to express detectable levels of their known target genes. Expression of mb-1 could, however, be induced in neuroblastoma cells after disruption of the chromatin structure by treatment with 5-azacytidine and Trichostatin A.These data suggest that transcription factors are able to selectively activate target genes in different tissues and that chromatin structure plays a key role in the regulation of this activity. This factor is, however, broadly expressed and directly involved in cell cycle regulation and apoptosis explaining the common involvement of the protein in tumor development. Many malignancies are characterized by specific chromosomal translocations that frequently affects the expression or structure of transcription factors with a more tissue specific expression pattern, often with an important function during development [Tal-1 is associated with T cell leukemia's [BCL-6 [c-myc [The complex process of tumor development often involves changes in the transcription regulatory networks. In human cancer, genetic changes involving the transcription factor elopment . Exampleukemia's  while mo6 [c-myc  is assocmb-1) [B29) [Chromogranin A (CGA) and SCG10 genes [To investigate mechanisms involved in lineage specific gene regulation and transcription factor target gene selection in tumor cells we have compared transcription factor expression in neuroblastoma and pre-B lymphoma cells. This revealed that both these highly divergent tumor types expressed the transcription factor EBF  that hasmb-1) ,11 and I1) [B29) . The EBF1) [B29)  and it a1) [B29) ,14. Pre-1) [B29)  while ne1) [B29) . There a10 genes .mb-1 and CD19. The expression of the mb-1 gene could, however, be activated by treatment of the neuroblastoma cells with chromatin disrupting agents. This suggests that chromatin structure is a key component in the regulation of transcription factor function, by restricting the accessibility of target genes, possibly contributing to the apparent link between a transcription factor and specific malignancies.In the present study we were interested in the mechanisms of tissue specific target gene activation by comparing transcription factor function and gene expression patterns in neuroblastomas and pre-B cells. We here report that even though neuroblastoma cells express both Pax-5 and E-proteins, both suggested to be crucial co-activators for EBF target genes in pre-B cells, the cells do not express the pre-B cell restricted target genes 2. SH-SY5Y, IMR-32 and SK-N-BE(2)c neuroblastoma cells were cultured in Eagle's Minimum Essential Medium (MEM) with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin in an atmosphere of 5% CO2 at 37°C (Life Technologies). KCN-69n and LA-N-1 neuroblastoma cells were cultured in RPMI 1640 medium supplemented with FCS and antibiotics as above. The SK-N-BE(2)c cells were differentiated with 10 μM all-trans-retinoic acid (RA) in MEM containing 10% FCS for 0, 2, 8, 24, and 96 hours. 5-azacytidine (5-azaC) treatments were performed on SK-N-BE(2)c and SH-SY5Y cells at a concentration of 400 nM. After 64 h, fresh medium containing 50 nM Trichostatin A (TSA) was added. The cells were harvested in PBS after 24 h.HeLa, THP-1, KM3 and Nalm6 cells were grown in RPMI 1640 medium supplemented with 7.5% fetal calf serum (FCS), 10 mM HEPES, 2 mM pyruvate and 50 μg/ml gentamicin (complete RPMI media) (Life Technologies) at 37°C and 5% CO+ nylon membranes (Amersham) using capillary blotting with 0.4 M NaOH. Membranes were pre-hybridized in 5X Denhardt's, 6XSSC, 0.1% SDS and 50 μg/ml Salmon Sperm DNA, at 57°C for 90 minutes and hybridized with γ[32P] labeled oligonucleotide for 12 hours at 57°C in the same solution. Membranes were washed at room temperature 2 times in 2XSSC supplemented with 0.1% SDS for 15 minutes.RNA was prepared from cells using Trizol (Life Technology) and cDNA was generated by annealing 1 μg of total RNA to 0,5 μg of random hexamers in 10 μl DEPC-treated water. Reverse transcriptase reactions were performed with 200 units of SuperScript Reverse Transcriptase (Life Technologies) in the manufacturers' buffer supplemented with 0,5 mM dNTP, 10 mM DTT and 20 units RNase inhibitor (Boeringer Mannheim, Bromma, Sweden) in a total volume of 20 μl, at 37°C for 1 hr. One-twentieth of the RT reactions were used in the PCR assays. PCR reactions were performed with 1 unit of Taq-polymerase (Life Technologies) in the manufacturers' buffer supplemented with 0.2 mM dNTP, in a total volume of 25 μl. GADPH was amplified by 25 cycles (94°C, 30 s, 55°C, 30 s and 72°C, 30 s) while 30 cycles were used to amplify CGA, SCG10, mb-1 and CD19 message (94°C, 30 s, 61°C, 30 s, 72°C 30 s). Primers were added to a final concentration of 1 mM. PCR products were blotted onto Hybond NOligonucleotides used for RT-PCR were:GADPH sense; 5'-CCACCCATGGCAAATTCCATGGCA;GADPH antisense; 5'-TCTAGACGGCAGGTCAGGTCCACC;CGA sense: 5'-GAAGATGAACTCTCAGAGGTTCCGA antisense: 5'-GGATCTCCTTGTAGCCAAGGCTCGCD19 sense: 5'-AGTCATTGCTGAGCCTAGAGCTGCD19 antisense: 5'-CTCGGAGTCCTCCTCACTGTCAGmb-1 sense; 5'-CCAGCATCATTGATGGTGAGCCmb-1 antisense: 5'-GACATCTCCTATGTTGAGGCTGCmb-1 hybridization; 5'-CCCGCACAATAGCAGCAACAACGCCAACGTSCG10 sense; 5'-ATGCTGTCACTGATCTGCTCTTGCSCG10 antisense; 5'-CAGGTTGAACTGTCTGGCTGAAG32P] ATP by incubation with T4 polynucleotide kinase , annealed and purified on a 5% polyacrylamide Tris-borate-EDTA (TBE) gel. Nuclear extract [in vitro transcribed-translated protein, was incubated with labeled probe (20,000 cpm, 3 fmol) for 30 min at room temperature in binding buffer (10 mM HEPES pH [7.9], 70 mM KCl, 1 mM Dithiothreitol, 1 mM EDTA, 2.5 mM MgCl2, 1 mM ZnCl2, 5% Glycerol) with 0.75 μg Poly(dI/dC) (Amersham Pharmacia). Antibodies  were added 10 min before the addition of the DNA probe. The samples were separated on a 6% polyacrylamide TBE gel, which was dried and subjected to autoradiography.DNA probes were labeled with γ[ extract , or in vOligonucleotides used for EMSA were the following:mb-1 sense 5'-AGCCACCTCTCAGGGGAATTGTGG;mb-1 antisense 5'-CCACAATTCCCCTGAGAGGTGGCT;CD19-BSAP sense 5'-GCAGACACCCATGGTTGAGTGCCCTCCAGG;CD19-BSAP antisense 5'-CCTGGAGGGCACTCAACCATGGGTGTCTGC;E5 sense: 5'-GGCCAGAACACCTGCAGACG;μE5 antisense: 5'-CGTCTGCAGGTGTTCTGGCC;μOct binding site sense 5'-CATCTCAAGTGATTTGCATCGCATGAGACG;Oct binding site antisense 5'-CGTCTCATGCGATGCAAATCACTTGAGATC;Lambda B (Pu.1 site) sense: 5'-GAAAAAGAGAAATAAAAGGAAGTGAAACCA AG;Lambda B antisense: 5'-CTTGGTTTCACTTCCTTTTATTTCTCTTTTTC;CRE (ATF5 site) sense: TCA TGG TAA AAA TGA CGT CAT GGT AAT TACRE antisense: TAA TTA CCA TGA CGT CAT TTT TAC CAT GARNA from Nalm6, SK-N-BE(2)c, SH-SY5Y, KM3, THP-1 and HeLa cells was extracted using Trizol™ . A common RNA control obtained by mixing a variety of cell lines was used for all hybridizations. RNA was concentrated to 50 μg total RNA (25 μg sample RNA and 25 μg control RNA) to generate aminoallyl-modified cDNA. Sample cDNA was labeled with Cy3-dCTP and control RNA was labeled with Cy5-dCTP using CyScribe Post-Labeling Kit (Amersham Pharmacia Biosciences). A hybridization solution was made by combining labeled cDNA with 20 μl Cot-1 DNA (1 mg/ml), 3 μl Poly dA (4 mg/ml) and 1.5 μl yeast t-RNA (4 mg/ml), dry down by speed-vac and resuspended in 40 μl Pronto! Universal Hybridization Solution™ . The hybridization solution was added to a pre-hybridized microarray slide (DNA microarrays were obtained from the SWEGENE DNA Microarray Resource Center, Lund University). The arrays were hybridized at 42°C for 18 hrs, washed according to the manufacturers recommendations , dried by centrifugation and scanned on Agilent microarray scanner. Scans were analyzed using GenePix Pro versions 4.0.1.9 and 4.1.1.4. BioArray Software Environment (BASE) :software0003.1–0003.6). The settings for the analysis presented were, Background Correlation: Mean FG – Mean BG, Spot filter: (Raw) SNR ch1 mean > = 2, (Raw) SNR ch2 mean > = 2, (Raw) Flags = 0, (Raw) Spot diameter > = 40, Normalization: Lowess, Reporter filter: in # of assays = 16, Analysis: Hierarchical clustering (reporter)N-myc gene, which in the clinic is correlated to adverse outcome of the disease, while the SH-SY5Y cell line carry an N-myc gene in germ line configuration [Knowing that neuroblastoma and pre-B cells share the expression of EBF ,13,14, wguration .CD19 [mb-1 [SCG10 and CGA [SCG10 and CGA message they did not express either CD19 or mb-1 message. The opposite pattern was observed in the pre-B cell lines where CD19 and mb-1 but not SCG10 or CGA message could be detected. This show that even though neuroblastoma and pre-B lymphoma cells to some extent share gene expression patterns, they do not appear to share the expression of specific EBF target genes.The finding that these two different types of tumors did display a degree of similarity in overall gene expression patterns opened the possibility that the previously identified EBF target genes would be expressed in both cell types. To investigate this possibility we extracted RNA from pre-B cell lines as well as neuroblastoma cells and investigated the expression of the pre-B cell EBF target genes 19 [mb-1  as well  and CGA  by RT-PCmb-1 and CD19 genes are genetic targets for both EBF and the paired domain protein Pax-5 (BSAP) [CD19 promoter [CD19 Pax-5 binding site. No such binding activity was detected in extracts from epithelial cells (Data not shown). To verify that the binding activity was due to the presence of Pax-5 we performed a super-shift experiment using a Pax-5 specific antibody, a control antibody or no antibody in the binding reaction  ,19-21. Tpromoter . These en Figure . While tn Figure . Since tn Figure . This inIn addition to provide information of overall relationships between different types of tumors, the micro-array analysis also yield preliminary information about differential gene expression patterns of individual genes (Appendix A). The data obtained in our experiments suggested differential expression of genes such as myb, CBFβ and HMG 1, 3 and 4 proteins with high expression restricted to the pre-B cells while Bcl-6 appeared to be over-expressed specifically in neuroblastoma cells (Appendix A). To further investigate other potential differences and similarities between pre-B and neuroblastoma cells with regard to expression of gene regulatory proteins we extracted expression data regarding additional genes encoding transcription factors from our data set Figure . SeveralTo further investigate the E-box binding activities in SH-SY5Y cells as compared to Nalm6 cells we performed super-shift assays using antibodies against E2-2, E47 or c-myc Figure . The majThe micro-array analysis did also suggest a difference in the expression of activator of transcription 5 (ATF5/ATF7/ATFX) ,26. ATF5EBF has also been suggested to share pre-B cell restricted target genes with Ets proteins and one factor belonging to this family of proteins suggested to contribute to B lineage identity is the Ets protein Pu.1 ,29. In omb-1 gene in neuroblastoma cells after disruption of the chromatin structure by culturing the cells in the presence of the DNA de-methylating agent 5-azaC and the histone deacetylase inhibitor TSA. RT-PCR analysis suggested that while the untreated control cells did not express any detectable amount of mb-1 transcripts the 5-azaC/TSA treated cells expressed significant amounts of mb-1 transcripts c cells treated with 5-azaC/TSA was about one twentieth of that observed in a pre-B cell, the SH-SY5Y cells were induced to express levels comparable to that observed in the pre-B cells. In contrast we were unable to observe an induction of CD19 expression (Data not shown). Thus, we conclude that the lineage specific regulation of the mb-1 gene may be directly dependent on chromatin status, while expression of other pre-B cell specific EBF target genes, such as CD19, must be explained by other mechanisms.These experiments suggested that there indeed are specific differences in transcription factor expression between the two cell types, but also that they share expression of several key regulatory molecules. This indicates that additional components of gene regulation may be of crucial importance for target gene selection. One potential barrier to gene activation could be the chromatin structure, that is, the epigenetic setting of the cells directs the transcription factors to their tissue specific target genes. To investigate this possibility we analyzed the expression of the s Figure . To obtain vitro data [We here report that neuroblastoma and pre-B lymphoma cells share the expression of transcription factors believed to be essential for normal B cell development while the expression of their B lineage target genes remains tissue specific. The finding that neuroblastoma cells expressed Pax-5 was somewhat surprising even though this factor has been shown to be involved in midbrain development. Mice carrying a homologous disruption of the Pax-5 gene lack both B-lymphoid cells and a fully developed central nervous system ,31. In ttro data  as well tro data -40 indicIn contrast to the stable expression of Pax-5 there appeared to be large differences in E-protein expression in the neuroblastoma cell lines investigated. In neuronal tissues a number of tissue specific bHLH proteins have been defined. Importantly, these pro-neuronal bHLH proteins require heterodimerization with E-proteins for the formation of DNA binding complexes. In the developing sympathetic nervous system HASH-1 is transiently expressed and is of pivotal importance for the formation of the autonomic and olfactory nervous system, neuroendocrine cells of the lung and specific regions of the telencephalon (reviewed in ). We havmb-1 promoter is has been shown that methylation of specific C residues in the promoter prevents the complex formation between Pax-5 and Ets proteins, thereby reducing functional activity of the promoter [mb-1 transcription in the SH-SY5Y cells then in the SK-N-BE(2)c cells  declare that they have no competing interests.Anna Lagergren has performed the EMSAs, the micro-array experiments and the PCR experiments under the supervision of Mikael Sigvardsson while Christina Manetopoulus has performed the cell culture work as well as 5-aza-Cytidine treatments and RNA purifications under the supervision of Håkan Axelson. All contributors have been involved in the writing of the manuscript.The pre-publication history for this paper can be accessed here:The data shows hierarchical clustering of the microarray data displaying differential gene expression patterns of individual genes in the different cell lines used in this study.Click here for file


The 4-day course was sponsored by WHO-EMRO with additional support from the Canadian Program in Genomics and Global Health. The overall objective of the course was to collectively explore how to best harness genomics to improve health in the region. This article presents the course findings and recommendations for genomics policy in EMR.While innovations in medicine, science and technology have resulted in improved health and quality of life for many people, the benefits of modern medicine continue to elude millions of people in many parts of the world. To assess the potential of genomics to address health needs in EMR, the World Health Organization's Eastern Mediterranean Regional Office and the University of Toronto Joint Centre for Bioethics jointly organized a Genomics and Public Health Policy Executive Course, held September 20The course brought together senior representatives from academia, biotechnology companies, regulatory bodies, media, voluntary, and legal organizations to engage in discussion. Topics covered included scientific advances in genomics, followed by innovations in business models, public sector perspectives, ethics, legal issues and national innovation systems.A set of recommendations, summarized below, was formulated for the Regional Office, the Member States and for individuals.• Advocacy for genomics and biotechnology for political leadership;• Networking between member states to share information, expertise, training, and regional cooperation in biotechnology; coordination of national surveys for assessment of health biotechnology innovation systems, science capacity, government policies, legislation and regulations, intellectual property policies, private sector activity;• Creation in each member country of an effective National Body on genomics, biotechnology and health to:- formulate national biotechnology strategies- raise biotechnology awareness- encourage teaching and training of biotechnology- devise integration of biotechnology within national health systems.The recommendations provide the basis for a road map for EMR to take steps to harness biotechnology for better and more equitable health. As a result of these recommendations, health ministers from the region, at the 50th Regional Committee Meeting held in October 2003, have urged Member States to establish national bodies of biotechnology to formulate a strategic vision for developing biotechnology in the service of the region's health. These efforts promise to raise the profile of genomics in EMR and increase regional cooperation in this exciting new field. In a recent study, University of Toronto researchers identified the "Top 10 Biotechnologies to Improve Health in Developing Countries" . Its misThere is a wide range of scientific capacity and health system development across the Eastern Mediterranean Region (EMR) , which cth–23rd, 2003, in Muscat, Oman. This 4-day course/workshop was sponsored by WHO-EMRO with additional support from the Canadian Program in Genomics and Global Health.In order to assess the potential of genomics to address health needs in the Region, the World Health Organization-Eastern Mediterranean Regional Office (WHO-EMRO) and the University of Toronto Joint Centre for Bioethics jointly organized a Genomics and Public Health Policy Executive Course, held September 20The overall objective of the Genomics Policy Executive Course was to familiarize participants with the potential of genomics and related biotechnologies to address health needs and to collectively address the question of how best to harness genomics to improve health in the region table .We based the program and designed the sessions and lecture topics of this course on prior courses held in Nairobi, Kenya in March 2002; in Toronto, Canada in May 2002; and in Kumarakom, India in January 2003. The sessions and presenters of the course are shown in table "How best to harness genomics and biotechnology to improve the health of the people in the Eastern Mediterranean Region?" The groups met frequently to discuss the presentations, and each participant was also provided a course reader with additional literature on the lecture topics (table 3). The overall task of these study groups was to draw upon the course material and their own experiences and propose a set of recommendations for genomics and biotechnology policy in the region. On the last day of the meeting each group presented these recommendations.The sessions dealt with a wide range of relevant topics, starting with recent scientific advances in genomics and stem cell research, followed by discussions on business models in genomics and biotechnology, intellectual property rights and regulatory frameworks, public engagement and an internet-based opinion leaders' network. The presentations were designed to be interactive and foster active discussion from, and among, the participants, and each presentation was followed by a moderated discussion period. Early in the course, the attendees were placed into one of five study groups – these groups were carefully designed to capitalize on the diverse backgrounds of the participants. Each group was assigned the task to on the deliberate the key question In order to assess the level of interest in the formation of an email-based opinion leaders' network to continue discussion among the participants following the course, a brief survey was conducted on the participants' internet access and their expectations of the network.Dr Peter A. Singer, Director of the University of Toronto Joint Centre for Bioethics, described the overall aim of the course "How to best harness genomics to improve health in the region", as well as the 4-day program. Dr Ali Jaffer Suleiman of the Ministry of Health in Oman acted as chairperson of the meeting.Professor Riad Bayoumi of Sultan Qaboos University highlighted new scientific developments that have resulted from the genomics revolution, such as proteomics; mapping of single nucleotide polymorphisms (SNPs) to understand human genetic variation and its relationship with disease; gene expression chips to monitor differential gene expression and identify drug targets; and bioinformatics as a new field that combines biology, mathematics, statistics and computer programming to mine large-scale biological data.Professor Alexander Capron, Director, Department of Ethics, Trade and Human Rights at WHO, described the 2002 report of the World Health Organization "Genomics and World Health" . He callProfessor Abdallah Daar described a recent study, conducted by the University of Toronto Joint Centre for Bioethics' Canadian Program on Genomics and Global Health, to identify the ten most promising biotechnologies for improving health in developing countries in the next five to ten years. These technologies offer guidance to those who can influence the direction of R&D in developing countries and challenge common assumptions about the relevance of biotechnology for these countries, as shown by the mapping of the Top 10 Biotechnologies onto the UN Millennium Development Goals [Professor Al Bar of King Fahad University, Saudi Arabia, illustrated Islamic perspectives on genetic testing, cloning, recombinant DNA technology and other genomics-related technologies. He showed that while Islam and science have always been aligned through history, today's religious leaders in the region must take the initiative to develop and formulate recommendations for genomics.Dr. Peter Singer described the concept of innovation systems and presented some observations of other countries' innovation systems and their successes. One definition of a national system of innovation (NSI) is the "network of institutions in the public and private sector whose activities initiate, import, modify and diffuse new technologies" -13. The Mr. Khalil Ahmed, Managing Director of Shantha Biotechnics, described the successes of this biotechnology company based in Hyderabad, India. Shantha was established in 1993, in part with seed money from the Government of Oman. Today, it is the first Indian company to receive WHO certification for a recombinant hepatitis-B vaccine Shanvac-B™, paving the way for UNICEF to buy 8.5 million doses for distribution globally. Hepatitis B vaccine is currently priced internationally as high as $8–10 per dose, while Shantha is selling it at $2 per dose ,16.Professor Richard Gold of McGill University's Centre for Intellectual Property Policy described the basics of intellectual property rights, patents and copyright issues, as well as policy options for developing countries in the international context. He outlined the characteristics of international agreements, which offer considerable flexibility to countries on how to apply patent laws to genomics and biotechnology. Developing countries have a number of options, such as compulsory licensing and research exemptions, to deal with property rights.Dr Jayasuriya, Director of UNAIDS in Pakistan described ways in which legal systems can facilitate best use of biotechnology. It is essential that legal reform keep up with the rapidly evolving science of genomics. Health law can facilitate best use of biotechnology – by providing for fast-track approval of biotechnology products; reducing import duties on health interventions; and allowing multiple channels of procurement and distribution to improve access and optimize prices.Dr Anwar Nasim, Chairman of Pakistan's National Council of Biotechnology, described the role of regulations both to promote useful biotechnologies and limit their risks for human health and the environment. A national-level regulatory body could provide guidelines for the use and release of biotechnology products, conduct biosafety reviews and risk assessments and formulate feedback mechanisms to improve the system through experience. The National Commission of Biotechnology of Pakistan, established in November 2001, focuses on biotechnology regulatory issues in health, agriculture, environment and industry. Similar commissions could be set up in other countries of the region and their interaction could further regional cooperation in biotechnology.Mr. Ehsan Masood of Scidev.net pointed out that active public engagement based upon knowledge can stimulate action to improve public health. Public engagement is far greater in today's world than it has ever been in the past. This is because of several factors including; a) the growing implications of research on public health, b) the increased awareness in civil societies to invest in health care and research, and to influence policy change and action, c) development and access of information technologies."How best to harness genomics and biotechnology to improve the health of the people in the Eastern Mediterranean Region?", were invited to present their findings. The main points of these group presentations are summarized in the recommendations that emerged from the course.On the last day of the course the five participant groups, who had been assigned the task to on the deliberate the key question The session discussions and group presentations underscored the urgent need for action to create the enabling environments  for research and development in genomics and biotechnology.The issue of awareness of biotechnology among political leaders to garner support at the highest level was raised early on in the course, and was reinforced throughout the course discussions. The participants felt strongly that political commitment is crucial to the advancement of biotechnology in the region. Political leadership is a critical factor in raising the profile of science in developing countries. A prominent example in the Eastern-Mediterranean Region is that of the Sultan Bin Mohammed Al-Qassimi, Shaikh of the Arab emirate of Sharjah. He has made a dedicated effort to change the face of science in the Gulf . For exaThe course attendees recommended that WHO-EMRO should take the responsibility to engage political leaders in the region's national governments. The participants shared experiences of the importance of government support for biotechnology – in Egypt there appears to be relatively strong support for biotechnology by the government, with biotechnology activity in academic and other research institutions as well as in the private sector. In Iran, considerable advances have been made in private and academic research centres, leading to a number of biotechnology products that are soon to reach the market, as well as well-respected scientific journals such as the Iranian Journal of Biotechnology . Other cIt will also be crucial to involve, inform and engage religious leaders in the region in order to promote genomics and biotechnology for improving public health. In doing so, it is worthwhile to note that Islam and science have always been aligned through history and it is well-established that Islam has historically contributed tremendous achievements to the advancement of science.  A recentThe active participation and involvement of organizations like COMSTECH was recommended to give support and guidance to WHO-EMRO's efforts. COMSTECH was established by the Islamic Summit in 1981 and includes among its objectives the building of indigenous capabilities in the fields of science and technology, promotion and continuing cooperation and coordination in scientific and technological areas of is member states and creation of effective institutional structure for planning research, development and monitoring of scientific and technological activities. COMSTECH has already launched networks across the region for exchange of information, and has valuable experience in the promotion of cooperation and coordination amongst the member states in science and technology activities in high technology areas .Bacillus thuringiensis [A major discussion point at the workshop was the effective assessment of existing capacity in the region. Several participants shared disappointment over the relative lack of participation of the region in global science and technology advances, citing, for instance, poor representation of the region at international scientific conferences. Many felt the need to evaluate and assess the level of scientific activity and capacity in individual countries in the region, in order to identify, among other things, strengths and weaknesses, entry points for countries, opportunities for regional collaboration, and areas for improvement. The workshop attendees felt it important to carry out this type of survey of each country's innovation system as a prerequisite to revising and revamping national and regional genomics policy. The factors identified by the participants as important for assessment in this survey – scientific capacity within public and private sector, private enterprise, religious and political leadership and intellectual property rights – can be considered to be part of the National System of Innovation (NSI). This proposed survey of NSI in the region can help to identify factors for successful growth of biotechnology sectors. Similarly, the recently completed study by the Canadian Program on Genomics and Global Health of the health biotechnology innovation systems of Brazil, China, Cuba, Egypt, India, South Africa and South Korea may also help to identify some of these factors . Workshongiensis . In IranAs highlighted by the 2004 report of the UN Commission on Private Sector and Development report , the proThe attendees observed that the key to effective participation in the genomics revolution is building scientific capacity, and expressed serious concern over the region's limited capacity to absorb and utilize genomics and biotechnology. Critical to capacity-building is access not just to technology but, more importantly, to scientific knowledge. One point-of-entry that was greeted with enthusiasm by the participants was bioinformatics, which is typically less-resource intensive compared with other genomics-related sciences, such as sequencing and proteomics. According to the report on the "Top 10 technologies to improve health in developing countries" countries of EMR can take advantage of genomic data and apply the power of bioinformatics to local health problems without having to invest heavily in the technologies used to produce them, A dedicated Genomics and Health Research Fund for the region may help to break down financial barriers to encourage scientific research and development in the region.The participants expressed enthusiasm for setting up National Biotechnology Commissions to address genomics policies at the national level and to contribute to the development of the above-mentioned national biotechnology strategy. A National Commission on Biotechnology (NCB) has been set up in Pakistan. The overall goal would be to help devise regulations to facilitate innovation in biotechnology. The NCB could help coordinate national biotechnology activities and policy, with activities including evaluation of biotechnology capacity (see above) to priority setting and public engagement. Accordingly, the NCB should have broad cross-sectoral representation in order to minimize the negative effects of inter-institutional rivalry. One of the main goals of the NCB , following the above-mentioned national biotechnology survey, will be to help develop and adopt a national biotechnology strategy, perhaps as part of a long-term science and technology policy. Other important objectives are discussed below.public awareness) of biotechnology and genomics in the region, and that this lack of awareness may be the biggest barrier to advances in genomics. Public awareness and engagement can help change the pace of research, leading to increased opportunities for greater societal involvement for improved health care. Media can be used as a catalyst to raise community awareness for social beneficence, equity and justice in health care. Recently, in May 2004, science journalists from across the region met in Cairo to discuss the hurdles they face in science reporting. The hurdles identified at this meeting included bureaucracy and poor access to scientific research taking place in the region [At the outset of the conference, participants voiced their concern about the low level of awareness  Request regional governments and policy makers at highest level to give priority to genomics for health and health biotechnologyb) Develop linkages with Organization of Islamic Conference Standing Committee for Science and Technology (COMSTECH) and other international partners to build Regional networking and cooperation for developing and utilizing health biotechnology.c) In collaboration with Member States undertake a national survey/situation analysis/needs assessment of health biotechnology innovation systems including resource capacities, government policies  In collaboration with COMSTECH initiate a research grant for applied  genomics and biotechnologyThe participants agreed that each member state would benefit from creating effective National Commissions on Biotechnology (NCB) to develop national biotechnology strategies. NCB should; a) develop national priorities, programmes and guidelines aimed at raising public education and awareness and b) collaborate with civil sectors to develop plans to integrate genetic and genomic products  build capacities for utilization and access of health biotechnology to the needy and e) ensure ethical safeguards against unwanted harm and exploitation and improve equity to improve health of the poor. The participants also felt that there was a need for strong personal commitment at individual level by experts and key actors to engage actively in the implementation of the recommendations to strengthen the initiative on genomics and biotechnology to improve the health and well-being of people in EMR. One concrete outcome of the workshop that may contribute to strengthening capacity in the region is the joint agreement between WHO/EMRO and the University of Toronto Joint Centre for Bioethics to provide scholarships to the Master of Health Sciences Program at the JCB. Furthermore, there seems to be growing need to establish a Regional Health Biotechnology Network for EMR, and WHO-EMRO is now planning to hold a Regional meeting to discuss this in Iran in July 2004.th Regional Committee Meeting held in October 2003, urged Member States to establish national bodies for genomics and biotechnology to formulate strategic vision for creating public awareness and for developing biotechnology for equitable health care in the region.WHO-EMRO has initiated follow-up of these recommendations. Ministers of Health from the region, at the 50Recently, a paper on harnessing genomics and biotechnology for public health was presented at the EM 28th Regional Consultative Committee Meeting (RCC) held in Cairo in April of this year. The recommendations in the paper were derived from those developed at the workshop. The paper will be presented at this year's Regional Committee Meeting to be held in October 2004.The methods and recommendations outlined in this paper demonstrate progress in bringing genomics and biotechnology to the forefront of science policy in developing countries. Our procedure has now led to the formation of three regional networks – the two previous ones being the African Genome Policy Forum, which encompasses participants from 10 African nations, the Indian Genome Policy Forum. We have also now held a course for Latin America and the Caribbean in association with PAHO and the UN University in Venezuela May 23–26 2004, and a similar network is being created of participants of that course. The next one will be held in the Southeast Asian region, most likely based in Hong Kong. These regional genome policy networks will provide models to establish a Global Genome Policy Forum.The author(s) declare that they have no competing interests.TA drafted the manuscript; PAS and ASD conceived of the course, and all authors participated in its design and coordination. All authors revised the manuscript for critical content and approved the final draft.


Duhuo Jisheng Wan (DJW) is perhaps the best known and most widely used Chinese herbal recipe for arthralgia, but the clinical study to verify its efficacy is lacking. The purpose of this study was to compare the efficacy of DJW versus diclofenac in symptomatic treatment of osteoarthritis (OA) of the knee.This study was a randomized, double-blind, double-dummy, controlled trial. The 200 patients suffering from OA of the knee, were randomized into the DJW and diclofenac group. The patients were evaluated after a run-in period of one week (week 0) and then weekly during 4 weeks of treatment. The clinical assessments included visual analog scale (VAS) score that assessed pain and stiffness, Lequesne's functional index, time for climbing up 10 steps, as well as physician's and patients' overall opinions on improvement.Ninety four patients in each group completed the study. In the first few weeks of treatment, the mean changes in some variables  of the DJW group were significantly lower than those of the diclofenac group. Afterwards, these mean changes became no different throughout the study. Most of the physician's and patients' overall opinions on improvement at each time point did not significantly differ between the two groups. Approximately 30% of patients in both groups experienced mild adverse events.DJW demonstrates clinically comparable efficacy to diclofenac after 4 weeks of treatment. However, the slow onset of action as well as approximately equal rate of adverse events to diclofenac might limit its alternative role in treatment of OA of the knee. Osteoarthritis (OA) is the most prevalent joint disorder characterized by articular cartilage degradation with an accompanying peri-articular bone response . OA affeThe use of Chinese and other foreign patent herbal medicines (pills and tablets) in arthralgia treatment is highly prevalent and increasing in Thailand, but importing these medicines from the People's Republic of China and other foreign countries is usually rather expensive. However, the cost of similar preparations can be minimized by using imported dried herbs available in Thailand as raw materials in the manufacturing process coupled with simple and inexpensive traditional drug manufacturing techniques. Thus, if clinical studies suggest that these herbal medicines are as effective and/or less toxic than conventional treatment, promotion of self-produced recipes in each community will lead to community-directed osteoarthritic treatment in Thailand.Bei Ji Qian Jing Yao Fang compiled by Sun Simiao in the Tang Dynasty (652 A.D.) [The herbal recipe used in this study was "Duhuo Jisheng Wan (DJW)", which means pill of Pubescent angelica root and Mulberry mistletoe combination, and it was quoted from the book 52 A.D.) ,12. Alth52 A.D.) , the cliThis randomized, double-blind, double-dummy, controlled trial was approved by the Medical Ethics Committee of the Faculty of Medicine, Chiang Mai University and was in compliance with the Helsinki Declaration.Two hundred out-patients of either sex were recruited. They were aged over 40 years, and had been suffering from unilateral or bilateral OA of the knee according to the criteria of the American College of Rheumatology  for moreDuring a run-in period of 1 week (week 0), patients considered eligible for the study were informed to discontinue all analgesics, anti-inflammatory drugs, and other modalities for the treatment of arthralgia and arthritis. At the beginning of week 1, patients who still met the eligible criteria were randomized into 2 groups (DJW and diclofenac group) and treated for 4 weeks (Table ®) were provided by Novartis (Thailand) Co., Ltd. In order to completely blind the patients, each diclofenac tablet was packed into a capsule with an appearance identical to its placebo. Either diclofenac or placebo was prescribed at 1 capsule, 3 times a day, immediately after meals.Twenty five mg film-coated tablets of commercially marketed diclofenac sodium , Radix Gentianae Macrophyllae (Qinjiao), Cortex Eucommiae (Duzhong), Radix Achyranthis Bidentatae (Niuxi), Radix Angelicae Sinensis (Danggui), Herba Taxilli (Sangjisheng), Radix Rehmanniae Preparata (Shudihuang), Rhizoma Chuanxiong (Chuanxiong), Cortex Cinnamomi (Rougui) and Radix Ledebouriellae (Fangfeng), 5% each of Radix Paeoniae Alba (Baishao), Radix Codonopsis (Dangshen), Radix Glycyrrhizae (Gancao) and Poria (Fuling), as well as 2.5% of Herba Asari (Xixin).Xixin, Niuxi, Shudihuang and Rougui were imported from the Shantou Traditional Chinese Medicine Factory, the People's Republic of China (PRC). The remaining herbs were imported from the Qixin Co., Ltd. (Hebei Province), PRC. Each pulverized ingredient was mixed thoroughly together according to the formula mentioned above and prepared into honeyed pills, which were baked in a hot air oven until completely dry, and then pulverized. The pulverized powder was finally filled into capsules of 500 mg per capsule. The quality control and standardization of DJW (i.e., assessment of weight variation, disintegration time, screening for microorganisms and aflatoxin) were conducted by using guidelines recommended by the Food and Drug Administration of Thailand . DJW andClinical assessments were evaluated for base-line data at the end of a run-in period (week 0) and then weekly for 4 weeks. These assessments included 100-mm VAS that assessed pain , 100-mm VAS that assessed stiffness , Lequesne's functional index that assessed the patient's daily activities (score ranging from 0–24) , and timAt the end of week 1–4, 100-mm VAS that assessed the physician's and patients' overall opinions on improvement were also evaluated. The assessment forms were designed so that the patients and evaluator could view their own previously recorded scores, but they were not allowed to view each other's VAS. Clinical assessments were evaluated by the same physician who had been blinded to the treatment. Complete physical examination and non-directive questioning for adverse events were also performed weekly for 4 weeks in order to acquire a safety assessment.In within the group analysis, the mean VAS and Lequesne's functional index between base-line and the following weeks were compared by a non-parametric Wilcoxon's signed-rank test, whereas, the average time for climbing up 10 steps was compared by the paired t-test.In the analysis between the groups, a non-parametric Wilcoxon's rank-sum test was used to determine whether the two groups differed in the physician's and patients' overall opinions on improvement. In addition, the mean changes in VAS that assessed pain and stiffness, as well as Lequesne's functional index were compared by the same test. The student's t-test was used to compare the mean changes in the time for climbing up 10 steps.A total of 429 patients were recruited into this study, of whom 229 were excluded Figure . The remThe VAS that assessed pain and stiffness at the end of week 1–4 decreased significantly when compared to their own base-line values , as did Lequesne's functional index and time for climbing up 10 steps . All adverse events reported were mild in intensity in both groups. The most common adverse events occurring in the DJW and diclofenac group were raised blood pressure (16% vs. 19%), central nervous system symptoms including dizziness, somnolence and drowsiness (16% vs. 11%), and gastrointestinal symptoms including nausea/vomiting, dyspepsia, diarrhea and constipation (12% vs. 5%). The least common adverse events were increased appetite, cramp, rash and flu. More than one adverse events might be occurred in some patients. However, the percentages of patients who experienced each adverse event in both groups were not significantly different.In summary, the VAS that assessed pain and stiffness, Lequesne's functional index and time for climbing up 10 steps at each time point decreased significantly in the DJW and diclofenac group when compared to their own base-line values. The mean changes in all VAS that assessed pain, except those for walking and standing, did not differ significantly between both groups. The differences in mean changes in the VAS that assessed walking pain, standing pain and stiffness were found only during week 0–1, whereas, those in Lequesne's functional index were found during week 0–1 and 0–2.Since the preparations and dosages of DJW and diclofenac were different, this study was designed as a randomized, double dummy, controlled trial in order to completely blind both patients and physician (double-blind). Therefore, the placebo of DJW was also prescribed for the patients in the diclofenac group, and vice versa, the placebo of diclofenac was prescribed for the patients in the DJW group.Among the 15 herbs used as raw materials in DJW, Xixin (Herba Asari) seemed to be the most toxic, due to its pungent taste and warm property . GeneralIn an ITT analysis , the mean changes in some variables between the two groups were significantly different after the first few weeks of treatment, and became no different afterwards. These differences suggest that the onset of DJW is significantly slower than diclofenac for at least 2 weeks  or 3 weeks . The reason why DJW needs a few weeks to exert its effect may be due to 3 possibilities. Firstly, from the pharmacokinetic point of view, the elimination half-life of the active ingredients in DJW might be too long, and therefore needs weeks to accumulate until a steady state concentration is reached  and its maximal therapeutic effect is evident. Secondly, from the pharmacodynamic point of view, DJW may exert its effects via several probable mechanisms (similar to many novel biologic treatments of arthropathy) involved modifications of cartilage metabolism, normalized viscosity and elasticity of synovial fluid, etc. These mechanisms of action might resemble many symptomatic slow acting drugs in osteoarthritis (SYSADOA) such as glucosamine sulfate, intra-artricular hyaluronan, and others. These interventions always need a period of time to exert their therapeutic action. Thirdly, the major effect of DJW might be the result from placebo effect and/or natural fluctuation of the OA symptoms. It could be simply that diclofenac worked quickly, but patients in both groups got better anyway by 2–3 weeks. Although the last possibility cannot be entirely ruled out, but it seems unlikely because even there is a tendency of OA symptoms to improve after placebo treatment, it has been reported that diclofenac was significantly superior to placebo in relieving pain, improving stiffness, and improving physical function after 4 weeks of treatment . FurtherSince this study demonstrated that approximately 30% of study subjects in each group experienced adverse events, this data suggest that the toxicity profiles of DJW are similar to diclofenac. Therefore, cautious use of DJW should be considered in the same manner as using diclofenac including other NSAIDs. However, the gastrointestinal adverse effects in the diclofenac group were quite low when compared to other short-term NSAIDs studies ,19. ThisDJW demonstrates clinically comparable efficacy to diclofenac after 4 weeks of treatment. However, the slow onset of action as well as approximately equal rate of adverse events to diclofenac might limit its alternative role in treatment of OA of the knee.The author(s) declare that they have no competing interests.ST carried out the randomization, supervised data collection and analysis, and drafted the manuscript. PK participated in the design of the study and performed the statistical analysis. NR participated in the selection of patients eligible for the study. KS carried out the outcome assessments. SP participated in the report of radiographic findings of knee. SL participated in the preparation of DJW and its placebo. SP carried out the screening for microorganism contamination in DJW and its placebo. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: