The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5′ and 3′ transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close  JAZF1 gene on chromosome 7p15 and the 3′ exons of JJAZ1  located on chromosome 17q11. In this case no SV could be detected The complex repertoire of RNAs found in cells from yeast to human is unexpected and at times seemingly daunting. In part, this complexity is composed of transcripts whose sequences are chimeras formed from sequences found in separate genes. The origins of such chimeric RNAs are derived from multiple biological mechanisms as well as technical artifacts. The biological sources of chimeric RNAs have been seen to stem from both DNA and RNA mediated events. DNA mediated event include such mechanisms as chromosomal rearrangements, gene duplications, retrotransposition and retrotransduction in vitroin vivoin vitro experiments leading to RT-mediated chimeric products (RT). With the development of high throughput RNA sequencing (RNAseq) methods that are reliant on RT for conversion of RNA into double stranded cDNAs, the template switching activity of RT has been observed in these sequencing methods by two Drosophila species mixture experiments Complicating the identification of chimeric RNAs is the clear possibility of technical artifacts caused by the template switching capabilities of reverse trancriptase (RT) in both The focus of the studies presented here was to systematically analyze the diversity of transcripts found within and extending from the annotated boundaries of genic loci in the human genome. These studies follow in the footsteps of a set of earlier works performed with a limited number of human genes that were located in the 1% of the human genome selected by the Encyclopedia of DNA Elements (ENCODE) project Protein coding genes encoded on human chromosomes 21 and 22 were interrogated using a combination of methods including rapid amplification of cDNA ends (RACE) and tiling arrays The products of the RACE reactions were then analyzed using chromosome 21 and 22 tiling arrays interrogating the non-repeat portions of these chromosomes at 17 nucleotide resolution. RACE reactions were pooled prior to hybridization to the tiling arrays. First, RACE reactions for a given primer originating from different tissues were pooled together  to the primer, consistency of connectivity across multiple samples, and support of connectivity by multiple primers see . After tThe identified and assigned RACEfrags displayed a length ranging from 47 to 4,493 base pairs, and a median length of 133 base pairs. As expected this is similar to the median length of exons (128 nt) in the Gencode The total 195,982 RACEfrags assigned to primers across all tissues and mixtures corresponded to 79,560 distinct loci of transcription. When overlapping RACEfrags found in different biological samples were melded, a total of 17,450 projected RACEfrags, covering 5.1 Mb in chromosomes 21 and 22 (15%), resulted. The distributions of detected RACEfrags both within the gene from which the RACE reaction started (index gene) and the genomic domains outside of the index gene are given in Interestingly, more than half of the gene anchored RACEfrags (59%) map outside of the annotated boundaries of the index genes . The medBecause of the systematic 5′ and 3′ RACE interrogations of the same exons within a gene, we often observe genes that are reciprocally connected through the RACE reactions. Specifically, we define this condition in the following way: two genes A and B are reciprocally connected if and only if, a RACE primer in A originates a RACEfrag within locus boundaries of B, and a RACE primer in B originates a RACEfrag in A , and detThe array detected chimeric transcripts were first characterized and validated by molecular cloning and sequencing. Starting from the RNA pools from which they were detected, a total of 200 RACEfrags were selected for RT-PCR amplification, full-length cloning and sequencing. These RACEfrags were comprised of 67 reciprocal gene to gene connections and 133 additional chimeric cases stratified according to increasing distances from the index gene to the RACEfrag . A total−16, While many gene to gene connections observed in this study are specific to a given cell line/tissue type, a majority can also be detected in more than one sample. Indeed, 9,647 gene to gene connections were detected in two or more samples (57.9%), 6,264 in three or more (37.6%) and 169 (1.0%) in all of the samples analyzed. Further the pairwise correlation involving reciprocal gene to gene connections observed between cell types was determined. These correlations range from 0.05 to 0.35, and were all found to be statistically significant  .In a second set of analyses we compared our RACEarray results with the results of paired end RNAseq analysis of total polyA+RNA from pooled human testes/prostate and brain tissues (Human Body Map 2.0 data). A total of 4,485 and 7,448 gene to gene connections  were detAs stated earlier, a serious concern is the potential that these observations are caused by the capability of RT to switch templates during primer extension leading to technically created chimeric artifacts. This capability of RT has been observed during the RNAseq protocol In order to evaluate further the origin of the observed chimeric transcripts, two sets of RT-independent validation experiments were performed.The first set consisted of RNAse protection assays (RPA). Although the level of sensitivity of this hybridization-based assay has conventionally been understood to be considerably lower than traditional RT-PCR The second set consisted of estimating the amounts of possible contribution of technical artifacts to our results. These experiments are based on the analysis of RNAseq libraries prepared from mixtures of human GM12878 cell and Drosophila melanogaster adult female RNAs. This strategy allowed us to analyze in isolation a control population of chimeric reads consisting exclusively of technical artifacts (inter-genomic reads), and to compare its properties to that of our populations of interest (intra-genomic chimeras).We first analyzed two libraries prepared from pure total RNA from either human or fly, and one library prepared from a 1∶1 mix of RNA from both sources. We obtained about 10 M reads per library that could map uniquely to either of the two reference genomes. Considering that this simple null model may not accurately describe the generation of artifactual chimeric reads, and that some factors – such as sequence similarity, for instance – may favor the formation of intra-genomic artifacts, we had also sequenced additional libraries designed specifically to distinguish between molecules of technical versus biological origin within the population of human chimeric reads. All were prepared from a mix of human and fly total RNA, keeping the total amount of RNA constant but varying the human-to-fly ratio from 1∶1 down to 1∶100. In this type of dilution series, molecules of biological origin and library preparation artifacts are expected to have drastically different behaviors. The number of the former should decrease linearly with the dilution factor, whereas that of the latter should decrease linearly with the square of the dilution factor. Using the most stringent mapping criteria (min 2×25 bases) the decrease is very close to linear . This number of connections is statistically greater than a median of 2 that is expected in a random network . More geSecond, a detailed examination of the gene to gene interconnections indicated that the network was enriched in “cliques”, that is, sets of genes that are all pairwise connected see . At totaSuch properties of non-random gene to gene connections and the structure of these interconnections to form cliques show characteristic of many biological network A set of 74 highly connected genes were identified in this analysis that exhibited a substantially larger number of interconnections than expected given their length and the underlying network structure . These gInterestingly, the identified hubs appear to be more highly expressed than other genes. To measure gene and transcript expression we performed hybridizations of the 16 RNA samples onto chromosomes 21 and 22 see . From thhttp://www.biomart.org), to identify potential orthologs of all our RACE interrogated genes. This was done by examining six different eukaryotic species representing a panel of increasing evolutionary distances from human species: Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Tetraodon nigroviridis, Gallus gallus and Mus musculus .We have found that transcriptional connected genes tend to have coordinated expression. For each gene, we monitored expression over 16 tissues ; we therefore computed the correlation of expression profiles for each gene pair. We found that connected genes exhibit a higher correlation of expression profile than non-connected genes   method −3) .Finally, a total of 208 cDNA sequences resulting from our RT-PCR validation experiments focused on the chimeric junctions were examined for open reading frames (ORFs) with the result that 42% (87) such sequences included at least one ORF in at least one of the six translational frames  p = 0.0, and 7 eThe data from these studies raises several questions concerning the potential biological function(s) and the mechanism of their formation. While a definitive answer to these questions is not available as yet, the significant enrichment of exonic sequences derived from coordinately expressed genes, which often are part of multigenic networks, seems striking and suggestive of an underlying biological function. However, it is important to emphasize the limitations of this primer pooling approach. Because the chimeric RNAs cover very large distances in genome space, unequivocal assignment of a RACEfrag to a primer/gene is problematic. Thus this approach should contain a non-negligible number of false positive assignments—artificially favoring proximal ones. This fact strongly mandates and emphasizes that after additional filtration  and biochemical validation (non-RT dependent experiments) is carried out on the candidate chimeric RNAs, the resulting observed chimeric transcripts should possess non-random and biologically meaningful characteristics.Additional experiments such as capturing the chimeric RNAs by hybridization to junction oligonucleotides followed by high throughput sequencing could be done, but this approach does not avoid the problem of RT template switch. The direct, RT-independent sequencing of RNA molecules, as recently described The functional attributes of the chimeric transcripts could be in theory evaluated by disruption or silencing of these RNAs. However, the phenotypic readouts of these experiments are largely unknown and unpredicted, and one could use a litany of cellular assays in order to identify a potential phenotypic signature.in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs all point to candidates that are likely not to be caused by technical artifacts and biologically important.The statistically significant interconnections of genes involved, the greater phylogenetic depth of the genes involved in many chimeric interactions, the coordination of the expression of connected genes and the close Importantly, the synthesis of chimeric RNAs can be envisioned to provide a molecular record of transcribed regions. Speculatively, functions of such an accounting system or the cellular processes could be myriad. One such function could be to provide a means to feedback information to regulate transcription or post-transcriptional levels of the transcripts involved in chimeric RNA production. Evidence from the 5C experiments pointing to co-localization of the genomic regions giving rise to chimeric RNAs suggests the opportunity that the origin of these RNAs may be associated with transcription factories Several features of the characterized chimeric RNAs suggest that the mechanism(s) involved in their formation is (are) likely not to be limited to the canonical splicing processes. These features include: 1) the very long genomic distances separating some of the exons found in the chimeric transcripts; 2) the presence of chimeric transcripts containing sequences from different chromosomes ; 3) the Finally, observations of the existence of chimeric RNAs in not novel The high-quality HAVANA annotation ftp://genome.crg.es/pub/Encode/ChimericRNAChr2122/suppl_materials_manual_annotation.zipRACE primers were designed within what we call Atomic Coding Exon Projections (ACEPs), namely, coding exon segments not interrupted by any annotated exon boundary. This ensured that all RACE primers would match an actual exon sequence, rather than a “collapsed” version of many.In order to limit cross-hybridization of RACE products, all ACEPs were searched in the entire human genome for possible repeats with BLAT To achieve an optimal, balanced final distribution of primers along genes (ref), the following procedure was applied, to both 5′- and 3′-RACE potential primer sets:for genes whose virtual CDS sequence  was shorter than 1.2 kb, only one putative RACE primer per gene was picked, as close as possible to the middle of it.when the virtual CDS sequence of a gene was longer than 1.2 kb, the two most extreme primers were selected. If these two were separated by more than 1.2 kb of virtual CDS sequence, a third primer was picked, as close as possible to the middle of it.http://gemlibrary.sourceforge.net/), we find that all of them have a unique hit in the genome with no mismatch, 3 of them have a hit in more than one location with 1 mismatch, and 31 with two mismatches.This resulted in a final set of primers, consisting in 844 5′-RACE and 824 3′-RACE oligo-nucleotides, targeting 492 genes in total . A cost effective strategy is to pool together RACE products prior to hybridization. This compounds, however, the assignment of RACEfrags to the originating primers. To facilitate the assignment, it is desirable to maximize the genomic distance between the primed regions of the RACE experiments that are pooled together, so that each resulting signal from the array can be assigned to its corresponding primer as unambiguously as possible. The composition of each pool in terms of RACE primers is thus designed with the aim of simultaneously achieving two goals: Minimizing the number of pools (and consequently the cost of the experiment), and maximizing the genomic distance between the genomic positions of the primers that are pooled together.In order to address this optimization problem, the pooling strategy is organized in two steps. First, i/Given the genomic positions of the primers, compute the minimum number of pools required in order to guarantee a minimum distance between two consecutive primers from the same pool. The longer the distance, the more the pools. ii/Given this total number of pools, distribute the primers within pools, in order to maximize the distance between each two consecutive primers. The constraint is the minimum distance between primers. This depends on the orientation of the primers. Indeed, as primers should only allow amplification from their 3′ ends, the minimum distance from their 5′ end can be lower than from the 3′. Two minimum distances can thus be defined: distance L in 3′ of the primers, and distance l in 5′ of the primers. In practice, primers boundaries are initially extended accordingly  before being input to the pooling process. The values that we employed were L = minimum distance from 3′: 900,000 bp, and l = minimum distance from 5′: 90,000 bp.The two steps of the algorithm are described in more details in In our case, given the distance constraints above, we ended up with 102 pools. 5′ and 3′ RACE reactions were performed as published RACE reactions were pooled in two ways. First, between 14 and 19 reactions (mean 16.4) from a single tissue/cell line were pooled together respecting the greatest possible genomic distance between the different primers used  to create a set of 102 pools for each tissue/cell line. Second, for cost efficiency the same pools from Prostate and Testis, Ovary and Placenta, Brain Frontal Lobe, Brain Hippocampus and Brain Hypothalamus, as well as Fetal Kidney, Fetal Spleen and Fetal Thymus were pooled before being hybridized to the tiling arrays. Thus this two-step procedure generated a set of 1,020 pools.RACE PCR reactions were pooled, purified using QIAquick PCR purification kit (Qiagen) following the vendor's instructions, ethanol-precipitated, labeled and hybridized to Affymetrix Chromosome 21/22 2.0R arrays (P/N 900936) as previously described http://www.affymetrix.com/support/developer/downloads/TilingArrayTools/index.affx). In order to address the issues of pool-unspecific RACEfrag  and unspecific probes (we observed that 7% of the probes in chromosome 21–22 tiling arrays had multiple exact matches in the genome), we discarded array signals coming from these two types of unspecific probes and optimized the parameters of the RACEfrag calling, using a RACEfrag caller developed in-house (software available upon request). The caller depends on four parameters: (1) the percentile intensity threshold I above which we consider a probe to produce a positive signal. Given this threshold, the set of positive probes can be defined for a given experiment, (2) the maximum number nucleotides that is allowed between two consecutive positive probes to be included in the same RACEfrag, (3) the minimum number of probes that are needed to call a RACEfrag, and (4), a binary flag that determines whether RACEfrags are called from the middle of the first probe to the middle of the last probe, or from the start of the first probe to the end of the last probe.RACEfrags are usually called from Affymetrix tiling array probe intensities using TAS  and artifactual ones . The RACEarray simulator is described in detail in In order to eliminate RACEfrags coming from unspecific priming and/or unspecific cross-hybridization, we used our in-house On the other hand, transcripts that have not been targeted by any RACE primer but that are present at high levels in a given tissue may provide artifactual signals on the corresponding tiling array, and thus give rise to unspecific RACEfrags in this tissue. In order to eliminate such RACEfrags, we performed negative control experiments, i.e., exactly the same RACEarray experiments without any specific RACE primer in the mixture on which RACE reactions are performed , and with more sensitive parameters for RACEfrag calling . These negative control experiments thus provide us with 10 RACEfrag sets, one for each of the 10 tissue pools, which are then subtracted to the RACEfrags remaining from the USPP filter in a tissue specific manner. More precisely, any RACEfrag overlapping a negative control RACEfrag of the same tissue is eliminated. On the 302,699 RACEfrags remaining from the USPP filter, 40,227 were eliminated by the negative control filter (14%), and we kept the remaining 262,472.As a given experiment (pool) corresponds to the hybridization onto a tiling array of a mixture of products obtained from RACE reactions performed with several different RACE primers, the information as to which RACEfrag comes from which primer of its pool is not known a priori. Still, it is crucial to be able to assign an originating RACE primer to each RACEfrag, in a pool-by-pool fashion.RACEfrag assignment is an optimization problem; however we lack a proper underlying model to solve it as such. We instead use heuristic rules based on the three following remarks or definitions: i) a given pool includes primers of different chromosomes and pointing in different directions (5′ and 3′ RACE primers located on genes on the forward and the reverse strands); ii) within a pool, a primer is said to be compatible with a RACEfrag if it is located on the same chromosome as the RACEfrag and is pointing in its direction; iii) a primer is said to be active in pool p and tissue t, if it is overlapped by a RACEfrag that appears in p and t.Given a RACEfrag r of pool p and tissue t, if the closest compatible primer of pool p is active in p and t, then we assign it to r, else no primer is assigned to r and r is eliminated from the rest of the study.The fact that several tissues or/and several primers per gene are used in the RACEarray strategy can be utilized to attribute a confidence score to the assignments . More precisely, for any RACEfrag r and locus l, we define the assignment confidence score of the pair , ACS, as:Where:R is the number of times RACEfrag r appears in total ,L is the number of times r is assigned to a primer of locus l,P is the number of primers of locus l to which RACEfrag r is assigned.L is raised in order to favor RACEfrags appearing in multiple experiments: it is raised to the power of P+1 to favor RACEfrags assigned to multiple primers from the same locus, while not penalizing too much RACEfrag-locus pairs co-occurring only in a small number of pools. Raising R to the power of one plus the number of times RACEfrag r is not assigned to l greatly penalizes “ubiquitous” (unspecific) RACEfrags, while taking into account the number of times r appears. As a result, a RACEfrag r is all the more confidently assigned to a locus l as the corresponding assignment score is low.The list of these RACEfrags is also provided in gff format at the following address:ftp://genome.crg.es/pub/Encode/ChimericRNAChr2122/suppl_materials_RACEfrags.zipPolyA+RNA from the same 16 cell types as described above hybridized to Affymetrix Chromosome 21/22.0R arrays (P/N 900936) as previously described ftp://genome.crg.es/pub/Encode/ChimericRNAChr2122/suppl_materials_gene_expression_profiles.zipWe performed RT-PCR, cloning and sequencing on 245 RACE connections , so as to validate them and determine their detailed exon structures. These 245 cases were selected as follows.All 67 reciprocal gene-gene connections supported by a direct primer-primer link were included in this part of the experiment, together with 178 simple  primer/RACEfrag pairs. The latter set underwent the following selection procedure: first, all 195,982 candidate connections from the complete RACEarray map were grouped into two primer/RACEfrag distance bins, (i) 0–350 kb (short) and (ii) more than 350 kb (long). 96 primer/RACEfrag pairs were randomly picked in the short-distance bin. Pairs from the long-distance bin were ranked according to their ACS (see above) and the 82 most confident ones  were selected for RT-PCR. Within the 245 test cases, 200 corresponded to chimeric cases, namely, connections involving two different annotated loci.To experimentally verify the RACE-array defined connections , nested PCR were carried out to increase sensitivity and specificity of the reactions. Amplification was done using two sources of templates: reverse-transcribed RNA from six adult human tissues  and the relevant set of original reverse-transcribed RNA used in 3′ and 5′ RACE reactions. Reverse transcription of adult tissue RNAs was done as previously described Eight colonies were picked for sequencing from either the RACE-template reactions or the six-tissue RT-PCR clones with priority given to the RACE-template reactions. For cases in which a positive product was not observed in the RACE-templated reactions but product(s) were present in the adult tissue RT-PCRs, the latter were chosen for further analyses. In each case, colonies were grown in deep 96-well plates containing 100 µg/ml spectinomycin LB. A small volume of these cultures served as template to amplify the cloned inserts using universal vector primers. The resulting PCR amplicons were sequenced at Agencourt Bioscience Corp. . Forward and reverse sequence reads were vector-trimmed with cross_match  and assembled using Phrap To monitor RT-PCR and cloning efficiency, we carried out amplification of canonical open reading frames from four genes using the six-tissue RNA and RACE templates. These ORFs were from TP53 , CIRH1A (2061 bp), ORC1L , and THRAP4 . Following Gateway recombinational cloning, 16 colonies were picked (8 from each of the 6-tissue RT-PCR and the RACE templated experiments) and end sequenced. For TP53 and CIRH1A, all colonies  generated expected sequences. For ORC1L and THRAP4, 5 out of 16 clones, and 8 out of 16 colonies yielded the expected sequences, respectively.Second round of RT-PCR .A second round of RT-PCR was performed using the same procedure as the one described in the above subsection. However, some clone inserts could not be completely sequenced.(i) – Genome mapping and RT-PCR success rate.In total, 3,503 RT-PCR clone sequences (both assembled contigs and singlet reads) were obtained. After trimming off vector sequences and filtering out sequences of less than 100 nucleotides, 1,572 of them remained and were mapped onto the human genome (NCBI 35/hg17 version) using BLAT The 208 clusters of RT-PCR products were fed into the HAVANA manual annotation pipeline Where the RT-PCR sequence was shown by any of the approaches described above to support locus-joining canonical splice sites , a novel transcript structure was annotated and tags indicating the coding potential of the variant were added A large proportion of the RT-PCR confirmed transcript structures were found to contain small genomic duplications around the locus-joining exon junction , a tendeIn order to evaluate the genuineness, RT-PCR independent nature of the chimeric connections, we randomly picked 15 RT-PCR clones arising from reciprocal RACE connections to undergo RNAse protection assays (RPA). Each RT-PCR clone to be tested was sub-cloned into the pDONR223 vector. DNA fragments covering each chimeric junction were synthesized by PCR (100–300 base pairs) using specific primers containing SP6 (forward primer) and T7 (reverse primer). Using this template, antisense and sense riboprobes were prepared with SP6 and T7 polymerase MAXIscript in vitro transcription kit (Ambion). Designed riboprobes were checked for uniqueness using BLAT on the hg18 version of the human genome.RNase protection assays were performed by using the RPA III kit from Ambion according to the manufacturer's instructions. 500 ng of each Poly(A+) RNA (Clontech) were used per reaction. Protected fragments were separated on a 10% denaturing (8 M Urea) polyacrylamide gel and signals were analyzed by autoradiography. On the 15 chimeric transcripts tested by this method, 3 were found positive, and 2 out of the 3 positive cases were found to include a small duplicated sequence around the tested junction, as shown in ftp://genome.crg.es/pub/Encode/ChimericRNAChr2122/tabS2.xlsxIn order to know whether some RACEarray reciprocal gene to gene connections could be due to the three dimensional proximity between the two genes involved in the connection, we used 5C studies (Chromosome Conformation Capture Carbon Copy) performed on chromosome 21 on cell lines GM06990 and K562. The 5C technique indeed provides pairs of genomic regions supposed to physically interact. In the rest of the paragraph and for simplification purposes, a reciprocal gene to gene connection will simply be called a gene to gene connection.Let us first introduce some definitions. A gene to gene connection g1–g2 is:Detectable by RACEarray if there exists a RACE primer in g1 pointing in the direction of g2 and a RACE primer in g2 pointing in the direction of g1;Validated by RACEarray if g1–g2 is a reciprocal gene to gene connection;Detectable by 5C if there exists a 5C pair  such that either g1 overlaps FW and g2 overlaps RV, or g1 overlaps RV and g2 overlaps FW;Validated by 5C if there exists a 5C pair  in the 10% best 5C pairs, such that either g1 overlaps FW and g2 overlaps RV, or g1 overlaps RV and g2 overlaps FW.Among the 785 gene to gene connections validated by RACEarray on chromosome 21, 638 are detectable by 5C, and 496 are validated by 5C, yielding a proportion of 77.7% gene to gene connections validated by 5C (496/638). We were thus interested in testing the following hypothesis: “from the gene to gene connections validated by RACEarray (638), is the number of gene to gene connections validated by 5C (496) higher than expected by chance?” To do this, we sampled 1,000 times 638 gene to gene connections within the 8,852 that we find detectable by both techniques while keeping the same distributions of distance between connected genes, and of length of connected genes as in the 638 connections validated by RACEarray (details not shown), and for each sample we computed the proportion of gene to gene connections validated by 5C , and to compare its properties to that of our populations of interest (intra-genomic chimeras).A series of control experiments were conducted to investigate a possible contribution of technical artifacts – from library preparation, sequencing, or mapping – to our sequencing results. These experiments are based on the analysis of RNAseq libraries prepared from mixes of RNA from different sources –namely, human GM12878 cells and http://gingeraslab.cshl.edu/STAR/We first analyzed two libraries prepared from pure total RNA from either human or fly, and one library prepared from a 1∶1 mix of RNA from both sources. We obtained about 10 M reads per library that our alignment algorithm . With our least stringent mapping policy (minimum mapped length of 2×20 bases) the decrease is less than linear, suggesting that the analysis is confounded by sequencing/mapping artifacts – which we know not to be filtered efficiently by this policy, as explained above. With more stringent mapping (min 2×25 bases) the decrease is very close to linear: we expected 2,050, 2,050/5 = 410 & 2,050/50 = 41 reads, and observed 2,050, 317 & 51. The most stringent mapping policy shows a slightly faster-than-linear decay, revealing a minor artifactual component. These results further support the notion that the human transcriptome does naturally include some chimeric molecules.Materials S1Supplementary text.(DOC)Click here for additional data file.Figure S1Distance between consecutive pooled primers. This figure represents the distribution of distances between consecutive primers of the same pool for each chromosome and each relative position of primer pairs: head to head (→ ←), head to tail (→→) and tail to tail (←→), as a box plot. The gene density of chromosome 22 being higher than that of chromosome 21, the distance between consecutive pooled primers is smaller for the latter than for the former. Also, as expected, the distance between consecutive pooled primers is higher for head to head than for head to tail configuration, and higher for head to tail than for tail to tail configuration.(TIFF)Click here for additional data file.Figure S2Two measures to assess RACEfrag sets. This figure describes the two measures used to assess RACEfrag sets while optimizing the parameters used for RACEfrag calling: the exonic accuracy and the splice site score. The exonic accuracy assesses the RACEfrag set with respect to a reference set: the projected internal exons. More precisely for each projected internal exon, considered as a reference, the exonic accuracy assesses the accuracy with which the RACEfrags overlapping this reference mimics this reference. This accuracy is measured in terms of intersection over union of the projected internal exons and the RACEfrags, i.e., for each projected internal exon with overlapping RACEfrags, the number of nucleotides in common between the two sets is divided by the number of nucleotides in either of the two sets. The exonic accuracy of a RACEfrag set is then the median of the exonic accuracy of projected internal exons with overlapping RACEfrags. Unlike the exonic accuracy the splice site score of a RACEfrag set does not depend on any reference but is rather intrinsic to the RACEfrag set. More precisely, the spice site score is divided into two sub-measures: the acceptor score and the donor score. Both scores involve the scanning of two windows around the RACEfrag boundaries, W1 around the left boundary and W2 around the right boundary, where both acceptor and donor sites have previously been found by the geneid program. The RACEfrag acceptor score is then defined as the score of the best acceptor site on the 2 windows, and the RACEfrag donor score as the one of the best donor site on the 2 windows.(TIFF)Click here for additional data file.Figure S3RACEfrag calling. This figure represents the exonic accuracy of 10 RACEfrag sets coming from 10 randomly chosen experiments, as a function of the intensity threshold (I) and the maxgap (M), for 3 different minrun values (m): 3, 4 and 5. The blue arrows indicate the maximum exonic accuracy found over all the possible values of the three parameters, and the red arrows the minimum exonic accuracy. The maximum is reached for I = 99.1%ile, M = 59 bp, m = 5 probes.(TIFF)Click here for additional data file.Figure S4USPP filter. This simulation involves two steps: (1) RACE from a set of primers and a set of known transcripts; (2) hybridization of the obtained RACE products on tiling arrays. The RACEarray simulator generates a set of tiling array probes that are highlighted by the RACE products and that we call simulated positive probes (SPPs). These SPPs can be further divided into two categories: (1) bona fide SPPs, i.e. overlapping an exon of the target locus; (2) unspecific SPPs, also called USPPs, i.e. mapping outside of the target locus exons. In our model these USPPs correspond to false positives that originate from RACE mis-priming and/or from array cross-hybridization (see text for a more detailed explanation).(TIFF)Click here for additional data file.Figure S5RACEfrag assignment. This figure is divided into three parts: 1) on the top, the annotations of a given chromosome are represented, which are here the different alternative transcripts of three loci: A, B and C; 2) in the middle, the primers and RACEfrags of three different pools in several tissues are represented; 3) on the bottom, the formula of the assignment confidence score is provided again as well as its application on 5 different  pairs (note that here two RACEfrags with the same coordinates are given the same identifier). The first two parts of the figure are thus dedicated to the description of the assignment method, while the third part shows how the assignment score behaves on already assigned RACEfrags associated to their locus. Primers are named and colored after the locus they are originating from, and RACEfrags after the locus they have been assigned to. In pool 1, primer C1 is active and points in the direction of all RACEfrags, so all RACEfrags of pool 1 are assigned to primer C1. In pool 2, it is the same with primer C2, and in pool 3 the same with primer A1. Then the ACS formula is applied to 5 different  pairs, and the lower the score the more confidence we have in the assignment of the RACEfrag to the locus. Here the  pair we are the most confident in is  since RACEfrag 3 appears 4 times in total and each time it appears it is assigned to locus C. Also, the fact that it is assigned to two different primers of locus C, primers C1 and C2, strengthens the confidence we have in this pair. The pair  is similar to the pair  except that RACEfrag 4 appears in 2 experiments instead of 4. It thus also has a good score, although less than the one of . The pair  is like the pair  except that RACEfrag 2 also appears in pool 3, tissue 1 where it is assigned to locus A. This makes it more uncertain we should assign RACEfrags 2 to locus C, as compared to RACEfrag 3, and this is why the score of  is lower than the one of . The pair  is similar to the pair  except that RACEfrag 5 is only assigned to 1 primer of locus C (primer C1), compared to two primers of locus C for RACEfrag 4 (primers C1 and C2). This explains the lower score of  with respect to the one of . Finally the pair  is given a very bad score since RACEfrag 2 appears 5 times but is assigned only once to locus A.(TIFF)Click here for additional data file.Figure S6Chromosome 21 transcriptional networks. RACE connection networks in all 10 assayed cell types are represented. In each plot, the chromosome is depicted as a circle, and RACEfrag connections as inner links between genomic regions . The circular tracks are, going inwards: (1) - chromosome scale , (2) - plus-strand annotated genes (green), (3) - plus-strand annotated pseudogenes, (4) - minus-strand annotated genes, (5) - minus-strand annotated pseudogenes.(JPG)Click here for additional data file.Figure S7Chromosome 22 transcriptional networks. See legend of (JPG)Click here for additional data file.Figure S8Reciprocal gene to gene connections in chromosome 21 (A) and 22 (B). All 2,324 pure and composite gene/gene reciprocal connections observed in the 10 cell types studied are represented as blue (connection involving two genes on the same chromosome strand) and orange (connection involving two genes on different strands) inner ribbons. See (JPG)Click here for additional data file.Figure S9Reciprocal gene to gene connections observed in each cell type on chromosome 21. Networks of reciprocal gene to gene connections observed in each of the 10 assayed cell types are represented as blue (connection involving two genes on the same chromosome strand) and orange (connection involving two genes on different strands) inner ribbons. See (JPG)Click here for additional data file.Figure S10Reciprocal gene to gene connections observed in each cell type on chromosome 22. See legend of (JPG)Click here for additional data file.Figure S11Pairwise correlations between cell types based on pure reciprocal gene to gene connections. This figure represents the pairwise correlations between the cell types used in the RACEarray experiments as a heatmap: the closer to the white, the more correlated. More precisely for each pair of cell types, the Pearson's product moment correlation between them was computed based on the number of reciprocal gene to gene connections commonly observed, in the universe of all possible reciprocal gene to gene connections. This number is the one indicated in the corresponding cell of the heatmap. Note that genes g1 and g2 form a possible reciprocal gene to gene connection if and only if there is a RACE primer in g1 pointing in the direction of g2 and a RACE primer in g2 pointing in the direction of g1.(PNG)Click here for additional data file.Figure S12Number of observed (left) and of expected (right) gene to gene connections on chromosomes 21 (top) and 22 (bottom). The shape of the observed distributions is similar for the two chromosomes, as well as the shape of the expected ones, however the distributions are decreasing much more rapidly for the expected connections compared to the observed connections.(PNG)Click here for additional data file.Figure S13Difference between number of observed and number of expected gene to gene connections on chromosome 21 (A) and on chromosome 22 (B). These two histograms (A and B) represent the distributions of the difference between the number of observed and the number of expected gene to gene connections for reciprocally connected genes on chromosomes 21 and 22 respectively. These distributions are shifted towards the positive values, and have a mean of 6 and 5 respectively. In our analysis the difference between the number of observed and the number of expected gene to gene connections of reciprocally connected genes is used as a score for those genes and is used to delineate a set of genes much more connected than we would expect given their length and number of primers: the hubs.(PNG)Click here for additional data file.Figure S14Different categories of genes used in the RACEarray experiments. Proportional Venn diagram representation of inclusion relationships between some of the most used sets of genes used in this study. The area highlighted in light blue corresponds to non-hub genes, which are all reciprocally connected.(PNG)Click here for additional data file.Figure S15Expected number of gene to gene connections found by RACEarray and RNA PET ditags in K562.(JPG)Click here for additional data file.Figure S16Expected number of gene to gene connections found by RACEarray and Illumina Human Body Map PE50 RNAseq (A) in Testes+Prostate and in Brain (B).(JPG)Click here for additional data file.Figure S17Interspecies chimeric RNAs used as a metric of technical artifacts. The number of reads/10 M total reads for intra-genomic and inter-genomic chimeric junction sites is plotted human and fly alone and various ratios of RNAs from human and fly (mixtures). A total of at least 25 nucleotides on each side of a chimeric junction site was chosen as a minimum to allow for unique mapping in each genome.(JPG)Click here for additional data file.Figure S185C data validates RACEarray gene to gene connections. This figure represents the distribution of the proportion of gene to gene connections validated by 5C in 1,000 sets of gene to gene connections detectable by RACEarray and by 5C with the same distributions of distance between connected genes and of length of connected genes as in the 638 connections detectable by both techniques that are actually observed. The mean of this distribution is 70.8 (standard deviation = 1.9), which is significantly lower than the observed proportion .(JPG)Click here for additional data file.Figure S19Domain organization for chimera OTTHUMP00000221101. Chimera OTTHUMP00000221101 results from the fusion of two receptors involved in immune response, Interferon-alpha/beta receptor 2  and Interleukin-10 receptor subunit beta . The resulting protein will have an extra-cellular domain that is double the size of the usual extra-cellular receptor domain and that is composed of a repeat of paired tissue factor (green) and alpha/beta interferon receptor (red) domains. The chimeric protein also conserves a signal peptide signal and a single trams-membrane helix. A similar domain configuration is recorded in Uniprot for the chicken interferon receptor (Q5XPI1_CHICK).(PDF)Click here for additional data file.Figure S20Model of possible structure of fused fragments for chimera OTTHUMP00000221101. Models for the N- and C-terminal sections have been obtained respectively from structures 2hym and 3g9v by comparative modeling . Linker region (shown as a gap in the structure) is located in flexible regions for both templates. Domain folds could then be maintained independently.(PNG)Click here for additional data file.Table S1Number of gene to gene and number of reciprocal gene to gene connections by distance. This table is similar to (JPG)Click here for additional data file.Table S2Validation results of chimeric transcripts by RNase protection assays. This table lists the RACE name, the pool of poly-A+RNA used, description of the probes, a summary of the RNase Protection Assay screening with a detailed interpretation of the results based on the autoradiography gel.(XLSX)Click here for additional data file.Table S3Names and characteristics of the hubs. For each of the 74 hubs the table provides: - the number of observed connections, - the number of expected connections, - the difference between the two, which could be seen as their connectivity score.(JPG)Click here for additional data file.Table S4Hubs have higher phylogenetic depth than non hubs. We consider four different gene sets . Note that both the proportions of genes with an ortholog in each of the 6 species for RACE interrogated (“raced”) genes, hubs and non hubs, and the significance of the Fisher tests mentioned here are provided on (JPG)Click here for additional data file.Table S5Over-representation of cliques in chromosomes 21 and 22. For each chromosome, we report the number of cliques observed, as well as the mean and the standard deviation of the number of cliques expected.(JPG)Click here for additional data file.Table S6Constitutive cliques. For each constitutive clique (the maximum size is 3), we provide: • the names of the genes involved in the clique, • the chromosome where the clique is observed, • the list of cell types in which the clique is observed.(JPG)Click here for additional data file.Table S7Overlap of maximal cliques. For each clique size, we report: - the number of cliques observed, - the number of corresponding edges if cliques were not overlapping, - the number of observed edges.(JPG)Click here for additional data file.


A new study now suggests maternal exposure to extreme heat may have an immediate effect on pregnancy duration .Pregnancy tends to make women more vulnerable to heat stress. Added fat deposits and the attendant decrease in the ratio of body surface area to body mass mean a woman’s body is less able to cool off by losing heat to the environment. Heat stress has been linked in earlier studies to induction of uterine contractions, increased secretion of the childbirth-related hormones oxytocin and prostaglandin F90), 95th (HI95), and 99th (HI99) percentiles for heat index for the longer period of 1983–2006.The Spanish-based research team analyzed birth data for 7,585 women who delivered at the Hospital Clinic of Barcelona between January 2001 and June 2005. They used national data on daily heat and temperature to calculate which days during that period exceeded the 90th (HI90 episode on the day before delivery (lag 1) was associated with a 1-day reduction in average pregnancy duration, a lag 1 HI95 episode was associated with a 2-day reduction, and a lag 1 HI99 event was connected to a 5-day reduction. There was little evidence of an association beyond the delivery date and the day before, suggesting that any effect heat stress may have on pregnancy duration is immediate.Pregnancies ranged from 22.2 to 43.5 weeks, with an average of 40 weeks. The results showed that all three HI percentiles were associated with a reduction in pregnancy duration. An HI99 episode on the day of delivery and an 8-day reduction in average pregnancy duration for European women compared with a 1-day reduction for non-Europeans.When the investigators assessed Europeans versus non-Europeans, they found a relationship between an HIThe study was limited by the inability to control for air-conditioner use, study the associations in ethnic groups besides Europeans, or examine how the length of extreme heat episodes affected pregnancy duration. With future climate projections including increases in the frequency and intensity of extreme heat conditions—and given that a reduction of even a week in the length of pregnancy has been linked to adverse health outcomes in newborns—the authors contend that future studies should consider these factors to help inform appropriate public health interventions.


Fingerprint recognition with identical twins is a challenging task due to the closest genetics-based relationship existing in the identical twins. Several pioneers have analyzed the similarity between twins' fingerprints. In this work we continue to investigate the topic of the similarity of identical twin fingerprints. Our study was tested based on a large identical twin fingerprint database that contains 83 twin pairs, 4 fingers per individual and six impressions per finger: 3984 (83*2*4*6) images. Compared to the previous work, our contributions are summarized as follows: (1) Two state-of-the-art fingerprint identification methods: P071 and VeriFinger 6.1 were used, rather than one fingerprint identification method in previous studies. (2) Six impressions per finger were captured, rather than just one impression, which makes the genuine distribution of matching scores more realistic. (3) A larger sample (83 pairs) was collected. (4) A novel statistical analysis, which aims at showing the probability distribution of the fingerprint types for the corresponding fingers of identical twins which have same fingerprint type, has been conducted. (5) A novel analysis, which aims at showing which finger from identical twins has higher probability of having same fingerprint type, has been conducted. Our results showed that: (a) A state-of-the-art automatic fingerprint verification system can distinguish identical twins without drastic degradation in performance. (b) The chance that the fingerprints have the same type from identical twins is 0.7440, comparing to 0.3215 from non-identical twins. (c) For the corresponding fingers of identical twins which have same fingerprint type, the probability distribution of five major fingerprint types is similar to the probability distribution for all the fingers' fingerprint type. (d) For each of four fingers of identical twins, the probability of having same fingerprint type is similar. Biometrics refers to the automatic identification of a person based on his or her physiological or behavioral characteristics. These methods have advantages over traditional token based identification approaches using a physical key or access card, and over knowledge based identification approaches that use a password for various reasons. First, the person to be identified is required to be physically present at the point of identification to provide his or her biometric traits. Second, identification based on biometric characteristics avoids the need to carry a card or remember a password. Finally, the biometric characteristics of identified person cannot be lost or forged. During the past few decades, a number of verification systems based on different biometric characteristics have been proposed There are two basic types of twins: dizygotic, commonly referred to as fraternal twins and monozygotic, referred to as identical twins This paper presents the continued investigations of the ability of fingerprint verification technology to distinguish between identical twins. The investigations are tested on a large identical twin fingerprint database which contains 83 twin pairs, 4 fingers per individual and six impressions per finger: 3984 (83*2*4*6) images. Comparing to the pioneers' work Compared to all the methods Compared to Jain's Compared to Sun et al.'s method A novel statistical analysis is conducted for five major fingerprint types, which aims at showing the probability distribution of the fingerprint types for the corresponding fingers of identical twins which have same fingerprint type. This is novel in our paper.A probability analysis is conducted for four fingers from identical-twins, which aims at showing which finger has higher probability of having same fingerprint type. This is also novel in our paper.The rest of this paper is organized as follows. Sec. 2 describes the twin fingerprint database and the state-of-the-art automatic fingerprint verification matcher for this study. Sec. 3 presents our experimental results and analysis. Sec. 4 provides a summary and conclusion.The database was collected on October 2, 2007 at the Beijing Chaoyang Park during the Fourth Beijing Twins Culture Festival. The fingerprint images were captured by a sweep sensor sw6888 0 with a resolution of 500DPI. The database includes 3984 (3984 = 83×2x×4×6) fingerprint images which from 83 pairs of identical twins, and four different fingers  were scanned for each person. The number of impressions for each finger was six. The finger was scanned consecutively six times to get the six impressions. All of the images were captured on the same day (single data captured session). In this paper, two state-of-the-art methods are used to identify the similarity of twin fingerprints: P071 n) and the mean distance difference of the matched minutiae pairs (d) in the process of similarity computing. Fuzzy features were used to represent n and d. Each character is associated with a fuzzy feature that assigns a value (between 0 and 1) to each feature vector in the feature space. The value, named degree of membership, illustrates the degree of similarity of the template and input fingerprints.P071 algorithm is first used for the identification of twin fingerprints which has been evaluated in the Fingerprint Verification Competition 2004 (FVC2004) and the performance was ranked No.3 among all of the participated algorithms. The detailed performance of the proposed algorithm on FVC2004 can be seen from the website n is represented by fuzzy feature Feature d is represented by fuzzy feature Feature In order to achieve the better classification performance, Similarity  = Cappelli et al. VeriFinger 6.1 SDK Enroll fingerprint. Fingerprint can be enrolled from the image or by using fingerprint the scanner.Enroll fingerprint with generalization. Using this option, several fingerprints can be enrolled and features generalized.Verification. Using this option, one fingerprint can be verified against the other (1:1 matching).Identification. Using this option, the fingerprint is identified against an internal database (1: N matching).VeriFinger fingerprint recognition algorithm follows the commonly accepted fingerprint identification scheme, which uses a set of specific fingerprint points (minutiae). However, it contains many proprietary algorithmic solutions, which enhance the system performance and reliability. Some of them are listed below:Adaptive image filtration algorithm allows to eliminate noises, ridge ruptures and stuck ridges, and extract minutiae reliably even from poor quality fingerprints;VeriFinger includes a fast template matching algorithm that is tolerant to fingerprint translation, rotation and deformation.VeriFinger does not require the presence of the fingerprint core or delta points in the image, and can recognize a fingerprint from any part of it.An example of fingerprint enhancement and minutiae extraction with user interface in VeriFinger 6.1 is shown in To study the similarity of identical twin fingerprints, three distributions were generated: genuine distribution, identical-twin imposter distribution and non-twin imposter distribution. The genuine distribution was obtained by matching each image of one finger against the remaining images of the same finger. The identical-twin imposter distribution was obtained by matching each image of a person with his/her identical twin. The non-twin imposter distribution was obtained by matching a person with everyone else except for his/her identical twin. To sum up, a total of 9 960 (83×2×4×6×5/2) genuine matching, 332 (83*4) identical-twin imposter matching and 435 584 (83*2*82*2*16) non-twin imposter matching runs were conducted. All of the experiments were conducted on a PC Intel Core2 E6500 @ 2.33 GHZ.Identical twins have the same chromosomes and similar physical characteristics and, therefore, they have a high class/type similarity in their fingerprints. It is well known that the class/type similarity is, to a certain extent, related to the fingerprint matching perfomance. It is our hypothesis that identical twins have higher class correlation, and the higher class correlation causes a higher similarity, i.e. it is more difficult to tell the difference between identical twins than non-identical twins. To prove our hypothesis, we manually classified 83 pairs of identical twin fingerprints in our database into five types . And the analysis for each type was then performed.The VeriFinger 6.1 SDK matching algorithm provides a value of the features matching score as a result. The higher the score, the higher the probability that the features obtained are from the same person. The different matching threshold is linked to the different false acceptance rate (FAR). The relationships can be found in All of the fingers were manually classified into five classes . The proportion of appearance of each of the five major fingerprint types in our database is shown in A statistical analysis is conducted for five major fingerprint types/classes, which aims at showing the probability distribution of the fingerprint types for the corresponding fingers of identical twins which have same fingerprint type. To the best of our knowledge, this is the first report about it. The results are shown in Another probability analysis is conducted for four fingers from identical-twins, which aims at showing which finger has higher probability of having same fingerprint type. This is also novel in our paper. The results are shown in In this paper, we have investigated the ability of the fingerprint verification matcher to discriminate between identical twins. The experimental results demonstrated that the identical twins can be distinguished by a state-of-the-art method P071 and the commercial fingerprint matcher VeriFinger 6.1.Our research is a continued investigation in In summary, our contributions are as follows:Compared to all the methods Compared to Jain's Compared to Sun et al. 's method A novel statistical analysis which aims at showing the probability distribution of the fingerprint types for the corresponding fingers of identical twins which have same fingerprint type has been conducted. The experimental results showed that the probability distribution of five major fingerprint types is similar to the distribution for all the fingers' fingerprint type.A novel analysis which aims at showing which finger from identical twins has higher probability of having same fingerprint type has been conducted. The results show that the probability of having same fingerprint type for each finger of identical twins is similar.


Purpose: The development of novel biomarkers is an unmet need in chronic obstructive pulmonary disease (COPD). Arterial blood comes directly from the lung and venous blood drains capillary beds of the organ or tissue supplied. We hypothesized that there would be a difference in levels of the biomarkers metalloproteinase 9 (MMP-9), vascular endothelial growth factor A (VEGF-A) and interleukin 6 (IL-6) in arterial compared with venous blood. Methods: Radial artery and brachial vein blood samples were taken simultaneously in each of 12 patients with COPD and seven controls with normal lung function. Circulating immunoreactive MMP-9, VEGF-A and IL-6 levels in serum were measured using quantitative enzyme-linked immunosorbent assays. Results were compared using a Student’s paired t test. The study was powered to determine whether significant differences in cytokine levels were present between paired arterial and venous blood samples.  Results: In the 12 patients with COPD, four were female, and age ranged 53-85 years, mean age 69 years. Three patients in the control group were female, with age range 46-84 years, mean age 64.7 years. In the COPD group, three patients had mild, five moderate and four severe COPD. No significant difference was found between arterial and venous levels of MMP-9, VEGF-A or IL-6. Conclusions: In this pilot study, levels of the measured biomarkers in arterial compared with venous blood in both COPD patients and healthy controls did not differ. This suggests that as we continue to chase the elusive biomarker in COPD as a potential tool to measure disease activity, we should focus on venous blood for this purpose. At present, the Global Initiative for Obstructive Lung Diseases (GOLD) divides patients into categories of mild, moderate, severe and very severe based on the forced expiratory volume in 1 second (FEV1). This classification has been shown to predict outcome and has been pivotal in guiding treatment of the disease3.Chronic obstructive pulmonary disease (COPD) is a major public health issue, predicted to become the 31 over time, usually measured over years. Indeed, the duration of the large trials that have evaluated the effect of pharmacological or surgical benefits in patients with COPD have ranged from 2 to 4 years7. It follows that defining disease activity or response to therapy with validated biomarkers that reflect disease progression would make potential future interventions easier to evaluate. Several studies have reviewed the roles of different potential biomarkers in COPD8. Our group has recently shown that serum levels of interleukin 6 (IL-6) and tumor necrosis factor alpha  were higher and matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) lower in patients with severe and very severe COPD compared with smoker and non-smoker controls without airflow obstruction9. Furthermore, the levels correlated with several phenotypic expressions of the disease including exercise capacity, quality of life, exacerbation and mortality9. Data from the ECLIPSE investigators, who studied a larger cohort of COPD patients, showed that several biomarkers correlated with baseline FEV1 but only one correlated with rate of decline in FEV1, the Clara-Cell Protein 16 (CC-16)10. While there is no question that a biomarker would be of great importance in COPD, the sample site having the greatest yield has not been clearly defined11.The progression of COPD has classically been determined by the change of the FEVBlood from the systemic circulation returns to the lung for gas exchange and, theoretically, the presence of biomarkers could be modified during the transient time blood spends in the pulmonary vessels. Arterial blood should provide a more direct window to events occurring in the lungs than venous blood, which is perhaps more reflective of events happening in the capillary bed of the organ/tissues that it supplies. This is most evident in the significant differences that exist between arterial and venous blood gas measurements.We conducted this pilot study to test the hypothesis that in patients with COPD (a primary disease of the lungs) there is a difference in serum concentrations of the biomarkers MMP-9, VEGF-A and IL-6 between simultaneously collected arterial and venous blood. Further, we compared the results with similar samples from patients without airflow obstruction that served as controls.1/FVC  ratio, < 0.7 after 400 μg of inhaled salbutamol. Patients had stable COPD and were not included if they had a history of an exacerbation in the last 3 months. The controls were patients attending the pulmonary clinic at the same institution with no history of COPD and normal lung function.This was a prospective pilot study. Samples were collected from patients attending the pulmonary clinic at St. Elizabeth’s Medical Center in Boston, Massachusetts. The study was approved by the Human Institutional Review Board of the institution and all patients signed the informed consent. The patients in the COPD group had smoking history ≥ 20 pack-years  and had post-bronchodilator FEV1, dyspnoea as quantified by the modified Medical Research Council dyspnoea scale and exercise tolerance, as measured with a 6 minute walk test12.Age, gender, smoking history and body mass index (BMI) were recorded for every participant. All subjects performed a spirometry according to the American Thoracic Society (ATS) recommendations and standard references and severity of COPD was categorized using GOLD staging. The BODE score is a composite score of BMI, degree of obstruction as recorded by FEVSimultaneous radial artery and antecubital venous samples were obtained in each participant. The blood samples were centrifuged immediately at 2500 rpm for 10 minutes and serum stored at -80ºC. Circulating immunoreactive MMP-9, VEGF-A and IL-6 levels in the serum were measured using commercially available quantitative enzyme-linked immunosorbent assays (ELISA) .13. If the true difference in the mean response of matched pairs is 0.3, we would need to study 10 pairs of subjects to reject the null hypothesis with probability (power) of 0.8. If the true difference in the mean response of matched pairs is 0.4, we would need to study 12 pairs of subjects to be able to reject the null hypothesis with a probability (power) of 0.8. The Type I error probability associated with this test is 0.05.We worked out a power/sample size calculation based on the premise that this was a study of a continuous response variable from matched pairs of study subjects. Prior data indicated that the difference in the response of matched pairs is normally distributed with standard deviation of 0.3 in the case of MMP-9 and VEGF-A, and 0.4 for IL-6Groups were compared using the Student’s t test for normally distributed variables and Mann-Whitney U test for variables not normally distributed. p ≤ 0.05 was considered statistically significant. Spearman’s rank order correlation coefficient was also used for non-parametric data. Statistical analysis was performed using a commercial statistical package .14. The seven patients  included in the control group had normal lung function and their mean age range was similar to the COPD group (46–84 years). The majority of subjects in both groups were ex-smokers, with only 3 patients in the control group being never-smokers.The characteristics of all participants are shown inMMP-9 : Serum MMP-9 levels were not normally distributed in all of the subjects (n =19). The median venous MMP-9 level was 40,706 pg/ml, . The median arterial level was 37,653 pg/ml . There was no significant difference between the venous and arterial levels, p = 0.812. There was no difference between COPD and control levels of venous or arterial MMP-9.VEGF-A : In the 19 subjects, the VEGF-A levels in venous blood had a median value of 67.24 pg/ml, with interquartile range 45.24 to 268.90 pg/ml whereas in arterial blood the median value was 89.88 pg/ml, with interquartile range 58.90 to 239.38 pg/ml. There was no statistically significant difference between arterial and venous samples (p = 0.249). No significant difference was observed between patients with COPD and controls.IL-6 : IL-6 levels were undetectable in many of the subjects. The median venous level of IL-6 was 0, with an interquartile range of 0 to 4.52 pg/ml and the median arterial level was also 0 pg/ml with an interquartile range of 0 to 3.61 pg/ml. There was no statistically significant difference (p = 0.986) between venous and arterial levels of this cytokine. As levels of IL-6 were undetectable in many of the patients, it was not possible to determine if a significant difference existed between those with COPD and the control group.This pilot study showed no difference in serum levels of MMP-9, VEGF-A or IL-6 between arterial and venous blood samples in a group of patients with COPD and controls without airflow obstruction. Our results indicate no advantage of obtaining arterial over venous samples for the determination of these biomarker levels in patients with COPD.1 value and its progression over time, usually measured in years. This approach has its limitations and fails to account for the so-called activity of the disease15. As more treatments for COPD are emerging, the importance of biomarkers that reflect disease activity becomes paramount. A biomarker, by definition, is “any molecule or material (e.g. cells and tissues) that reflects the disease process”16. Because of its ease of accessibility, work has focused on peripheral blood biomarkers. Many have been investigated with initial work showing C reactive protein (CRP) levels to be associated with degree of airway obstruction, although subsequent work in patients with moderate to severe COPD found no relationship between CRP levels and reduced survival19. Other promising biomarker candidates previously studied include circulating levels of Clara cell secretory protein-16 (CC-16)20, surfactant protein (SP)-D21 and serum amyloid A (SAA)22. Serum levels of CC-16, a marker of Clara cell toxicity, are reduced in patients with COPD10, while SP-D is increased in smokers with and without COPD21, and SAA may be a potential biomarker of COPD exacerbation22.At present, the most important determinant of COPD severity and progression is the FEV24. In a study of the effect of moderate hypothermia on systemic and internal jugular plasma IL-6 levels after traumatic brain injury in humans, IL-6 was found to be significantly higher in internal jugular venous blood than in arterial plasma25. In the setting of recent neuro-trauma this is understandable. Having stressed the importance of suitable potential biomarkers in COPD, we hypothesized that determining the optimal source of the sample could be of potential value. Our most important finding was that for the biomarkers studied, levels did not differ between arterial and venous samples. This was not due to the nature of the biomarkers selected because the markers we investigated in this study were chosen based on prior work demonstrating the level of IL-6 was higher and MMP-9 and VEGF levels lower in patients with more advanced COPD compared with controls27. Although the host response to insult is, to a large extent, compartmentalized to the affected lung, cytokine spillover into the systemic circulation has been shown to occur28 and be measurable in the systemic circulation28. We did not find differences in serum biomarker levels between patients with COPD and those without airflow obstruction; however, the study was not powered to explore this hypothesis.There are no prior published results on cytokine measurement in arterial blood in COPD patients. Studies where arterial vessels are accessed for cardiopulmonary bypass or for extracorporeal liver support have measured arterial blood levels of various cytokines but only within narrow inclusion criteria, and arterial and venous level comparisons were not publishedOur pilot study has some limitations. First, the number of patients recruited could be considered small, but it was powered to address whether there was a difference between arterial and venous levels of MMP-9, VEGF-A and IL-6. The use of matched samples allowed for accurate interpretation of the results with these subject numbers. Second, the blood samples were drawn from the radial artery and from a peripheral antecubital vein. Possibly, to obtain more accurate sampling of blood immediately leaving the lung, pulmonary arterial sampling would have been optimal. However, sampling of central venous blood is invasive and would not offer any practical advantage. Third, it is possible that the 3 selected analytes are not "exclusively" produced in the lung (as it is the case for SPD and CC16) and represent the systemic compartment and not just the lung milieu. This possibility requires the simultaneous measurement of these biomarkers in future studies.In summary, this pilot study shows that there was no difference in levels of MMP-9, VEGF-A or IL-6 when measured in blood samples from the radial artery compared with peripheral venous samples. This suggests that as we continue to chase the optimal biomarker in COPD as a potential tool to measure disease activity, focusing on venous blood for this purpose remains a valid option. In this pilot study, the authors compared 3 potential biomarkers between arterial and venous blood samples among normal and COPD patients. Although the methods as well as statistical analyses are sound, there exist some limitations, as discussed by the authors; such as, small number of samples, unclarified differences between pulmonary arterial and venous blood, and limited number of biomarkers. I believe this study becomes a milestone for the future larger-scaled study for these important topics.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for your comments. In this pilot study the authors evaluate whether there are differences in arterial versus venous blood in 3 potential biomarkers of COPD. Since the bulk of prior work on COPD biomarkers has been focused on venous blood and there are potential limitations to not using arterial blood. This is an appropriate study question.The methods are appropriate, statistical analyses are sound. There is the limitation of sampling pulmonary arterial and venous blood to understand the “direct” lung effects on blood markers, but this is likely neither feasible nor practical in biomarker discovery as its clinical use would be limited.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for your comments. Although the article is generally well written and the methods and results are likely to be correct, there is one major flaw that really should have been addressed before embarking on this negative project; Trans lung changes in mediators means you absolutely have to obtain pulmonary arterial and peripheral arterial blood. Even then differences could reflect a “cleaning” of the blood which is a non respiratory function of the lung and is most notable in the clotting differences. Peripheral arterial and venous blood will if anything reflect changes in the local limb capillary circulation and clearly misses changes to more systemic venous blood that is fully mixed in the pulmonary artery. This concept was recognised over 30 years ago and although the Authors mention it in discussion as a potential limitation I think this is a definite limitation. The study also fails to include the likelihood of some shunt in COPD lungs. Finally it is not clear whether the blood was allowed to clot but as serum is mentioned the assumptionis that  it was and this would have the effect of activating local cells during the clotting process so ideally plasma should be studied.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Thank you for taking the time to review our work. We acknowledge the limitations of this work and agree that there would be advantages to taking pulmonary arterial samples to give more physiological data. We share your concerns that the peripheral arterial sample may better reflect the occurrences in the distal capillary bed and we have raised these issues in the discussion. We were endeavouring to investigate the site of biomarker testing that could be used in real-life clinical scenarios and therefore chose radial arterial sampling. Serum was the tested sample in this study. Future work could give answers for these remaining physiological questions.


Evidence indicates that supervised home exercises, combined or not with manual therapy, can be beneficial for patients with non-specific chronic neck pain (NCNP). The objective of the study is to investigate the efficacy of preventive spinal manipulative therapy (SMT) compared to a no treatment group in NCNP patients. Another objective is to assess the efficacy of SMT with and without a home exercise program.Ninety-eight patients underwent a short symptomatic phase of treatment before being randomly allocated to either an attention-group (n = 29), a SMT group (n = 36) or a SMT + exercise group (n = 33). The preventive phase of treatment, which lasted for 10 months, consisted of meeting with a chiropractor every two months to evaluate and discuss symptoms (attention-control group), 1 monthly SMT session (SMT group) or 1 monthly SMT session combined with a home exercise program (SMT + exercise group). The primary and secondary outcome measures were represented by scores on a 10-cm visual analog scale (VAS), active cervical ranges of motion (cROM), the neck disability index (NDI) and the Bournemouth questionnaire (BQ). Exploratory outcome measures were scored on the Fear-avoidance Behaviour Questionnaire (FABQ) and the SF-12 Questionnaire.Our results show that, in the preventive phase of the trial, all 3 groups showed primary and secondary outcomes scores similar to those obtain following the non-randomised, symptomatic phase. No group difference was observed for the primary, secondary and exploratory variables. Significant improvements in FABQ scores were noted in all groups during the preventive phase of the trial. However, no significant change in health related quality of life (HRQL) was associated with the preventive phase.This study hypothesised that participants in the combined intervention group would have less pain and disability and better function than participants from the 2 other groups during the preventive phase of the trial. This hypothesis was not supported by the study results. Lack of a treatment specific effect is discussed in relation to the placebo and patient provider interactions in manual therapies. Further research is needed to delineate the specific and non-specific effects of treatment modalities to prevent unnecessary disability and to minimise morbidity related to NCNP. Additional investigation is also required to identify the best strategies for secondary and tertiary prevention of NCNP.NCT00566930ClinicalTrials.gov:  Non-specific neck pain is frequent, with an annual prevalence estimated to be 30% to 50%. Often pChiropractic intervention is usually directed toward neuromusculoskeletal problems, with aroThe objective of the study is to investigate the efficacy of preventive SMT compared to a no treatment group in NCNP patients. Another objective is to assess the efficacy of SMT with and without a home exercise program. It was hypothesised that during a 10-month period of preventive care, participants with NCNP receiving SMT combined with a home exercise program would have less pain and disability with improved function compared to patients given only SMT or no treatment.Study participants were recruited if they had NCNP, defined as pain of mechanical origin located in the anatomical region of the neck, with or without radiation to the head, trunk or limbs, as described by Guzman et al. The incParticipants were recruited through radio and printed advertisements aimed at the French-speaking community of Trois-Rivières, Québec . The study was carried out entirely at the chiropractic clinic and human research laboratory of the Department of Chiropractic at the Université du Québec à Trois-Rivières. All participants gave their informed written consent according to the research protocol approved by the local ethics committee.Three research assistants undertook standardised phone screening regarding major inclusion and exclusion criteria for study participation. Three other research assistants, who were experienced chiropractors and were blinded to future treatment allocation, performed the preliminary standardised history-taking and physical examination of eligible participants. Radiographs of the cervical spine were ordered when indicated. All preThe trial was divided into 2 phases. The first was the non-randomised, symptomatic phase during which all eligible participants received a short course of SMT. After completing the symptomatic phase, participants were randomly assigned to one of three parallel groups. The second phase, the preventive phase, lasted 10 months.Participants had an equal probability of assignment to any 1 of the 3 groups. Each study participant was assigned a number by the principal investigator (PI) who put these numbers on individual cards in an opaque envelope. An assistant, not involved in the research project and blinded to the process, drew numbers from the original, opaque envelope and arranged them in 3 different opaque envelopes which were numbered 1 to 3. Randomisation was concealed until the beginning of the preventive treatment phase. No block or stratification strategy was used in the randomisation process.Randomisation was carried out after acceptance of the appropriate number of participants into the study and before beginning the non-randomised, symptomatic phase. The PI opened the sealed, opaque envelopes after the symptomatic phase was over, and baseline (second set) outcome measures were collected. Each participant was assigned to his/her appropriate group before beginning the preventive phase of the trial. They remained on the same allocation throughout the entire period if they continued in the trial.A credible placebo for SMT of patients previously administered this type of therapy does not exist, especially if its specific and non-specific effects are considered-24. TherDuring the symptomatic phase of the trial, all eligible participants received a short course of SMT designed to relieve symptoms. A team of 3 chiropractors using high velocity low amplitude spinal manipulation and withThe preventive phase of the trial lasted 10 months during which the participants attended the clinic regularly. A chiropractor with at least 3 years of experience was in charge of the interventions for each group. The interventions were standardised beforehand. Medication(s) and co-intervention such as other manual therapies, physiotherapy, massage therapy as well as any other common neck pain treatment were discouraged. Before adopting any such pain control strategy, the participants were instructed to first call their treating chiropractor to discuss the problem. Patients in all 3 groups received identical verbal and written instructions regarding co-interventions and were asked to record any co-interventions in a personal diary. Patients were given an ice pack that could be used whenever pain intensity reached a level of 5/10. Again all 3 groups received identical instructions concerning ice application.At each visit, the chiropractor asked the participants for a standardised, short health history regarding symptoms during the last period and had them complete a visual analog scale (VAS) for current symptoms. The chiropractor also performed standardised passive palpation of the cervical and upper thoracic spine. No advice or educative strategy was allowed during this phase, nor was any type of soft tissue therapy. Diaries were distributed at these visits. Other interventions during each visit were specific for each of the 3 groups:This group received a maximum of 4 spinal manipulations to the cervical and upper thoracic areas. They were given 1 treatment per month that lasted 10 to 15 minutes.This group received a maximum of 4 spinal manipulations to the cervical and upper thoracic areas (down to T4). They were dispensed with 1 treatment per month, and each of them lasted 10 to 15 minutes.Participants were advised to perform a home exercise program at least 3 times a week. The program was designed by an experienced kinesiologist and required low technology equipment . It included general range of motion (ROM) exercises that served for warm-up and cool down purposes, followed by 4 stretching/mobilization and 4 strengthening exercises (concentric and isometric contractions) of the cervical and upper thoracic spine, principally flexion/extension, lateral flexion and rotation of the cervical spine. Three series of each exercise were performed during a training session, with a 30- to 60-second rest period between each series. A complete training session lasted between 20 to 30 minutes.All participants were instructed to follow the same exercise routine. However, exercise volume was tailored to each participant's strength and flexibility as well as his/her ability to complete the routine with minimal neck pain. Each patient received a written copy of the program. At trial onset, a kinesiologist met each participant individually on 2 different occasions to instruct and correct them on execution of the exercises. Between these 2 meetings, follow-up was conducted through phone interviews to answer questions and confirm that the exercises were not triggering cervical pain. The kinesiologist met the participants individually every 2 months (during the regular treatment visit) to ensure full understanding, appropriate execution and compliance. Minor changes were made only when exercises produced symptoms or when a patient was unable to perform a specific exercise. During the 10 months of the preventive phase, exercise type and volume could be modified under the following conditions: exercise-related pain described by the patient or major difficulty in carrying out an exercise.This group received no treatment (no SMT or exercise program) but each participant attended the clinic once every 2 months. To give all trial participants the same attention, each visit lasted twice as long as the other two groups treatment time, specifically 20 to 30 minutes. The same procedures as for the other 2 groups were performed at these meetings  but with much slower flow. As with the 2 other groups, no advice or educative strategy was allowed.©). Many studies have demonstrated acceptable validity and reliability of this instrument[The primary outcome measure throughout the trial was pain level. Pain was scored with a 10-cm VAS. The psychometric properties of the VAS have been studied extensively. The numnstrument. Active nstrument,28 have nstrument.Exploratory outcome measures included health-related quality of life (HRQOL), fear and avoidance phenomena, exercise adherence and co-intervention. HRQOL and fear avoidance phenomena were scored with the SF-12 Questionnaire and the Exercise adherence and co-intervention were measured through a diary that patients completed on a weekly basis. Use of co-intervention, registered by the patients, was calculated by adding up the number of co-intervention episodes from one visit to another. Also recorded were the frequency of ice application due to pain intensity greater than 5/10 and the use of analgesics or consultation with other professionals for pain management. Patients performing home exercises also recorded the dates of their 3 weekly exercise sessions.Preliminary data were collected after participants signed an informed consent form, before the beginning of the non-randomised, symptomatic phase of the trial. Baseline data were gathered once the symptomatic phase was over, on specific appointment. During the 10 months of the preventive phase, all outcomes were noted every 2 months. For the groups receiving treatments, these measurements were recorded before the treatments.For preliminary and baseline data, the assessors were blinded to group allocation. The assessors were not blinded to the data during the preventive phase but were trained before the trial to ensure standardisation of the outcome measures.Thirty-five participants per group were required to have a 90% chance of detecting a significant difference between groups in the mean of VAS scores (i.e. a 2-point difference on the VAS at the 2-sided 5% level) with an assumed standard deviation of 2.5 and a loss to follow-up of 20%. Since no RCT on the efficacy of preventive spinal manipulation for chronic cervical pain has been performed in the past, the 20% loss to follow-up level was estimated from the results of a similar RCT on the efficacy of preventive spinal manipulation for chronic lumbar pain. The effAll data were analysed according to a pre-established experimental design using Version 6.1 of Statistica software. One-way ANOVA was performed for baseline values of continuous variables. T-tests for dependent samples were conducted on primary and secondary outcomes to analyse data from the symptomatic phase of the trial (pooled data).The main analysis was undertaken on an intention-to-treat basis. Missing values were imputed on the basis of the last-observation-carried-forward technique and included all randomly-assigned participants who stayed in the study until the first visit of the preventive phase. All clinical variables were analyzed using ANCOVA, with treatment and time intervals representing the main factors. Gender, age and pain improvement in the symptomatic phase were used as covariates in the analyses. Data were adjusted for gender and pain improvement in the symptomatic phase because these 2 variables were deemed to play an important role in possible further improvement of the patient's condition. One-way ANOVA served to compare co-intervention data across the 3 groups.Whenever factorial analyses revealed significant effects, post hoc analyses were performed with the least significant difference test. For all analyses, statistical significance was set at p < 0.05.Patients were recruited during the months of August and September 2007. One hundred and seventeen participants were assessed for eligibility through an initial evaluation in September 2007. Preliminary data were collected at this time. All included participants n = 108) received treatments during the symptomatic phase of the trial, which began in September 2007. Ten patients  dropped out of the study. This phase ended in December 2007, at which point baseline data were collected. After completing symptomatic treatments, participants were assigned to their groups. Drop outs from the symptomatic phase led to unequal group sizes: SMT (n = 36), SMT + exercise (n = 33) or attention-control (n = 29). The preventive phase lasted from January 2008 to October 2008, at which point all participant follow-up ended , presented in Table During the non-randomised, symptomatic phase of this trial, the average pain level of participants decreased by 1.2 cm on the VAS, the disability level declined by 4.9 points on the NDI and by 6.5 points on the BQ. Function, measured as active cROMs, improved by 6.5° in the axial plane (rotation), 2.4° in the coronal plane  but decreased by 14.1° in the sagittal plane (flexion-extension). These differences were all statistically significant, except for lateral flexion  did not yield significant between group differences for the primary and secondary outcome measures. Table Significant improvements in FABQ scores  over time were noted in all groups. Statistical analysis of HRQOL  did not yield any significant effect. Table The overall compliance of each participant for all groups  was 96% (8.2) for the attention-control group and 96% (6.3) and 93% (9.8) for SMT and SMT+ exercise groups respectively. Patients' diaries were consulted to verify exercise adherence in the combined intervention group and co-intervention along with ice application in all patients. Mean adherence to the home exercise program  was 48.8%. Out of 26 patients in the combined intervention group, 16 (61.5%) completed more than 50% of the exercise sessions, 6 (23.1%) finished less than 50%, and 4 (15.4%) did not adhere at all to the program.Significant between group differences were noted for both the number of co-intervention episodes (p = 0.006) and ice application p = 0.032). The attention-control group required significantly more co-intervention (7.6 ± 1.1) compared to the other 2 groups (4.1 ± 0.9 and 2.9 ± 0.9). Moreover, the attention-control group chose to apply ice as a pain-relieving modality significantly more often (20.8 ± 4.7) that the other 2 groups (7.8 ± 4.0 and 4.5 ± 4.2). Figure 2. The atThis study hypothesised that participants in the combined intervention group would have less pain and disability and better function than participants from the 2 other groups during the preventive phase of the trial. In fact, all 3 groups showed primary and secondary outcomes scores similar to those obtained following the non-randomised, symptomatic phase. No significant change in HRQOL was associated with the preventive phase, but the 3 groups demonstrated statistically significant improvement in their fear avoidance behaviour scores over time. Overall spinal manipulation or spinal manipulation combined with exercises did not yield significant advantages when compared to the no treatment strategy.The course of neck pain is usually described as episodes occurring over a lifetime with variable degrees of recovery between episodes,3,37. FuAnother plausible explanation for these results is the placebo effect, which refers to the outcome attributable to a procedure but not to its specific properties. Any perceived therapeutic action includes its specific and non-specific effects. The non-specific effects, also termed the "context of treatment", represent the psychosocial aspect of every treatment. In our study, different clinicians were responsible for each of the 3 groups, and therefore the treating clinician (not only the treatment used) may have influenced the clinical outcomes. Many factors contribute to these outcomes, the 3 most often described being the patient, the provider and patient-provider interaction-41. FromExercise adherence in the combined intervention group was 48.8%. This compares with previous estimates of adherence in home exercise programs for neck and low back pain, converging around 50%,43. ThisBefore the preventive phase of this study, all participants received a short course of treatment to relieve symptoms: SMT to the cervical and upper thoracic areas and myofascial soft tissue therapy when appropriate. All primary and secondary outcomes  demonstrated statistically significant improvement, except lateral flexion. The results of this non-randomised, symptomatic phase compare favourably to the existing literature on SMT for NCNP-48. The Some trials have assessed the long-term efficacy and effectiveness of SMT for non-specific neck pain,49-51. TThe limitations of the present study include the absence of blinding of participants or treating chiropractors. This was inevitable due to the trial design  and the impossibility of designing an adequate placebo for SMT,24. IndeA crucial aspect of any clinical trial is the equivalency of group sample size. In our RCT, randomisation was performed before the beginning of the trial's symptomatic phase, and an unequal number of drop-outs generated a smaller attention-control group than the experimental groups. It should also be noted that since the sample size of 35 subjects per group was not achieved, the pre-established statistical power was not reached. Because of the fluctuating nature of NCNP the patients included in this trial presented with low pain scores, at the time of enrolment. Such low levels of pain and related disabilities, may have limited the ability to detect statistically and clinically significant difference between the groups.Another limitation is that during the preventive phase, participants in the attention-control group consulted only once every 2 months for a period of 20 to 30 minutes. This contrasts with the 2 experimental groups who consulted on a monthly basis for 10 to 15 minutes. The difference in protocol was deemed necessary to minimize drop-outs in the attention-control group. The actual dropout rate of the 3 groups was 11% for the SMT only group, 3% for the SMT combined with exercise group, and 10% for the attention-control group. Since the attention-control group did not incur a higher drop-out rate compared to the experimental groups, it can be concluded that this was an adequate strategy. However, the impact of such a strategy on clinical outcomes cannot be assessed independently of the present study.Despite these limitations, this small-scale RCT demonstrates the feasibility of implementing controlled trials to investigate preventive strategies to improve prognosis or affect the natural history or recurrence of NCNP.The results of our study are important because there has been minimal previous research regarding the prevention of NCNP. Our results suggest that the mere fact of taking charge of and managing a patient for this condition might decrease the recurrence of pain episodes and, therefore, change the course of the disease. Considering the societal burden of NCNP, the issuOur results also indicate that there is no additional benefit for patients with NCNP to receive monthly preventive SMT or monthly preventive SMT with a home exercise program compared to meeting a chiropractor once every 2 months to discuss neck problems. In view of the rare but possible adverse reactions to cervical SMT, this tends to reject CPC when SMT is the main intervention. However, the premise of CPC stating that regular treatments, designed to preserve optimum health, will also minimize the recurrence of clinical problems, might hold true when intervention is geared towards reassurance, patient education, help with self-management and active care strategies. Further research in this domain has to be conducted.A final implication of these results is the equivalence between the SMT and combined intervention groups. The actual, best evidence regarding treatment for NCNP is a combined approach involving manual therapy and exercise. It is pThe authors declare that they have no competing interests.MD contributed to trial design and protocol development, had overall responsibility for conduct of the trial, and contributed to recruiting patients, deciding the statistical analysis, interpreting the data and preparing the manuscript. CD contributed to trial design and protocol development, statistical analysis, data interpretation and manuscript preparation. JM was responsible for day-to-day management of the trial, follow-ups of the attention-control group, statistical analysis and manuscript preparation, and contributed to data interpretation. JDD participated in the development of the home exercise program used throughout the trial, was responsible of supervising the program execution by patients allocated to the SMT + exercise group and contributed to data interpretation and manuscript preparation. All authors had full access to all data  in the study and take full responsibility for data integrity and the accuracy of data analysis. MD is the guarantor. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/41/prepub