Numerous studies examining the relationship between CD44 expression and prognostic impact in patients with osteosarcoma have yielded inconclusive results. The aim of this meta-analysis was carried out to investigate the relationship between CD44 expression and the survival in patients with osteosarcoma.We therefore conducted a meta-analysis to provide a comprehensive evaluation of the prognostic role of CD44 expression on the overall survival rate and metastasis, which compared the positive and negative expression of CD44 in patients of the available studies.A detailed search was made in MEDLINE and EMBASE for relevant original articles published in English. Finally, a total of six studies with 329 osteosarcoma patients were involved to estimate the relationship between CD44 expression and metastasis of tumor and overall survival. Positive expressions of CD44 did not predict neoplasm metastasis , and the results indicated that higher expression of CD44 could not predict poorer survival in osteosarcoma with the pooled HR of 0.55 .The findings from this present meta-analysis suggest that CD44 expression is not associated with overall survival rate and metastasis in osteosarcoma.http://www.diagnosticpathology.diagnomx.eu/vs/1373995521295618The virtual slide(s) for this article can be found here:  Osteosarcoma is the most common malignant primary bone tumor and the majority of these tumors occur among children and adolescents –3. DespiCD44 is the major hyaluronan (HA) receptor , and CD4Most of earlier studies suggested CD44 high expression was associated with high risk of tumor metastasis and worse survival in patients with osteosarcoma. However, some other studies showed insignificant or opposite results. Therefore, the aim of this meta-analysis was carried out to investigate the relationship between CD44 expression and the survival in patients with osteosarcoma. We also discuss the possibility of using CD44 as a prognostic marker in osteosarcoma.The PubMed, EMBASE, and MEDLINE databases were searched, in addition to the Cochrane Central Register of Controlled Trials, to locate articles (published between January 1994 and January 2014), including articles referenced in the publications. The search strategy included the following keywords variably combined by “CD44”, “osteosarcoma”, “bone tumor” and “prognosis”. Internet search engines were also used to perform a manual search for abstracts from international meetings, which were then downloaded and studied.Studies met the inclusion criteria if they studied the patients with osteosarcoma, measured the expression of CD44 in cancer tissue and investigated the association betweenCD44 expression levels and survival out come. When a study reporting the same patient cohort was included in several publications, only the most recent or complete study was selected. Studies of case reports, letters, and reviews without original data; non-English papers; animal or laboratory studies; and studies of nondichotomousCD44 expression levels and absence of survival outcome were excluded. If any doubt of suitability remained after the abstract was examined, the full manuscript was obtained .Two review authors assessed the methodological quality of potentially eligible studies, without consideration of the results. Extracted data were then crosschecked between the two authors to rule out any discrepancy. Data regarding the following for each included studies were extracted independently: first authors’ surname, publication year, origin country, sample size, CD44 assessment methods and the cut-off definition, and HR of CD44 expression for overall survival (OS) as well as corresponding 95% confidential interval (CI) and P value. Multivariate Cox hazard regression analysis reported in the article was included in the present analysis. Disagreements were discussed by the authors and resolved by consensus.2 statisticswas used to evaluate the between-study heterogeneity analysis in this meta-analysis [2 > 50%). The fixed effects model was used when there was no significant heterogeneity between the included studies (I2 ≤ 50%). Publication bias was estimated using a funnel plot with an Egger’s line arregression test; funnel plot asymmetry on the natural logarithm scale of the HR was measured by a line arregression approach.The statistical analysis was carried out using the Review Manager (RevMan) software version 5.0. All these HRs and 95% confidence interval(CI) were calculated following Tierney’s method. Pooled HR was calculated using a fixed-effects model or random-effects model to evaluate the relationship between CD44expression and overall survival. Ianalysis . The ranThe initial search retrieved a total of 86 references, and after screening titles and abstracts of identified articles, 38 were excluded because they were not related to the current study. Upon further review, 34 were excluded because they were either laboratory studies or records without survival data. Then we evaluated 14 potential candidate studies in full text. It was found that the survival data of one article could not be used in this study. Finally, 6 –24 studiI2 = 0%), so the fixed-effect model was used to calculate the pooled HR with corresponding 95% CI. The result indicated that positive expressions of CD44 did not predict neoplasm metastasis , and higher expression of CD44 could not predict poorer survival in osteosarcoma with the pooled HR of 0.55 . , there was no between-study heterogeneity among those six studies for CD44 I = 0%, soFunnel plot and Egger’s test were used to evaluate the publication bias of the literatures. The shape of the funnel plot did not reveal any evidence of obvious asymmetry .Osteosarcoma is a life-threatening bone malignancy that often occurs in teenagers. It is the second leading cause of cancer-related death in pediatric age group and young adults . CurrentCD44 was previously thought to be a transmembrane adhesion molecule, which also played a role in the metabolism of its principal ligand hyaluronan. It may exist in three distinct physical phases, as a transmembrane cell surface receptor, an integral component of the matrix and in a fluid phase, each with the potential for being functionally significant. CD44 is known to be a major hyaluronic acid receptor in vitro, although it is not known how often CD44 is expressed or what role it plays in normal bone tissues. CD44 proteins have been observed in osteoclasts and osteocytes by means of immunohistochemical analysis on bone tissue, but there have been some controversial reports that CD44 proteins were found in osteoblasts . As CD44The present meta-analysis showed that high CD44 expression did not indeed predict poor survival and metastasis in patients with osteosarcoma. However, it should be circumspect to make a verdict of the association with CD44 and osteosarcoma, because there are still several issues should be considered. First, since the number of included studies in this meta-analysis was only six, it might weaken the reliability of our results. More well-designed clinical studies with large cases of osteosarcoma should be performed in the future to validate the relationship between CD44 expression level and prognosis of osteosarcoma patients. Second, lack of abundant CD44 expression data in global population makes it difficult to set a standard value for the measurement of CD44. Third, the methods used for the evaluation of the levels of markers in osteosarcoma patients and the use of standard threshold, are both likely to impact on our results. Although immunohistochemistry was the most commonly applied method, the cut-off value was defined differently in inclusion studies. Therefore, we strongly suggest conducting more prognostic studies for high CD44 expression in osteosarcoma.Moreover, recently studies have demonstrated that microRNAs might influence chemoresistance of osteosarcomas with different pathways including CD44, resistance to chemotherapeutic agents is still one of the major reasons for the failure of osteosarcoma treatment, while we did not excluded the role of CD44 in chemoresistance of osteosarcoma. The identification of cancer-specific miRNAs and their targets is pivotal for understanding their role in tumorigenesis and metastasis, and may be important for the discovery of novel therapeutic targets. CD44 which contained the corresponding binding site of microRNAs’ 3′UTR, was regulated by some microRNAs, such as miR-34a, miR-140 and miR-215 –35. The On the other hand, many researchers have reported that high expression of some markers indicates bad clinical features. Such as clinical stage, positive distant metastasis and poor response to chemotherapy , 37. In Despite the inherent limitations of meta-analysis on prognostic literature, this meta-analysis, representing a quantified synthesis of all published studies of CD44, has shown that the high expressed CD44 is not significantly associated with poor survival and metastasis in patients with osteosarcoma. For better analysis the relationship between CD44 expression and prognostic with the osteosarcoma, it is necessary to improve the experimental methods and detection methods, and to clear a unified quantitative standard. Future adequately multi-center designed prospective with larger sample size were of great value to confirm these findings and more clinical studies should be carried out before the application of CD44 in prognosis of osteosarcoma.The findings from this present meta-analysis suggest that CD44 expression is not associated with overall survival rate and metastasis in osteosarcoma. CD44 may not be a useful marker to predict prognosis of osteosarcoma.Yu Liu and Yongwei Wu: co-first authors.


Perceptual learning has been shown to produce an improvement of visual acuity (VA) and contrast sensitivity (CS) both in subjects with amblyopia and refractive defects such as myopia or presbyopia. Transcranial random noise stimulation (tRNS) has proven to be efficacious in accelerating neural plasticity and boosting perceptual learning in healthy participants. In this study, we investigated whether a short behavioral training regime using a contrast detection task combined with online tRNS was as effective in improving visual functions in participants with mild myopia compared to a 2-month behavioral training regime without tRNS . After 2 Perceptual learning has been found useful in improving visual abilities such as visual acuity (VA) and contrast sensitivity (CS), both in participants with deficits in visual cortical processing such as amblyopia  and in pAlthough many studies support the view that a high degree of specificity of perceptual learning for simple stimulus attributes , more reNeuromodulation techniques such as transcranial magnetic stimulation (TMS) or transcranial electrical stimulation (tES) have also been tested for the restoration of visual functions in people with abnormal cortical processing and are also considered useful in the understanding of visual functions at the cortical level. While TMS has been found useful in increasing CS up to 1 Log CS on medium–high frequencies, both temporarily  and for Online transcranial random noise stimulation  has recently been proven to be the most efficacious type of electrical stimulation for boosting perceptual learning in healthy participants .To date, no techniques of neuromodulation have been used in an attempt to improve visual functions in participants with optical defects such as myopia. Thus, in the present study a short perceptual training regime in a contrast detection task using a single Gabor patch joined with tRNS was administered in order to investigate the effects of this combined approach on UCVA and UCCS in participants with mild myopia.Sixteen participants with mild myopia were recruited from the University of Padova . The first group of eight participants carried out a 2-week (eight sessions) behavioral training using a contrast detection task combined with online high-frequency tRNS (hf-tRNS). The second group of eight participants (control group) underwent the same training protocol but without tRNS. This was done in order to compare the effect of the combination of behavioral training + tRNS with the effect of the behavioral training alone (without tRNS) on UCVA and UCCS.The participants fit the following inclusion criteria: refractive error up to –2 diopters (D) in either eye (minimum was –0.75 D), with astigmatism not exceeding –0.5 D in either eye. All tests as well as the training were administered binocularly and with no optical corrections. The participants had a stable refractive index for the 6 months prior to training. Exclusion criteria included any other ocular condition or cause for reduced VA other than simple myopia and/or mild astigmatism; these include diabetes mellitus, pregnancy, presence of myopia-related ocular complications and any previous ocular surgery. To ensure the inclusion and exclusion criteria, prior to training the participants carried out a detailed assessment by an optometrist. This assessment was repeated at the end of the training. Additionally, each participant in the tRNS group also filled in a questionnaire ensuring that all were eligible to undergo non-invasive brain stimulation. Any participant with a history of seizures, internal metal objects, or previous traumatic brain injury was excluded from our study. This study was approved by the local ethics committee.Before (pre-tests) and after the training , UCVA and UCCS were assessed for each participant by using Landolt C and Grating tests of the Freiburg Visual Acuity Test FrACT; . After 3°. Grating orientations used were 0°, 45°, 90°, or 135°. The task of the participant entailed discriminating the orientation of the grating at different spatial frequencies, ranging from 1 to 15 cpd, in separate blocks.Stimuli (UCCS assessment) consisted of sinusoidal gratings presented in a circular window with a narrow Gaussian taper. Size of the gratings was 3The Landolt C test was used to assess UCVA. The task of the participants was to indicate, in every trial, the orientation of the gap of the Landolt C out of eight possible orientations.For both FrACT tests, the Best-Pest adaptive procedure was used to calculate the absolute threshold for each of these tests. Stimulus duration lasted until the participants’ response. An auditory cue was presented upon stimulus presentation and a different auditory cue was implemented as feedback for error responses.The following behavioral paradigm described was used in an earlier study by 2.Stimuli were displayed on a 22-in Philips Brilliance 202P4 monitor with a refresh rate of 60 Hz. The monitor was luminance-calibrated with gamma = 1 by means of a professional monitor calibrator (Datacolor Spyder 4 Elite). The stimuli used in the training were generated with the Matlab Psychtoolbox , whereasHigh-frequency tRNS was delivered using a battery-driven stimulator  through a pair of saline-soaked sponge electrodes. The tRNS consisted of an alternating current of 1.5 mA intensity with a 0-mA offset applied at random frequencies. The frequencies ranged from 100 to 640 Hz.2 and was placed over the occipital cortex measured at ∼3 cm above the inion. The reference electrode had an area of 60 cm2 and was placed extracephalically on the upper right arm. The current density was maintained well below the safety limits : participants trained on a contrast detection task for eight sessions with concurrent tRNS significantly improved their UCVA by 0.15 LogMAR , that is from 0.33 to 0.18 LogMAR, and this improvement was maintained at the follow-up, where VA (0.15 LogMAR) was still significantly different from pre-test , 5 cpd , 7 cpd , 9 cpd , and 11 cpd . The largest improvements were seen at intermediate spatial frequencies (3 and 5 cpd), where UCCS increased, on average, by five to six times at the post-test, with respect to the pre-test. Given that participants were mainly trained at higher spatial frequencies , this means that learning mainly generalized from higher to lower spatial frequencies, consistently with previous studies  did not improve neither in UCVA  in early childhood. Such a mismatch would decrease the perceived contrast, especially at high spatial frequencies, thus degrading UCVA . LearninSince eight sessions of perceptual training with no tRNS does not seem to induce any improvement, it could be argued that perceptual learning here is not playing a role at all, and that the observed improvements are solely due to tRNS. In fact, previous studies found that anodal tDCS over the visual cortex can improve CS even in the absence of perceptual training . It mustFuture studies are needed to assess whether more efficacious training protocols  can also benefit of a concurrent tRNS for improving visual abilities in participants with cortical (such as amblyopia) or non-cortical visual deficits , and whether the improvement is long-lasting, as found with longer trainings without brain stimulation . AdditioIn sum, these preliminary findings suggest that coupling a short contrast detection training with tRNS in participants with mild myopia results in an increased UCVA and UCCS, similar to or even larger than that seen with no tRNS and with a longer training using a similar training paradigm and stimuli parameters, but in the absence of brain stimulation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A combination of microarray data with extensive genome annotations resulted in a set of 28,456 experimentally supported transcripts, providing the first experiment-driven annotation of the human genome. Computational and microarray-based experimental approaches were used to generate a comprehensive transcript index for the human genome. Oligonucleotide probes designed from approximately 50,000 known and predicted transcript sequences from the human genome were used to survey transcription from a diverse set of 60 tissues and cell lines using ink-jet microarrays. Further, expression activity over at least six conditions was more generally assessed using genomic tiling arrays consisting of probes tiled through a repeat-masked version of the genomic sequence making up chromosomes 20 and 22.The combination of microarray data with extensive genome annotations resulted in a set of 28,456 experimentally supported transcripts. This set of high-confidence transcripts represents the first experimentally driven annotation of the human genome. In addition, the results from genomic tiling suggest that a large amount of transcription exists outside of annotated regions of the genome and serves as an example of how this activity could be measured on a genome-wide scale.These data represent one of the most comprehensive assessments of transcriptional activity in the human genome and provide an atlas of human gene expression over a unique set of gene predictions. Before the annotation of the human genome is considered complete, however, the previously unannotated transcriptional activity throughout the genome must be fully characterized. The completion of the sequencing of the human, mouse and other genomes has enabled efforts to extensively annotate these genomes using a combination of computational and experimental approaches. Generating a comprehensive list of transcripts coupled with basic information on where the different transcripts are expressed is an important first step towards annotating a genome once it has been fully sequenced. The task of identifying the transcribed regions of a sequenced genome is complicated by the fact that transcripts are composed of multiple short exons that are distributed over much larger regions of genomic DNA. This challenge is underscored by the widely divergent predictions of the number of genes in the human genome. For example, direct clustering of human expressed sequence tag (EST) sequences has predicted as many as 120,000 genes , and a het al. [Several recent studies have highlighted the limitations of relying solely on computational approaches to identify genes in the draft of the human genome -13. Furtet al.  also indet al. . They deab initio prediction programs operate by recognizing coding potential in stretches of genomic sequence, where the recognition capability of these programs depends on a training set of known coding regions [ab initio prediction programs or assembled from EST data are also inaccurate or incomplete much of the time [ab initio prediction programs perform well at identifying known genes, predictions that do not use existing expressed sequence and protein data often miss exons, incorrectly identify exon boundaries, and fail to accurately detect the 3' and 5' untranslated regions UTRs [Most current methods in widespread use for identifying novel genes in genomic sequence depend on sequence similarity to expressed sequence and protein data. For example,  regions . Therefothe time -12. Whilons UTRs . Similarons UTRs . These dons UTRs , but cloons UTRs .Arabidopsis thaliana and Escherichia coli genomes. The recent sequencing and analysis of the mouse genome indicates extensive homology between intergenic regions of the human and mouse genomes, further highlighting the potential for other classes of transcribed regions [Recently, several groups have used microarrays to test computational gene predictions experimentally and to tile across genomic sequence to discover the transcribed regions in the human and other genomes -12,15-17 regions . Interes regions .In the study reported here, we describe hybridization results generated from two large microarray-based gene-expression experiments involving predicted transcript arrays spanning the entire human genome and a comprehensive set of genomic tiling arrays for human chromosomes 20 and 22. mRNA samples collected from a diversity of conditions were amplified using a strand-specific labeling protocol that was optimized to generate full-length copies of the transcripts. Analyses of the resulting hybridization data from both sets of arrays revealed widespread transcriptional activity in both known or high-confidence predicted genes, as well as regions outside current annotations. The results from this analysis are summarized with respect to published genes on chromosomes 20 and 22 in addition to our own extensive set of genome alignments and gene predictions. Combining computational and experimental approaches has allowed us to generate a comprehensive transcript index for the human genome, which has been a valuable resource for guiding our array design and full-length cloning efforts. In addition, the expression data from the 60 conditions provides a comprehensive atlas of human gene expression over a unique set of gene predictions .ab initio gene-finding algorithms tend to have a high false-positive rate when applied at a low-stringency setting to cast as broad a discovery net as possible. Second, gene-finding algorithms are trained on known protein-coding genes, which may limit their ability to detect truly novel classes of transcribed sequences.Figure The second step towards the CTI is the use of two different types of microarrays to address these limitations Figure . First, ab initio gene predictions. The process resulted in mapping 91% of the well characterized genes found in the RefSeq database [To generate the PTI, three distinct computational analysis steps were executed in parallel: predictions based on similarity to expressed sequences from human and mouse; predictions based on similarity to all known proteins; and database , a percedatabase ,3. The mab initio predictions. The categories, in decreasing order of support, are as follows: (1) known genes, taken as the set of 11,214 human genes represented in the RefSeq database when the arrays were designed; (2) ab initio gene models with expressed sequence and protein support; (3) ab initio gene models with expressed sequence support; (4) ab initio gene models with protein support; (5) alignments of expressed sequence and protein data; (6) alignments of expressed sequence data, requiring at least two overlapping expressed sequences; (7) ab initio gene models with no expressed sequence or protein support; and (8) alignments of protein data. Because of the limitations discussed in the previous section, we considered predictions with a single line of evidence (categories 6-8) as low confidence.All locus projections were classified into one of eight categories on the basis of the level of underlying evidence from expressed sequence similarity, protein similarity and Table ab initio gene-finding algorithms [We previously described a high-throughput, experimental procedure to validate predicted exons and assemble exons into genes by using co-regulated expression over a diversity of conditions . Here wegorithms ,25,26 agWe used an enhanced version of a previously described gene-detection algorithm to analyze the predicted transcript array dataset . BasicalThe sensitivity of our procedure was assessed by computing the EVG detection rate for those Sanger genes that overlap predictions (locus projections) represented in our PTI Table . The aveThis 20% false-negative rate is the result of a complex mixture of issues, including limitations in our EVG-detection algorithm, limitations in the probe design step, lack of expression in the conditions profiled, and/or alternative splicing events. While the EVG-detection algorithm provides an efficient method to assemble probes into transcript units, the detection capabilities of this model could be expected to improve as the number of samples and the number of probes targeting any given transcript increases. The use of four probes per predicted transcript was determined to be sufficient for detection of most transcripts, as supported by the overall detection rate of known genes, although in many cases the probe design step was limited by our ability to find four high-quality probes per transcript. For many transcripts, there were not four nonoverlapping probes predicted to have good hybridization characteristics for the microarray experiment carried out here. The 60 samples were chosen to represent a broad array of tissue types, as an exhaustive list of human tissues is impossible to obtain. Because no replicate tissues/cell lines were run for any of the 60 chosen samples, we relied on the replication inherent in monitoring the same transcripts over 60 different conditions. In this case, genes expressed in multiple samples provide the replication necessary to increase our confidence in the detections. However, there are clear limitations in not replicating tissues/cell lines, as genes may be expressed in only a single condition or may be switched on only under certain physiological conditions or only during a certain stages of development. In such cases, we would have reduced power to detect these genes.Genes in the lower-confidence categories of our PTI annotations, which are not typically considered genes by Sanger, were detected at a significantly reduced rate. Interestingly, of the 337 (188 +149) higher-confidence transcripts on chromosomes 20 and 22 that did not intersect with Sanger genes, 47 (or 14%) were detected as EVGs ).However, before we can make claims to the discovery potential for this method over the entire genome, we need to assess the false-positive detection rates. To this end, we defined as false positives all detections made in regions with support by only a single gene model that fell outside Sanger-annotated genes on chromosomes 20 and 22. Applying this definition over all transcripts in our PTI leads to a false-positive rate of 3% (11 out of 406). Because we cannot exclude the possibility that some of the transcripts supported by a single gene model represent real genes, we consider this false-detection rate as an upper bound on the actual false-positive rate. Accepting that the Sanger annotations represent the gold standard for chromosome 22, we detected 70% of all Sanger-annotated genes, while only 4% of the chromosome 22 locus projections that did not intersect Sanger genes were detected by our procedure, highlighting the sensitivity and specificity of this approach. In addition, the enrichment for EVG detections in Sanger genes versus the non-Sanger PTI on chromosomes 20 and 22 was extremely significant with a ab initio gene predictions ever undertaken. We can use the false-positive and negative rates derived above to assess the discovery potential on that part of the genome that has not been as extensively characterized as chromosomes 20 and 22. First, we note that our detection rates over the genome were similar to that given for chromosomes 20 and 22. That is, 75% of the category 1 genes (RefSeq genes) were detected over the entire genome, compared to 80% for chromosomes 20 and 22. In total, 15,642 genes in the PTI were experimentally validated using this array-based approach. Assuming the false-positive rate of 3% defined above and a conservative false-negative rate of 30%, defined as the percentage of Sanger genes we failed to detect on chromosomes 20 and 22, these data suggest there are close to 21,675 potential coding genes represented in our PTI set. Because our PTI misses close to 10% of the Sanger genes, we corrected this number for those genes not represented in this set and provide an estimate of the total number of protein-coding genes in the human genome supported by our data to be approximately 25,000. This number is consistent with estimates given in the current release (22.34d.1) of the Ensembl database [Summarizing EVG data over the entire genome and assessing the discovery potential. The last column of Table database ,29.However, we caution that the estimate provided is based solely on the data described here, and that orthogonal sources of data  continueab initio prediction). These 2,093 transcripts represent a rich source of potential discoveries in our PTI. To assess the potential biological functions of this novel gene set, we annotated translations of this set by searching the domains represented in the Protein Families database (Pfam) [From Table e (Pfam) . The seae (Pfam)  codes aset al. [Because multiple probes in each of the approximate 50,000 predicted genes in the human genome have been monitored over 60 different tissues and cell lines, the EVG data represent a significant atlas of human gene expression that is now publicly available . For eacet al.  to proviTo complement the use of PTI arrays, we constructed a set of genome tiling arrays comprised of 60 mer oligonucleotide probes tiled in 30 base-pair steps through both strands of human chromosomes 20 and 22. Repetitive sequences identified by RepeatMasker were ignored for probe design. These genome tiling arrays allow for an unbiased view of the transcriptional activity outside of known and predicted genes on these two chromosomes. mRNA from six (chromosome 20) or eight (chromosome 22) conditions was amplified and hybridized to the tiling arrays .Figure e Figure , the tilEWSR1 gene. In contrast to the first example, this gene has intense transcriptional activity outside of the annotated exons. Specifically, the EWSR1 gene has 43 potentially false-positive calls out of 203 intron probes. However, the EST data and alternative splicing predictions strongly suggest that these probes represent biologically relevant transcriptional activity. As with the KDELR3 gene, EWRS1 is annotated by RefSeq as having two transcripts: NM_005243 and NM_013986. The Acembly predictions identify four additional alternative splice forms; most noteworthy among these are EWSR1.b and EWSR.g, shown in Figure EWSR1 gene that essentially divide the largest transcript into two transcripts, suggesting that multiple promoter and transcription-stop signals are present in this gene. The tiling data depicted in Figure EWSR.b and EWSR.g predicted alternative splice forms, providing experimental support that these predictions represent actual isoforms of this gene. In fact, these data may provide a more accurate representation of the putative structure of this gene, as they support multiple alternatively spliced transcripts in this gene, beyond what has already been annotated in the RefSeq database. In all, 5% of the probes detected as expressed in intronic sequence mapped to predicted alternative splice forms. Given the extent of alternative splicing that is yet to be characterized [Figure cterized , we belip-value for this enrichment using the Fisher exact test is less than 10-15). To estimate the upper bound of false-positive calls, we counted as false-positive events each probe identified as expressed by the detection process, but falling within an annotated intron of the RefSeq genes we detected as expressed. This resulted in an estimated false-positive rate of 1.3%.Our genome tiling arrays consisted of 2,119,794 and 1,201,632 probes for chromosomes 20 and 22, respectively. Of these, 1,615,034 probes fell into Sanger gene regions, with 239,542 probes actually overlapping Sanger exons. Under stringent criteria 64,241 probes were detected as expressed, with 34,245 of these falling within Sanger exons, 18,551 falling within Sanger introns, and 15,835 probes falling completely outside all Sanger annotations. This widespread transcriptional activity outside annotated regions of the human genome is consistent with other reports from multiple species ,12,15,16As indicated in Figure For those probes falling outside all Sanger genes, we again made use of our custom genome annotations to help interpret the extent of transcriptional activity in these regions. Table One further advantage of the tiling data is that they can be used to discriminate between transcribed and non-transcribed sequences conserved between human and mouse, or between any other pair of species. Figure ab initio methods, sequencing of EST libraries, full-length gene cloning projects, and comparative analyses between fully sequenced genomes of different species. However, we are still a long way from having a comprehensive set of annotations for the human and other genomes. There is need for new high-throughput experimental approaches to accelerate the process of annotating sequenced genomes in a comprehensive and accurate fashion. Toward this goal, we have used two microarray-based experimental approaches to provide evidence of widespread transcription activity outside of any known or predicted genes in the human genome. We have also provided experimental support for many ab initio predicted genes that have no other or minimal experimental sequence support, suggesting a small but significant class of genes that have evaded all other forms of experimental detection. Similar identifications have been made recently in the first extensive comparative analysis between mouse and human genomes [A complete understanding of the human genome will only come after all genes have been identified and the functions of those genes have been determined. There has been much recent progress in defining the human transcriptome with  genomes . Despite genomes ,40. FinaWe have used the expression data for the approximate 50,000 predicted transcripts hybridized to 60 diverse conditions in combination with genomic tiling data to generate a CTI containing 28,456 experimentally supported transcripts. The transcripts represented in the CTI include all computational predictions with two or more lines of evidence from our PTI , in addition to the overlapping set of 15,642 transcripts detected as EVGs. This resulting comprehensive list of known and predicted transcripts provides the starting point for large-scale systematic studies to determine the biological function of genes in both normal and disease states. The primary goal of the CTI is to allow researchers to focus experimental efforts on a comprehensive set of genes that are likely to be real.It is of note that between the time the predicted transcript arrays were designed and annotated using the custom genome annotations described above, and the time this work was published, more than 6,000 genes were added to the RefSeq collection. These newer RefSeq genes were represented by 5,100 locus projections in our original PTI that were not classified in the RefSeq category. Interestingly, 4,212 were detected as EVGs in the present analysis and had already been included in our CTI, a validation rate slightly greater than 82%. Only 19% of the non-RefSeq genes in our PTI had been detected as EVGs  and a mouse gene index (MGI). Clustering and alignment were performed with the DoubleTwist Clustering and Alignment Tools (CAT) . Input d-20 for human sequences and 10-8 for mouse sequences. For human UniGene and HGI, we refined only those BLAST hits where the target sequence showed greater than or equal to 92% identity to the genomic sequence over 75 bp. For human RefSeq, we refined hits with greater than or equal to 95% identity, and for MGI, RefSeq, and UniGene, we refined hits with greater than or equal to 80% identity. These thresholds were empirically determined to provide good sensitivity in aligning most sequences to the genome while limiting multiple alignments past those expected from paralogs present in the human genome. In all cases percent identity was measured over 75 bp. Individual sim4 exons of questionable confidence were then removed on the basis of percent identity and length thresholds. All sequence databases were downloaded from GenBank August, 2001.Human and mouse UniGene and RefSeq, MGI, and HGI sequences were aligned with the genome first by BLASTN 2.2.1 , followe-5. Adjacent protein alignments from a single protein were grouped together as a prediction whenever the protein sequence coordinates of the alignments were consistent in direction and did not significantly overlap.The GenBank nonredundant protein database (downloaded 25 August 2001) was aligned to the genomic sequence with BLASTX 2.2.1  using anab initio gene-prediction algorithms were run independently across the entire genome assembly to augment alignment-based gene identification methods. GrailEXP 4.0, GENSCAN 1.0, and FGENESH version 1.c were run with default parameters for human sequence. GrailEXP used expressed sequence evidence from RefSeq, UniGene and DoubleTwist HGI to refine gene predictions. FGENESH+ was run with protein sequences from BLASTX with E-score lower than 10-5. When multiple protein alignments overlapped, all overlapping protein sequences were clustered with BLASTClust [GrailEXP 4.0 , GENSCANASTClust  and the Locus projections contained the union of all exons from all overlapping predictions in a contiguous region of the chromosome that were derived from sequence alignments or gene-finding algorithms. Predictions to a given strand of the genomic sequence that overlapped by even a single nucleotide were grouped into a single locus projection (antisense transcripts were not considered in defining the locus projections). The criteria for grouping predictions were intentionally kept loose, given that the intent was to include as many potential exons as possible in a given genomic region, and then use the experimental microarray-based approach to elucidate the actual gene structure. These merged overlapping predictions defined the 5' and 3' ends of the locus projections. Overlapping predicted exons were merged to form an exon prediction of maximal extent. Low-quality predicted exons from sim4 alignments that contained a high percentage of A or T were removed. We also removed sim4-predicted exons that overlapped two or more predicted exons from another sim4 alignment. Additionally, 3' sim4 and 3' or 5' FGENESH+ predicted exons that were short and/or distant from internal predicted exons were removed. Finally, locus projections that contained mRNAs from RefSeq were split at the 5' end of the RefSeq sequence.ab initio gene model.Locus projections supported by expressed sequences alone could be portions of 3' or 5' UTRs of genes included in the other gene-prediction categories described in the text. To minimize the consequences of this potential artifact, we used a UTR filter to exclude locus projections from the expressed sequence alone category that were within 20 kb of a locus projection supported by an ab initio; expressed sequence + ab initio; protein + ab initio; expressed sequence + protein; ab initio alone; protein alone; expressed sequence alone. FGENESH+ predictions were counted as protein + ab initio. For the ab initio category, predictions from at least two of FGENESH, GENSCAN and GrailEXP were required to overlap in at least one exon to be merged.All data were loaded into a relational database to count and categorize locus projections. At least one type of evidence was assigned to each predicted exon for each locus projection. Multiple types of evidence were assigned to a merged predicted exon if there was overlap between predicted exons of different types for at least 1% of the length of the merged exon prediction. One of the eight evidence categories discussed in the text was assigned to each exon on the basis of the combination of types of evidence. Locus projections inherited the highest-ranking evidence category of their constituent exons. Evidence categories were ranked in the following order: Refseq (highest); expressed sequence + protein + Escherichia coli contamination and human non-coding RNA and mitochrondrial DNA contamination using Scylla (Paracel). For genomic tiling arrays, 60 mer probes were then selected from unmasked regions of both forward and reverse complement strands at uniform 30-base intervals. For predicted transcript arrays, up to four oligonucleotide probes were selected from the unmasked regions of each transcript using a multistep process.Input sequences for probe selection were masked for vector, interspersed repeats, simple repeats, poly(A) tails, Saccharomyces cerevisiae were added to the 3' ends of probes shorter than 60 nucleotides so that they had a total length of 60 bases when printed onto the arrays.The first step in the probe-selection process was the generation of a pool of candidate probes 60 nucleotides long (60 mers), where each probe was required to fall entirely within an exon from the set of exons under consideration. If there were fewer than four 60 mers then all 50 mers were considered as well. If there were fewer than four 50 mers or 60 mers then all 40 mers were considered, and so on. Stilts composed of sequence from The second step in the probe-selection process was the classification and reduction of the probe pool on the basis of base composition and related filters. Probes were sorted into four classes on the basis of several criteria, including A, G, C and T content, GC content, the length of the longest homopolymeric run and the number of A residues at the 5' end. For example, a probe had to have GC content between 35 and 45% to be in class 1, between 15 and 55% to be in class 2, and between 10 and 60% to be in class 3. After all classifications were made, probes from lower-quality classes were discarded, keeping the number of probes per gene greater than 15. In cases where a pair of probes was overlapping by more than 50 bases, only a single probe was chosen.The final step in the probe-selection process identified probes with minimal overlap, and predicted cross-hybridization and desirable positions in the transcript sequence. Cross-hybridization prediction was based on BLAST searching of the full collection of transcript sequences . Probes All arrays included a set of standard control probes which were used for image processing and quality control. Each array also included 30 randomly distributed copies of each of 51 negative-control probes. These probes were selected for their low intensities in previous human hybridizations. The negative controls local to each experimental probe were used for background correction. Non-control probes were added to each array such that all probes for a given input sequence were grouped together and ordered by their position on the sequence.Hybridization material was generated through a random-priming amplification procedure using primers with a random sequence at the 3' end and fixed motif at the 5' end. This amplification procedure has been fully described  and has 10 (expression ratio) where the expression ratio was taken to be the ratio between normalized, background-corrected intensity values for the two channels (red and green) for each spot on the predicted transcript arrays. An independent normalization routine was carried out on the tiling data as described [Array images were processed as described  to obtaiescribed  to correProbes from each computationally determined locus were analyzed for coordinated expression over 60 tissues by adapting an additive, probe-specific model initially developed to estimate gene expression indices . The modyij = μ + φj + θi + εj,yij represent the mlratio measurements for sample pair i and probe j in the current transcriptional model, μ is the grand mean term, φi is the probe-specific term for probe j in the model, θi is the sample-specific term for sample i, and εj is the probe-specific error term, which is taken to be normally distributed with mean 0 and variance . Given the above representation for an observed mlratio value, the likelihood for a single probe over N condition pairs is simplywhere the M probes comprising the current model:From this, the likelihood for a given transcriptional model, where a transcriptional model in this context is defined as a set of probes that are adjacent to one another in the genomic sequence and that co-regulate over a number of conditions, is easily seen to be the product of the individual probe likelihoods defined above over the The maximum likelihood estimates for the parameters of this model are obtained using standard optimization techniques.ab initio prediction that fell outside annotated Sanger genes on chromosomes 20 and 22. False negatives were defined as Sanger genes on chromosome 20 and 22 that were not detected. Probe sets with a maximum likelihood statistic greater than 100 and an r2 value for fit of data to the model greater than 0.8 were considered high-confidence candidates for EVGs.With the likelihood model described above, probe groups making up a transcriptional model were formed by iteratively considering whether neighboring probes (within a PTI member based on genomic location) of a given probe improved the fit of the model just described. This was determined by examining the likelihood ratio statistics between the current, best transcriptional model with or without an additional probe included in the model. Thresholds for the likelihood ratio test statistic and the different model parameters were empirically determined to minimize false-positive and false-negative rates. False positives were estimated by the detection of PTI members supported by only a single p-value < 0.01) in at least 10% of the samples; or significantly differentially expressed  in at least 10% of the condition pairs, were considered validated.For each high-confidence EVG candidate, probes were further assessed by considering the number of conditions in which the absolute intensity of the probe was seen to be significantly above background, and the number of times the probe was seen significantly differentially expressed. Candidate EVGs with at least one probe that was: significantly above background .Hidden Markov model Pfam (HMMPfam) domain predictions were run on six-frame translations of the PTIs using the HFRAME software from Paracel with an E-value cutoff of 0.01 and frameshift penalty of -12. Information on Pfam  domains The following additional data is available with the online version of this paper and at . AdditioA complete list of 48,614 transcripts in the PTI that were represented on the set of predicted transcript arraysClick here for additional data fileA complete list of 60 tissues and cell lines hybridized to the predicted transcript arraysClick here for additional data fileA list of six tissues and cell lines hybridized to the chromosome 20 genomic tiling arraysClick here for additional data fileThe eight tissues and cell lines hybridized to the chromosome 22 genomic tiling arraysClick here for additional data fileA comparison of EVG predictions with RefSeq sequencesPClick here for additional data file


Mammalian ribosomes contain 79 different proteins encoded by widely scattered single copy genes. Coordinate expression of these genes at transcriptional and post-transcriptional levels is required to ensure a roughly equimolar accumulation of ribosomal proteins. To date, detailed studies of only a very few ribosomal protein (rp) promoters have been made. To elucidate the general features of rp promoter architecture, I made a detailed sequence comparison of the promoter regions of the entire set of orthologous human and mouse rp genes.2C+1TY(T)2(Y)3. At least 60 % of the rp promoters contain a largely conserved TATA box or A/T-rich motif, which should theoretically have TBP-binding capability. A remarkably high proportion of the promoters contain conserved binding sites for transcription factors that were previously implicated in rp gene expression, namely upstream GABP and Sp1 sites and downstream YY1 sites. Over 80 % of human and mouse rp genes contain a transposable element residue within 900 bp of 5' flanking sequence; very little sequence identity between human and mouse orthologues was evident more than 200 bp upstream of the transcriptional start point.A striking evolutionarily conserved feature of most rp genes is the separation by an intron of the sequences involved in transcriptional and translational regulation from the sequences with protein encoding function. Another conserved feature is the polypyrimidine initiator, which conforms to the consensus (Y)This analysis has provided some valuable insights into the general architecture of mammalian rp promoters and has identified parameters that might coordinately regulate the transcriptional activity of certain subsets of rp genes. Ribosomes are vital organelles, which catalyze protein synthesis in all living organisms. Eukaryotic ribosomes consist of four RNA molecules (rRNAs) and 79 different proteins. The mammalian genes encoding the rRNAs are multicopy and clustered at a few loci, whereas those encoding the ribosomal proteins (rp genes) are single copy and scattered throughout the genome . In addiCoordinated expression of the rp genes at transcriptional and post-transcriptional levels is required to ensure a roughly equimolar accumulation of ribosomal proteins. Transcriptional run-on measurements with nuclei of rapidly proliferating cells indicated equivalent loading of RNA polymerases on three unlinked mouse rp genes , consistDespite the obvious importance of rp gene expression for cell viability, there have been very few experimental studies of rp promoter architecture and transcriptional regulation in higher eukaryotes. To date, efforts to identify functionally relevant cis-acting regulatory elements and transcription factor binding sites have been made for only 9 mammalian ,10-23 anBecause the previous experimental studies were limited to a small subset of rp genes, it is important to know whether the results of those studies can be used to predict special features of promoter architecture that characterize the entire set of rp genes or that could be used to sort the rp genes into classes with similar features. This applies not only to transcription factor-binding sites, but also to the polypyrimidine tract that spans the tsp. As an initiator element, this tract is atypical because the tsp of initiators is normally an adenine residue, flanked on both sides by pyrimidines .To address these issues, I have applied the principle of "phylogenetic footprinting", which holds that important regulatory sequences will have a tendency to be evolutionarily conserved and thus revealed by a sequence comparison of corresponding regions of orthologous genes . Some reTo make a study of the evolutionarily conserved features of ribosomal protein gene promoters, I first had to assemble a set of orthologous human and mouse rp gene sequences, in which the transcription start sites are reasonably well defined. For all of the human rp genes, annotated sequences are now available owing to the studies of Kato, Kenmochi and coworkers ,33, who In contrast to the situation with the human rp genes, accurate determinations of the tsp's of mouse rp genes have been made in only a few cases, and therefore the annotations of rp genes in the mouse genome databases are generally incomplete. To overcome this problem, I used the mVISTA alignment program  to comparpS16, rpL30 and rpL32 ). These genes were selected because the mouse orthologues have been previously studied extensively in my laboratory. Scans with a 50 bp window indicated that the conserved (> 75 % identity) flanking sequences are largely confined to regions within a few hundred base pairs of the tsp. There was very little alignment of the upstream region sequences and many gaps caused by the presence of numerous insertion elements. With this analysis, I detected only one short block of conserved sequence at -2 kb in rpL30 (77 % identity over 84 bp), one short block at -1.5 kb in rpL32 (82 % identity over 60 bp), and none in rpS16 . Neither of the conserved blocks contained any recognizable transcription factor binding sites or other remarkable characteristics. The lack of any credible long-range regulatory elements is consistent with earlier conclusions based on transient transfection experiments with these genes [In a preliminary analysis, I used the mVISTA sequence alignment program to examine six to ten kb of 5' flanking sequence of three pairs of orthologus rp genes  in the 5' flanks of a very high proportion of the rp genes see . Indeed,To evaluate the quality of TATA box motifs, I established criteria based on rules derived from a crystallographic analysis of TBP-DNA complexes . With thEmploying stringent criteria Figure , I used rpL13a (panel d) and within exon 2 of rpL30 (panel b), whereas in rpS18 (panel a) and rpS4 (panel c), it is at the extreme 3' end of exon 1. Many rp genes have this latter feature, which may be relevant to the evolution of vertebrate rp promoters (see below).Illustrative examples of four annotated promoter sequence comparisons are shown in Figure rpS18 promoter contains a conserved TATA (+) motif, while the TATA motifs of rpS4 and rpL13a were scored as (+/-) and that of rpL30 as (-). Well-conserved  consensus binding sites for GABP and/or Sp1 are located upstream of the tsp in all four examples; downstream YY1 sites are evident in rpL30 and rpL13a . The rpS18 and rpL13a promoters also contain conserved consensus binding sites for the ubiquitous AP1/ATF factors. The promoter elements that were found to have functional significance in experimental studies of the mouse rpL30 gene [rpL30 and rpL13a .The L30 gene  are all rpL29 ). In 29 % of the rp genes the AUG codon is at the extreme 3' end of exon 1 and in 47 % it is in exon 2. Thus, in 3/4 of the rp genes, the genetic elements involved in transcriptional/translational regulation are spatially separated from those with protein encoding function.The results of this type of analysis for the full set of rp promoters are tabulated in Table Contrary to previous impressions based on an incomplete set of rp genes, the rp promoters cannot generally be classified as "TATA-less". Thirty-five percent of the promoters contain, in the -25 region, a TATA box that should theoretically bind TBP with high affinity. An additional 25 % have an A/T-rich tract in this region, which might bind TBP with lower affinity, and the remainder would not be predicted to bind TBP without help from other proteins. For the most part, the TATA box quality is well conserved between the two mammalian species: the assessed quality of human and mouse TATA motifs was the same in 82 % of the rp promoters.The prevalence in rp promoters of evolutionarily conserved GABP, Sp1 and YY1 binding sites is readily apparent from these results. Conserved upstream GABP and Sp1 sites are present in 54 % and 48 % of the rp promoters, respectively. Conserved downstream YY1 sites are present in 52 % of the rp promoters. The ratio of aligned to unaligned sites in the human rp promoters is approximately 2:1 for GABP, 1:1 for Sp1 and 4:1 for YY1. The occurrence of unaligned sites in the mouse rp promoters is similar to that in the human rp promoters see . It is n2C+1TY(T)2(Y)3. A striking result is that T's are strongly preferred over C's at positions +2, +4 and +5. This preference, which is graphically illustrated in Figure The existence of a novel polypyrimidine initiator sequence in which the conventional A residue at the tsp is replaced by a C residue is well known. Moreover, the roles of this sequence in rp gene transcription and rp mRNA translation have been previously demonstrated experimentally for a few rp genes ,40-42. WrpS3  for mammals and lower vertebrates. Yet, the particular C residue within the initiator that is used as the major tsp may differ slightly. Also the adjudged quality of the TATA box for TBP binding is not always the same. Nevertheless, except for the location of upstream transcription factor-binding sites, the general features of rp promoter architecture are usually well conserved over large evolutionary distances.To determine the features of rp promoter architecture that are conserved over large evolutionary periods, I compared the promoter sequences of five chicken, six amphibian and five fish rp genes to their mammalian orthologues using the Clustal W multiple sequence alignment program. An example of such an alignment for 3 Figure  shows st3 Figure  indicategas5 and U17HG ). Annotated aligned sequences of the nine human and mouse non-rp TOP genes are presented on pages 1 through 9 of a supplementary file [see It is of interest to know which features of rp promoter architecture are specific and which might be common to other ubiquitously expressed genes. To this end, I examined the promoters of two additional sets of genes. One set consisted of non-rp genes that also produce translationally controlled mRNAs with 5'-terminal oligopyrimidine tracts (non-rp TOP genes). At the present, there are nine known genes in this category for which the tsp of at least one orthologue has been experimentally determined see . The nonfile see  and the There are some notable differences in the promoter architecture of rp genes and non-rp TOP genes. First, GABP- and YY1-binding motifs, which are prevalent in the rp promoters, are rarely found in the non-rp TOP promoters Table . Second,The second set of ubiquitously expressed genes consisted of 20 housekeeping genes randomly selected from the eukaryotic promoter database see . The proThe foregoing analysis of 79 mammalian ribosomal protein genes has revealed several features of rp promoter architecture, some of which are largely conserved over long periods of vertebrate evolution (about 450 million years) and others that are strongly conserved only in mammals. (about 90 million years). One highly conserved feature, present in over 3/4 of the rp genes, is the separation by an intron of the sequences involved in transcriptional and/or translational regulation from the sequences with protein-encoding function. In 47 % of the rp genes, the AUG translation initiation codon is in exon 2, and in 29 % of the genes, it is at the extreme 3' end of exon 1. It would seem that at an early stage of vertebrate evolution, these regulatory sequences were appended as discrete units to the loci containing the protein-encoding sequences.2C+1TY(T)2(Y)3. Thus, in addition to the C at the tsp, there is a clear preference for T over C at positions +2, +4, and +5. This preference may reflect a structural bias for transcription, e.g., ease of strand separation, or for translation, e.g., affinity of rp mRNAs for a putative repressor.The polypyrimidine tract that spans the tsp is present in all vertebrate rp genes. This tract can function as a transcriptional initiator ,41 and aThe presence of transposable element residues in the 5' flanks of the rp genes is noteworthy. Half of the rp genes contain an element  within 500 bp of the tsp, and over 80 % of the genes contain an element within 900 bp. Some elements are moderately conserved between mouse and human, but most appear to be lineage-specific. These elements are unlikely to have any specific role in rp promoter function. They may be passively tolerated because the vast majority of conserved 5' sequence is confined to within 200 bp of the tsp. Within a segment from -200 to +100, the sequence identity between human and mouse rp orthologues ranges from 50 to 75 % with an average of 61 %, whereas the sequence match beyond -200 is of borderline significance.rpL32 , might bind TBP weakly without help from an additional protein [The observation that 35 % of the rp promoters contain a TATA box motif at -25, which would be predicted to bind TBP with high affinity, and that an additional 25 % possess A/T-rich motifs, which might bind TBP with lower affinity, was unexpected. The assessed quality of these motifs for TBP binding, made according to rules established by a detailed structural study of TBP-DNA complexes , was lar protein ,44, so tWhen human and mouse rp promoter regions from -200 to +100 were scanned for conserved  transcription factor-binding sites with the rVISTA program , which irpL30 and rpL32 genes [rpS14 and rpS6 genes [Xenopus rpL18 gene [rpS16 gene, GABP binding at the initiator decreases transcription activity both in vivo and in vitro [GABP is a heteromeric protein consisting of an ETS family member, which has DNA-binding capability, and an ankyrin repeat-containing subunit, which greatly improves the stability of GABP-DNA interactions ,46. Two-32 genes ,14, the S6 genes ,22, and L18 gene . The GABin vitro . For simrpL32 [rpL30 , a downstream YY1 interaction also contributed positively to transcriptional activity, but only when a strong upstream interaction with GABP was eliminated [Xenopus rpL18 was shown to interact with the frog homologue of YY1, but the functional significance of this interaction was not demonstrable with the reporter constructs that were used in these experiments [YY1 is a zinc finger-containing protein with a variety of gene-specific functions, including transcriptional activation and repression, positioning of RNA polymerase II, and chromatin modification ,48. FiftrpL32 . In mousiminated . Intereseriments . The diveriments , have leBased on the results of the analysis of rp promoter sequences, I have sorted the different rp promoters into eight classes according to whether they possess conserved binding sites for the three prevalent transcription factors, GABP, Sp1 and YY1 Figure . Only 10RACK1 gene is embedded in a polypyrimidine tract that conforms perfectly to the rp initiator consensus sequence. Moreover, the promoter contains a TATA box of +/- quality, an aligned upstream binding motif for Sp1 and an aligned downstream motif for YY1, but no readily detectable motifs for other ubiquitous transcription factors. Interestingly, the features of this promoter resemble those of the rpL13a gene, which also encodes a protein with apparent pleiotropic function.It is worth noting that some ribosomal proteins can also have extraribosomal functions ,51. AmonThis analysis has highlighted features of rp promoter architecture that are shared by a high proportion of the rp genes. The evolutionary conservation of these features lends strong support to their functional relevance. Yet, superimposed on this general design are variations that confer certain idiosyncratic characteristics on each promoter. There does not seem to be a single master switch that co-regulates all rp genes at the transcriptional level. Rather, the rp promoters are tuned to respond to a combination of factors, including components of the general transcription machinery, a relatively small group of sequence-specific transcription factors, and modifiers of chromatin structure. The inherent functional redundancy and lack of dependence on any single factor are useful design features for genes that must be expressed in a broad spectrum of cell types and environmental situations.2C+1TY(T)2(Y)3. Contrary to previous impressions based on studies of a small subset of rp genes, the majority of rp promoters contain a TATA box or an A/T-rich motif at -25 that should theoretically have TBP-binding capability. Similarly, approximately half of the rp promoters contain conserved binding motifs for transcription factors previously implicated in rp gene expression, namely upstream GABP and Sp1 sites and downstream YY1 sites. Conserved motifs for other ubiquitous factors occurred much less frequently. Transposable element residues within 900 bp of 5'-flanking sequence were present in over 80 % of the rp genes; very little sequence conservation was evident more than 200 bp upstream of the tsp. Some of these architectural features were seen to be specific for rp promoters. From the results of this analysis, it was possible to sort the rp promoters into eight classes according to their possession of putative binding sites for GABP, Sp1 and YY1, and also to specify which promoters should have intrinsic affinity for TBP. This classification might prove useful for interpreting the results of experiments in which cell-specific or physiologically induced variations in the expression of different subsets of rp genes are observed.A sequence comparison of the promoter regions of all 79 orthologous human and mouse ribosomal protein genes has revealed several evolutionarily conserved features that are characteristic of a high proportion of the rp gene set. One such feature, which is also evident in the rp genes of lower vertebrates, is the separation by an intron of the sequences involved in transcriptional and translational regulation from the sequences with protein encoding function. Another conserved feature is the polypyrimidine initiator, which in mammals conforms to the consensus (Y)The rp gene sequences were extracted from three database sources see . The vasrRNA, ribosomal RNA; rp, ribosomal protein; tsp, transcriptional start point; TOP, terminal oligopyrimidine sequence; TBP, TATA-binding protein.ST1: Identification of the transcriptional start points (tsp's). Comparison of experimental determinations of the tsp's of human and mouse rp genes.Click here for fileST2: Promoter region sequences of rp genes. Sources of rp sequences, amount of analyzed sequence 5' of tsp, and locations of insertion elements nearest the tsp.Click here for fileAligned promoter sequences of rp genes. Annotated sequence alignments of 79 orthologous human and mouse rp promoters and a key to the annotation.Click here for fileST3: Characterization of the rp initiator. Occupancy matrix for determination of the consensus sequence of the mammalian rp initiator.Click here for fileST4: Promoter region sequences of non-rp TOP genes. Sources of non-rp TOP gene sequences, the amount of analyzed sequence 5' of the tsp, and location of insertion elements nearest the tsp.Click here for fileAligned promoter sequences of non-rp TOP genes. Annotated sequence alignments of 9 orthologous human and mouse non-rp TOP promoters.Click here for fileST5: Housekeeping gene promoters. List of 20 housekeeping gene sequences extracted from the Eukaryotic Promoter Database, which were analyzed for GABP, Sp1 and YY1 binding sites and for TATA box quality.Click here for file


The non-invasive nature of laser biostimulation has made lasers an attractive alternative in Medical Acupuncture at the last 25 years. However, there is still an uncertainty as to whether they work or their effect is just placebo. Although a plethora of scientific papers published about the topic showing positive clinical results, there is still a lack of objective scientific proofs about the biostimulation effect of lasers in Medical Acupuncture. The objective of this work was to design and build a low cost portable laser device for stimulation of acupuncture points, considered here as small localized biosources (SLB), without stimulating any sensory nerves via shock or heat and to find out a suitable method for objectively evaluating its stimulating effect. The design is aimed for studying SLB potentials provoked by laser stimulus, in search for objective proofs of the biostimulation effect of lasers used in Medical Acupuncture.The proposed biostimulator features two operational modes: program mode and stimulation mode and two output polarization modes: linearly and circularly polarized laser emission. In program mode, different user-defined stimulation protocols can be created and memorized. The laser output can be either continuous or pulse modulated. Each stimulation session consists of a pre-defined number of successive continuous or square pulse modulated sequences of laser emission. The variable parameters of the laser output are: average output power, pulse width, pulse period, and continuous or pulsed sequence duration and repetition period. In stimulation mode the stimulus is automatically applied according to the pre-programmed protocol. The laser source is 30 mW AlGaInP laser diode with an emission wavelength of 685 nm, driven by a highly integrated driver. The optical system designed for beam collimation and polarization change uses single collimating lens with large numerical aperture, linear polarizer and a quarter-wave retardation plate. The proposed method for testing the device efficiency employs a biofeedback from the subject by recording the biopotentials evoked by the laser stimulus at related distant SLB sites. Therefore measuring of SLB biopotentials caused by the stimulus would indicate that a biopotential has been evoked at the irradiated site and has propagated to the measurement sites, rather than being caused by local changes of the electrical skin conductivity.A prototype device was built according to the proposed design using relatively inexpensive and commercially available components. The laser output can be pulse modulated from 0.1 to 1000 Hz with a duty factor from 10 to 90 %. The average output power density can be adjusted in the range 24 – 480 mW/cm2, where the total irradiation is limited to 2 Joule per stimulation session. The device is controlled by an 8-bit RISC Flash microcontroller with internal RAM and EEPROM memory, which allows for a wide range of different stimulation protocols to be implemented and memorized. The integrated laser diode driver with its onboard light power control loop provides safe and consistent laser modulation. The prototype was tested on the right Tri-Heater (TH) acupuncture meridian according to the proposed method. Laser evoked potentials were recorded from most of the easily accessible SLB along the meridian under study. They appear like periodical spikes with a repetition rate from 0.05 to 10 Hz and amplitude range 0.1 – 1 mV.The prototype's specifications were found to be better or comparable to those of other existing devices. It features low component count, small size and low power consumption. Because of the low power levels used the possibility of sensory nerve stimulation via the phenomenon of shock or heat is excluded. Thus senseless optical stimulation is achieved. The optical system presented offers simple and cost effective way for beam collimation and polarization change. The novel method proposed for testing the device efficiency allows for objectively recording of SLB potentials evoked by laser stimulus. Based on the biopotential records obtained with this method, a scientifically based conclusion can be drawn about the effectiveness of the commercially available devices for low-level laser therapy used in Medical Acupuncture. The prototype tests showed that with the biostimulator presented, SLB could be effectively stimulated at low power levels. However more studies are needed to derive a general conclusion about the SLB biostimulation mechanism of lasers and their most effective power and optical settings. Nowadays lasers are widely used in therapy and diagnostics. They have been adapted to many medical procedures ranging from surgery, oncology, physiotherapy, dentistry, dermatology and biostimulation. The non-invasive nature of laser biostimulation have made lasers an attractive alternative in Medical Acupuncture at the last 25 years. Unfortunately, there is still an uncertainty as to whether they work or their effect is just placebo. Although a plethora of scientific papers published about the topic showing positive clinical results, there is still a lack of objective scientific proofs about the biostimulation effect of lasers in Medical Acupuncture.The properties of acupuncture points, considered here as small localized biosources (SLB), have been extensively studied over the past 50 years. Research has shown SLB to be small area body regions, which exhibit unique, electrical, physiological and anatomical properties . They are considered to form groups, each group being arranged along a line, called meridian and related to an internal organ -4. SLBs The objective of this work was to design and build a low cost portable laser device for effectively stimulation of SLB without exciting sensory nerves, and to find out a suitable method for objectively evaluating its efficiency. The attempt to define the optimal device parameters was based on the SLB properties, data about existing devices for low level laser therapy and on preliminary measurements performed in our laboratory. The latter suggest that the effect of SLB stimulation is also dependent on the polarization of the coherent emission in addition to its intensity, wavelength and modulation frequency. Therefore the device should provide a polarization adjustment, wide range of modulation frequencies, precise power settings and to have minimum size and cost.The block diagram of the proposed biostimulator design is shown in Fig. The variable parameters of the laser output are: average output power, pulse width, pulse period, and sequence duration and repetition period. After each input parameter is selected, the total energy that would be delivered at the end of the stimulation session is automatically calculated and displayed. When defining the stimulation protocol, the software program reads the selected parameter value and automatically re-calculates the possible set of the other parameters, so that the user could not select inconsistent values or ones that would result in a total energy delivered that exceeds a certain safety limit. In stimulation mode the laser stimulus is applied according to the pre-programmed protocol.θ between the electric field vector of the incident linearly polarized beam and the quarter-wave retarder principal plane is +45 degrees, the emergent beam is circularly polarized as shown in Fig. θ to -45 degrees reverses the sense of the circular polarization. The output from a single cavity laser diode is mainly linearly polarized, parallel to the laser junction. Although, spontaneous emission with a random polarization and with a polarization perpendicular to the laser junction is also present. For a diode operating near its maximum power the polarization ratio is typically greater than 100:1 but when operating near the threshold point, the ratio is considerably lower as spontaneous emission becomes more significant. Therefore a collimating lens followed by a linear polarizer is used since the retarder requires linearly polarized and normally incident light upon its plate over the whole power range. A single collimating lens, with good anti-reflection coating and large numerical aperture to efficiently capture the widely divergent perpendicular axis of the laser diode, is the most efficient and cost effective solution for the current application. Polarizers typically utilize birefringence, dichroism, optical activity, and polarization by reflection or by a metallic thin film [A quarter-wave retardation plate realizes the laser output polarization change, as shown in Fig. The application can tolerate an elliptical beam shape and waveform aberrations, so circularization of the laser beam or correction of the waveform aberrations is not required.The best way for testing the device efficiency is to obtain a biofeedback from the site of stimulation. A suitable non-invasive method is to measure the surface biopotential of the irradiated SLB site. However simultaneous stimulation and biopotential recording from a single SLB is technically difficult and inadequate, since the record may contain sham potentials due to local changes of the electrical conductivity of the irradiated skin. So a new method is proposed to avoid this problem. The method uses separate stimulation and measurement sites. Thus the laser stimulus is applied at SLB situated at the beginning of the meridian (e.g. the first point), where biopotential records are obtained from all the other easily accessible SLB lying distantly on the same meridian see Fig. . TherefoThe schematic of the practical biostimulator circuit, built according to the proposed design is shown in Fig. • RA0-RA2 – outputs used as LCD control signals• RA3-RA4 – outputs, used as DAC control signals• RB0-RB3 – either inputs or outputs, shared between the input control buttons B1-B4 and the LCD data bus DB4-DB7• RB4-RB6 – outputs, used as control signals for the laser diode driverThe LCD is implemented with the dot matrix alphanumeric character module U4 (Seiko Instruments L1682). It features low power consumption, high contrast, wide viewing angle, on-board controller and LSI driver (Samsung S6A0069). All functions required for the LCD drive are internally provided on the chip. Its internal operation is determined by signals sent from the microcontroller. These signals include:• Register select – RS• Read/Write – R/W• Data bus – DB4-DB7 (configured as inputs)• Read/Write Enable – EWhen ports RB0-RB3 are configured as inputs, the LCD data inputs DB4-DB7 have no practical influence on the logic levels set by push buttons B1-B4. LCD operation is also not affected since DB4-DB7 content is read only on logic high at E (U4-pin 6), set by the microcontroller . When pobias and the active power levels of the laser are set [bias level input (U1-pin 14) and at the active level input (U1-pin 3). These inputs drive track and hold amplifiers with hold capacitors C4 and C5. The input voltage range on both inputs ranges from Vref to Vref + 1.6 V, requiring an offset of Vref to be created for common based signals. The bias level is chosen to be equal to Vref, where the active level is determined by the circuit realized with op-amp U5. It performs the level shift and scales the DAC output from Vref to Vref + 1.6 V. This solution is attractive because both DAC and op-amp can run off a single 5 V supply, and the op-amp does not have to swing rail-to-rail. The op-amp U5 output voltage level is given by:The laser diode driver is implemented with the highly integrated circuit U1 , which combines a very fast output current switch with onboard analog light power control loops. It gets feedback current from the laser diode built-in photo detector (U1-pin 8), feeds it to a transimpedance amplifier (TZA) and then to two analog feedback loops where the  are set . The twobias control loops is periodically calibrated via U1-pin 15, where the active control loop is continuously calibrated via U1-pin 1. Resistors R3 and R4 are used to avoid floating of inputs U1-pin 15 and U1-pin 2 when microcontroller ports are in a high impedance mode. The laser pulse modulation is done by switching between the bias and the active power levels according to the logic level at U1-pin 2, where logic high turns the modulation current on. The gain resistor R5 matches the feedback loop transfer function to the laser/photo diode D1. Capacitor C6 optimizes the TZA response, with larger values to slow TZA response. Lower values increase TZA bandwidth but may cause oscillations. When input U1-pin 16 is logic high, the onboard disable circuit turns off the output drivers and returns the light power control loops to a safe state. It is used during initial power up of U1 and when the laser is inactive. In case that input U1-pin 16 floats (after POR or other reset conditions) the driver is disabled. When U1 is re-enabled the control loops are recalibrated.Since the monitor current is proportional to the laser diode light power, the feedback loops effectively control the laser power to a level proportional to the analog inputs. The dd as reference (U3-pin 8) filtered by R12 and C9. The DAC's full-scale output voltage ranges from 0 to Vdd. Special attention was paid to the PCB layout design to minimize the crosstalk between analog inputs and digital outputs.The DAC is implemented with the single 8-bit voltage output MAX517 (U3). It is controlled by the microcontroller via 2-wire serial interface , operates from a single power supply and swings rail-to-rail. POR ensures the DAC output is at zero volts when power is initially applied. It uses the power supply V2), optimized for 670 nm. The polarizer used is a linear polarizing film produced by aligning long chain polymers, which is then laminated in cellulose acetate butyrate (CAB) for durability and stiffness [The drawing of the practical optical system assembly is shown in Fig. tiffness . The quaA prototype device was built according to the proposed design using inexpensive and commercially available components. Its optical and electrical characteristics are given in Table The biostimulator was tested on the right Tri-Heater (TH) meridian of ten subjects according to the proposed method see Fig. . The lasThe noise present in the signals is mainly composed of electromyographic signals and noise from the electrode-skin interface. The first two signal records (TH-8 and control) contain ECG artifacts and additional electromyographic noise since those two electrodes were positioned relatively distant from the reference electrode. The frequency bandwidth was limited to 200 Hz by sixth order low-pass Bessel filter and the signals were sampled with 1 kHz.Recording to the EU regulation  this device falls under class IIb in order to obtain the CE mark. The biostimulator prototype was categorized as Class IIIb laser product according to the International Standard for the Safety of Medical Laser Products IEC 601-2-22, as stated in Table The best solution for building a compact hand held biostimulator would be to design a custom made integrated circuit, but the cost would be much higher. We found a good alternative in using surface mount technology (SMT), commercially available integrated laser diode driver and a RISC Flash microcontroller. This solution resulted in a reduction in parts, size and power consumption. The proposed method for testing the device efficiency is very sensitive to precise electrode and stimulus positioning. Even a deviation of 3 mm from the exact SLB location may prevent the recording electrode from capturing signals from the source. The same deviation of the stimulus position also results of ineffective excitation of the targeted SLB and thus no SLB evoked potentials can be recorded. The method is also susceptible to the electrode-skin pressure, but not only due to its strong influence on the contact impedance. It was observed that the excessive electrode-skin pressure led to diminishing or even disappearing of the SLB signal, although the contact impedance was lower. This is most probably due to the pressure exerted on the SLB source that may affect the signal generation or transduction. Alternatively insufficient electrode-skin pressure led to excessive contact impedance and noise from the electrode-skin interface. The preliminary results suggest that a circularly polarized laser emission is most effective when used on the so-called Yang acupuncture meridians but not on Yin types. However more studies are needed to validate or disprove this observation.The specifications of the prototype, built according to the proposed design, were found to be better or comparable to those of other existing devices. It features small size and low component count and power consumption. Because of the low power levels used the possibility of sensory nerve stimulation via the phenomenon of shock or heat is excluded. Thus senseless optical stimulation is achieved. The optical system presented offers simple and cost effective way for beam collimation and polarization change. The novel method proposed for testing the device efficiency allows for objectively recording of SLB potentials evoked by laser stimulus. Based on the biopotential records obtained with this method, a scientifically based conclusion can be drawn about the effectiveness of the commercially available devices for low level laser therapy used in Medical Acupuncture. The prototype tests showed that with the biostimulator presented, SLB could be effectively stimulated at low power levels. However more studies are needed to derive a general conclusion about the biostimulation mechanism of lasers in Medical Acupuncture and their most effective power and optical settings.The authors contributed equally to this work


RSV is recognized as the most important cause of serious lower respiratory tract illness in infants and young children worldwide leading to hospitalisation in a great number of cases, especially in certain high-risk groups. The aims of the present study were to identify risk groups, outcome and incidences of hospitalisation for RSV bronchiolitis in Norwegian children under two years of age and to compare the results with other studies.We performed a population-based retrospective survey for the period 1993–2000 in children under two years of age hospitalised for RSV bronchiolitis.822 admissions from 764 patients were identified, 93% had one hospitalisation, while 7% had two or more hospitalisations. Mean annual hospitalisation incidences were 21.7 per 1.000 children under one year of age, 6.8 per 1.000 children at 1–2 years of age and 14.1 per 1.000 children under two years of age. 77 children (85 admissions) belonged to one or more high-risk groups such as preterm birth, trisomy 21 and congenital heart disease. For preterm children under one year of age, at 1–2 years of age and under two years of age hospitalisation incidences per 1.000 children were 23.5, 8.7 and 16.2 respectively. The incidence for children under two years of age with trisomy 21 was 153.8 per 1.000 children.While the overall hospitalisation incidences and outcome of RSV bronchiolitis were in agreement with other studies, hospitalisation incidences for preterm children were lower than in many other studies. Age on admission for preterm children, when corrected for prematurity, was comparable to low-risk children. Length of hospitalisation and morbidity was high in both preterm children, children with a congenital heart disease and in children with trisomy 21, the last group being at particular high risk for severe disease. Respiratory tract infections due to respiratory syncytial virus (RSV) are very common in young children worldwide. In temperate climates the infection occurs in yearly winter epidemics, and by two years of age most children have been infected . ReinfecLocal epidemiological studies are important when considering new strategies for the prevention of RSV bronchiolitis. An example of such a strategy is the monthly injection of the humanised murine monoclonal antibody palivizumab to certain high-risk groups, which in many studies has shown a significant reduction in the incidence of hospitalisation for RSV bronchiolitis .The aims of the present study are to identify risk groups, outcome and incidences of hospitalisation for RSV bronchiolitis in Norwegian children under two years of age, and to compare these results with other studies.. The number of children under two years of age born before completed 37 weeks of gestation in the service area during the study period and still alive after the neonatal period was identified from the Norwegian Medical Birth Registry . All children with trisomy 21 have a program for follow-up from birth in our hospital and the number of children under two years of age with trisomy 21 was therefore identified from in-patient and out-patient hospital records. The number of children in the general population with a CHD was not known in the present study and hospitalisation incidences could therefore not be estimated. Overall mortality after the neonatal period as well as the number of children moving out of the service area during the study period were low and have not been corrected for.Akershus is a large suburban and countryside region located around the capital of Oslo with approximately 10% of the total population in Norway. The Paediatric Department at Akershus University Hospital (Ahus) is the only hospital for children under 15 years of age living in the 20 northern, southern and eastern communities of the region and serves all kinds of hospital services, except for paediatric surgery. Deliveries have been rather stable at approximately 3.500 per year in the service area during the last decade. Due to a local agreement infants from approximately 500 unselected term deliveries from our service area are born at Rikshospitalet in Oslo each year, but these infants are admitted to our hospital in case of disease during childhood. The number of children under two years of age living in the service area at any time during the follow-up period were identified from official Norwegian Population Statistics th 1993 to January 31th 2000 and diagnosed as positive with RSV in nasopharyngeal aspirate (NPA). RSV was diagnosed by an enzyme-linked immunosorbent assay (ELISA) for RSV (Abbott TestPack RSV). The test is simple and easy to perform giving a result within 30 minutes. The sensitivity, specificity, positive and negative predictive value is stated to be 74–92%, 86–100%, 81% and 93% respectively [The present study is a population-based retrospective survey on children under two years of age admitted to the hospital with a diagnosis of bronchiolitis ICD 9 & 10) during the period February 5 & 10 dur764 children who fulfilled the diagnostic criteria were identified. A total of 77 children belonged to one or more well known high-risk groups. Of these, 58 children had been born before completed 37 weeks of gestation, some with additional diseases such as CLD (12 children) and CHD (four children). 12 children had a diagnosis of CHD as the only additional disease and seven children had trisomy 21, four of these also diagnosed with CHD. These 77 children were regarded as a high-risk group for subgroup analysis. No children with other well-known risk-factors  were identified. The remaining 687 children were born at term and healthy and regarded as a low-risk group for subgroup analysis.Data were analysed with the statistical programme SPSS, version 11. For analysis of gender we used the Chi square test and for comparisons between low-risk and high-risk groups we used the independent sample T-test. A p-value <0.05 was used as limit for statistical significance.During this seven years follow-up period 764 children (822 admissions) were hospitalised for RSV bronchiolitis. 93% (707 children) had only one hospitalisation and 7% (57 children) had two or more hospitalisations for RSV bronchiolitis during the first two years of life. The highest number of admissions was recorded during the winter months December-April, but cases were also found in late spring and early autumn. Both the number of admissions and peak-time varied between seasons but with no specific pattern of variability figure . A signiThe majority of children  were hospitalised within the first year of life with children less than six months old being responsible for 45% of all admissions. Median age at hospitalisation was 6 months (range 0–23 months) and a median length of stay of 4 days range 1–41 days) was observed (table –41 days During the study period nine children (1.2%) developed severe respiratory distress and needed mechanical ventilation and two of them (0.3%) died. Four of these children had no known risk-factors for severe disease. However, the two children who died were both considered as high-risk patients, one of them was diagnosed with CHD and the other with trisomy 21 as well as CHD.Of all RSV hospitalisations during the first two years of life 58 children (64 admissions) were born before completed 37 weeks of gestation with a median gestational age of 30 weeks (range 24–36 weeks). A significant male predominance of 68% was also observed in this group (P = 0.006). Median age on admission, after postnatal age, was 8 months (range 0–23 months). When corrected for prematurity (corrected age), median age on admission was 5.4 months (range -2.5–21.3 months). Also, a median length of hospitalisation of 8 days (range 2–27 days) was observed (table Seven children (eight admissions) diagnosed with trisomy 21 were hospitalised for RSV bronchiolitis during this follow-up study. Four of them also had a CHD. Two children needed mechanical ventilation during hospitalisation and one of them subsequently died.A median age on admission of 9.0 months (range 1–20 months) and a median stay of 7.5 days (range 2–34 days) were recorded for children in this particular risk group  and median stay of 6.0 days (range 2–14 days) was observed  were hospitalised for RSV bronchiolitis, giving a hospitalisation incidence under two years of 153.8 per 1.000 children.In this retrospective study, as in many other comparable epidemiological studies, we found that RSV bronchiolitis appears as an annual winter epidemic with relatively high hospitalisation incidences, predominance of boys, young age, short length of stay, few complications and low mortality . The ratSeveral international studies have shown an increased incidence of RSV-related hospitalisations over the last two decades. A Norwegian study for the period 1972–78 showed a mean hospitalisation incidence of 9.5 per 1.000 children for children under one year of age compared to our much higher incidence of 21.7 per 1.000 children for the period 1993–2000 in the same age group . Also, fOur study showed that only 1.2% of the children in the study population needed mechanical ventilation. This result is lower than in many other studies . On the Another observation in our study was that among the mechanically ventilated children 44% had no underlying risk-factors for severe disease. Further research into why some otherwise healthy children have a severe course of RSV bronchiolitis is therefore very important, not least when trying to define risk groups even better . ResearcFor the population at large our study showed, as in other studies, a low mortality. A total of two children (0.3%) died, one had trisomy 21 with an atrioventricular septum defect (AVSD) and the other had an underlying CHD.The number of high-risk children in our study is relatively small and the results should be interpreted with caution. The mean hospitalisation incidences for preterm children were lower than in many other studies, even though 20% of the preterm children suffered from CLD as well and had a low median weeks of gestational age (wGA) of 30. One possible explanation is that the population data for this particular risk group was not corrected for migration out of our service area and mortality after the neonatal period. However, it is unlikely that such corrections would significantly influence the results. Another explanation for the low incidence among preterm infants might be that most of them are hospitalised up to almost 40 wGA and are discharged only when the child has reached a weight of more than 2.000 g and have normal oral feedings. This means that they are more or less completely protected from RSV for a long period after birth. Our routines with focus on good parental information on how to protect the newborn infant from RSV after discharge from hospital might also be important. Median length of hospitalisation for preterm children was in our study significantly longer when compared to the low-risk children and is in accordance with results from many other larger studies.Children with CHD are a wellknown risk-group for severe RSV disease. Children with trisomy 21 with or without a concomitant CHD should also be considered a high-risk group. Our study showed a mean hospitalisation incidence per 1.000 children under two years of age with trisomy 21 more than 11 times the whole study population. Even more important, out of a total of seven children with trisomy 21, two needed mechanical ventilation and one child died. We would, however, recommend a larger study to meet these observations for children with trisomy 21.One of the findings in our study was the large proportion of otherwise healthy children needing mechanical ventilation. Our numbers are small, but when new policies to prevent severe RSV bronchiolitis are discussed this aspect should also be considered. The research for improved identification of risk-groups and development of an effective vaccine for immunization should therefore be given high priority.While the overall hospitalisation incidences and outcome of RSV bronchiolitis were in agreement with other studies, hospitalisation incidences for preterm children were lower than in many other studies. Age on admission for preterm children, when corrected for prematurity, was comparable to low-risk children. Length of hospitalisation and morbidity was high in both preterm children, children with a congenital heart disease and in children with trisomy 21, the last group being at particular high risk for severe disease.The author(s) declare that they have no competing interests.Our individual contributions to the study have been as following: HOF had primary responsibility for protocol development, outcome assessment, data acquisition and analysis and writing of the manuscript. TF participated in the development of the protocol and analytic framework of the study, and contributed to the writing of the manuscript. DB participated in the data analyses and the writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: