Now, available data show that re-treating patients with other tyrosine kinase inhibitors after they progress on sunitinib or sorafenib, or both, is beneficial. A large phase iii trial recently showed that, as compared with placebo, treatment with everolimus, an inhibitor of the mammalian target of rapamycin (mtor), almost halved the risk of progression (37% vs. 65%) and doubled the median progression-free survival (4 months vs. 2 months). Overall survival was not improved in that study, likely reflecting treatment crossover in the placebo arm, but these data position everolimus as the second-line standard of care. A consistent and growing body of literature also suggests that re-treatment with other kinase inhibitors that the patient has not previously encountered is a reasonable option. Outcomes of initial treatment with sunitinib or sorafenib (or both) should not deter the use of second-line targeted therapy, because the first-line use of targeted agents does not appear to be predictive of outcomes with second-line therapy. However, in view of poor absolute outcomes after second-line treatment and the benefits seen with rationally developed targeted agents in the first-line setting, enrolment of second- and subsequent-line patients in further trials would be preferable.The development of molecularly targeted agents that inhibit pathways critical to the development of renal cell carcinoma has significantly improved outcomes in patients with these cancers. Compelling scientific and phase  In addition to effects on tumour proliferation, angiogenesis, and apoptosis, activation of mtor may also potentiate the activity of hiftor results in tumour inhibition in vitroin vivoLoss of the von Hippel–Lindau  pathway is also important to renal oncogenesis. Expression of egfr occurs at high frequency in rcc (70%–90%) vhl causes increased expression of transforming growth factor α hif-2α–dependent mechanisms vhl–/– rcc cell lines Interestingly, the epithelial growth factor receptor : 0.42; 95% confidence interval (ci): 0.32 to 0.54; p < 0.001), an improved objective response rate , and improved quality of life. Similar benefits were also seen in patients who had received prior cytokine therapy pfs  and the disease control rate . Quality of life was also improved in patients receiving sorafenib as compared with those receiving placebo egfr inhibitors failed to deliver substantial therapeutic benefit, either as single agents Treatment with a number of targeted agents i is now 2.3Until recently, little evidence was available to guide therapy once patients had progressed on first-line treatment with a targeted agent. There is certainly a need to offer treatments to this patient population, not uncommonly encountered in clinical practice and often of appropriate performance status to tolerate more therapy. Re-treatment with another targeted agent has become commonplace practice despite the lack of prospective data Prior response to first-line targeted treatment did not predict response to second-line treatment et al. iii trial in this clinical setting. Patients who had progressed on sunitinib or sorafenib (or both) were randomized to either the mtor inhibitor everolimus  or to placebo. Although patients from all prognostic groups were enrolled into the trial, approximately half the patients had an intermediate prognosis. The primary endpoint was pfs as determined by independent reviewers. The trial was halted early when an interim analysis indicated a substantial and significant difference in pfs in favour of the everolimus group. The progression rate was 37% in the everolimus group as compared with 65% in the placebo group . Median pfs was 4.0 months (95% ci: 3.7 to 5.5 months) in the everolimus group as compared with 1.9 months (95% ci: 1.8 to 1.9 months) in the control group. Median overall survival (os) had not been reached in the everolimus arm (in excess of 10 months as compared with 8.8 months in the placebo group). The difference in os did not reach statistical significance, likely as a result of the planned crossover from placebo to everolimus on study. Although more stomatitis (40% vs. 8%), rash (25% vs. 4%), and fatigue (20% vs. 16%) occurred in the everolimus group, these side effects were mostly mild or moderate in severity. Pneumonitis (any grade) was detected in 22 patients in the everolimus group (8%), but only 8 patients The best evidence to date is provided by Motzer os is always preferable as the primary endpoint in phase iii trials, the use of pfs in the Motzer study is an acceptable surrogate, and the overall case supporting the efficacy of everolimus as a second-line treatment is strong rcc, temsirolimus could be considered in this setting, given that it is currently approved in Canada and has a similar mechanism of activity. Once completed, two phase iii trials currently in progress may affect this choice. The first randomizes patients to temsirolimus or sorafenib as second-line therapy after progression on sunitinib . The second randomizes patients who failed a previous systemic treatment, which may be a tyrosine kinase inhibitor, to either sorafenib or axitinib .At this time, everolimus can reasonably be considered to be the preferred second-line treatment after initial failure of sunitinib or sorafenib. Although 3.rcc. When disease progression occurs after such therapy, there is clearly more benefit to be gained by re-treating these patients with another targeted agent. Everolimus is the drug of choice at the present time, and we expect it to obtain approval for this indication in Canada soon. Until such time as approval is forthcoming, temsirolimus (if available) could be substituted, given its similar mechanism of action. It would also not be unreasonable to use any of the targeted agents in rcc who have not previously been exposed to those agents. However, the benefits seen with rationally-developed targeted agents in the first-line setting strongly suggest that it is more appropriate to enrol those patients into clinical trials. Research priorities include the evaluation of predictive biomarkers to allow for patient enrichment, optimization of drug sequencing , and identification of other effective drugs—for example, histone deacetylase Inhibitors of multiple kinases such as sunitinib and sorafenib are now established as standard first-line therapy in patients with


High-throughput cDNA synthesis and sequencing of poly(A)-enriched RNA is rapidly emerging as a technology competing to replace microarrays as a quantitative platform for measuring gene expression.S. cerevisiae.Consequently, we compared full length cDNA sequencing to 2-channel gene expression microarrays in the context of measuring differential gene expression. Because of its comparable cost to a gene expression microarray, our study focused on the data obtainable from a single lane of an Illumina 1 G sequencer. We compared sequencing data to a highly replicated microarray experiment profiling two divergent strains of S. cerevisiae that indicate full length cDNA sequencing can discover heterozygosity and measure quantitative allele-specific expression simultaneously.Using a large number of quantitative PCR (qPCR) assays, more than previous studies, we found that neither technology is decisively better at measuring differential gene expression. Further, we report sequencing results from a diploid hybrid of two strains of  RNA sequencing has been one of the first applications of the revolutionary ultrahighthroughput DNA sequencing technologies -9. This S. cerevisiae [S cerevisiae: BY4716 and RM11-1a[In this study, we examined the use of full length cDNA sequencing for measuring differential gene expression in two divergent haploid strains of revisiae . BecauseIn contrast to gene expression microarrays, which measure gene expression by quantifying the hybridized and labeled cDNA, the Illumina 1 G measures gene expression by sequencing fragments of cDNA. The sequencing reads are aligned to a known reference genome sequence, and quantitative gene expression values are obtained by counting the number of these fragments matching a known open reading frame (ORF). One of the consequences of the adapter ligation cloning approach used in this study is that strand origin of the expressed transcript is lost. If two genes overlap, one on the Watson and the other on the Crick strand, their expression levels will be indistinguishable for the region of overlap.The primary limits of the Illumina 1 G sequencer are short read length and total number of reads obtainable in a single lane of a sequencing reaction. Read length at the time of our study was 32 base pairs, and the total number of reads obtainable in a sequencing reaction was about 5 million. Short read length increases the likelihood of reads that map to multiple locations. If these multiple-mapping reads are not removed, they will contribute mixed signal that does not distinguish expression of different genes. The second limitation, the number of reads, is more problematic given that a small subset of transcripts are highly abundant . These highly abundant transcripts will be sequenced more frequently, leaving few reads for transcripts that are in low abundance. As a result, the low abundance transcripts will be more affected by sampling error. Microarray analysis is much less limited by the presence of highly abundant transcripts, because each mRNA binds independently to its complementary sequence feature on a microarray.In addition to quantitative measurement of gene expression, we report initial results showing that full length cDNA sequencing can discover heterozygosity and measure allele specific expression (ASE) in diploid strains of yeast. Many organisms are primarily found in nature as highly heterozygous, diploid individuals. Further, many species, including some major crop plants, are descended from multiple hybridization events and are highly polyploid . The larS. cerevisiae, RM11-1a and BY4716, in two different growth media was measured using six biological replicates (GEO accession number GSE9376) [The basis of our comparison was a DNA microarray array experiment in which the gene expression in two different haploid strains of S. cerevisiae sequence [see Additional file Differential gene expression information from the Illumina 1 G sequencer was obtained by aligning reads to a reference genome and counting the number of reads that overlap each ORF. Samples were denoted BYg, BYe, RMg, RMe where BY indicates strain BY4716 and RM indicates RM11-1a. The lower-case e and g denote two nutrient conditions: ethanol and glucose as carbon sources. We used a simple alignment strategy that assumed a low error rate and determined the alignment with the entire reference genome with the fewest number of differences  of the reads. Between 91.6% and 96.2% of the reads obtained from a single lane of the Illumina 1 G sequencer aligned with the reference We separated reads into unique reads that aligned to a single location in the genome and reads that aligned to multiple locations. Among the reads that mapped to a unique position, we separated reads into those that mapped to an ORF, a ribosomal RNA (rRNA) gene, or a transfer RNA (tRNA) gene. We observed that the vast majority of the unique reads originated from ORFs [see Additional file 2 ratio of RMe to RMg counts, normalizing for the different total number of read counts between the two samples. For the 4697 genes that passed quality criteria for both the microarrays and sequencer, the resulting ratio was highly correlated  between microarrays and the sequencing data  as well as significantly lower average array intensity , suggesting that differential expression of genes with low expression levels is detected more readily by arrays. The subset of genes that were called significantly differentially expressed by sequencing alone had higher read counts than the set of all genes examined  as well as higher array intensities , indicating that differential expression of genes with high expression is detected more frequently by sequencing than by arrays.We identified the subset of differentially expressed genes for each platform. We detected significant differential expression for 1027 genes by arrays and 1303 genes by sequencing, with 566 genes detected as differentially expressed by both platforms Fig. . The subWe modeled the effect of counting noise on sequencing-based measurements of differential expression (see Methods). The scatter plots comparing differential gene expression determined from arrays and from simulated sequencing results predicted from our noise model were similar to what was experimentally observed Fig. . Both thTo better understand the disagreement between the cDNA sequencing and array results, we performed qPCR on a large subset of genes. We randomly selected 192 genes from genes significantly differentially expressed in the following categories: in the Illumina 1 G sequencing data only, in microarrays only, and as measured by both technologies. Of the 192 candidates, 12 genes that showed non-specific amplification or unexpected size amplification products were removed from the analysis. qPCR results are highly correlated with both microarrays .As a prelude to detecting heterozygosity and measuring allele specific expression (ASE), we examined the capability of the Illumina 1 G sequencer to locate cDNA single nucleotide polymorphisms (SNPs) in our haploid RM11-1a strain samples. Alignments of ORFs from the reference sequence with the RM11-1a unfinished genome sequence provided the known position of 24,751 SNPs. We tested how accurately these known RM11-1a SNPs were rediscovered using the Illumina 1 G reads aligned with the divergent S288C reference sequence. We tested a total of 5,926,474 bases covered by at least one read. 180,628 bases showed at least one discrepancy between an Illumina base-call and the reference sequence. Considering all discrepancies gives more than a sevenfold overestimate of the known SNPs, so we calculated a likelihood ratio statistic for each discrepant base to more accurately identify SNPs (see methods). With this additional filter we were able to detect 11,608 known SNPs while only falsely calling SNPs for 457 bases  for this in silico data set. We calculated simulated ASE expression by separating reads directly aligning over the SNP base into two groups based on which base they contain, counting the total reads in each group, and calculating the log2 ratio to determine differential gene expression. ASE measurements from the in silico BYg/RMg diploid for genes with over 10 sequencing reads were significantly correlated  with the differential expression values calculated by summing all reads aligning to an ORF from the haploid samples.Next, we focused on detecting heterozygosity and quantifying ASE simultaneously in data from diploid organisms. First, we simulated the results that could be expected from a diploid strain by combining the haploid BYg and RMg data sets together. This S. cerevisiae strain grown in glucose to discover sites of heterozygosity and simultaneously quantify allele specific expression. We tested 5,072,257 bases covered by at least one read, and 270,898 bases showed at least one discrepancy with the reference sequence. Using the likelihood ratio statistic, we were able to detect 3,691 known sites of heterozygosity while falsely calling 868 bases . We calculated ASE at these heterozygous sites by calculating the ratio of the number of reads containing the BY base and the number of reads containing the RM base. Considering only the heterozygous sites assessed by at least 10 sequencing reads, we observed limited correlation  with the ASE previously quantified for those transcripts in a BY/RM diploid by allele-specific RT-PCR [Last, we used sequencing data obtained from an actual BYg/RMg diploid c RT-PCR . Althougcis, such that local-linkage effect sizes in a sample of transcripts have been observed to correlate with ASE as quantified by allele-specific qPCR [As another assessment of ASE, we compared our ASE predictions to results from genetic linkage analysis of gene expression levels. Using 112 haploid segregants from a cross of the BY and RM strains, expression levels were linked to both local and distant loci . Local lfic qPCR . TherefoQuantitative measurement of RNA-cDNA hybridization kinetics demonstrates that the eukaryotic transcriptome is dominated by a relatively small number of highly expressed genes. 20% of We found that a large fraction of Illumina 1 G reads originated from highly abundant ribosomal rRNAs. Between 30.9%–60.4% of the total number of reads in our six samples originated from rRNAs. Even though we depleted each cDNA sample of ribosomal RNA before priming cDNA synthesis using the mRNA polyA tail, the remaining highly structured ribosomal RNAs self-primed a significant amount of reverse transcription, so that the final sample contained a large amount of cDNA originating from ribosomal RNA. This problem of limited mRNA coverage can be addressed in one of three ways: by combining data from several lanes of the sequencer (at increased cost), by developing new techniques in order to selectively and efficiently deplete abundant rRNAs, and through advances in sequencing technology that increase the number of reads per run.2 value of 0 . This suggested that the Illumina 1 G was less subject to saturation effects than microarray analysis.Based on our qPCR assessment of transcript levels for a large number of genes, we find both sequencing and array technologies have similar overall accuracy for measuring differential gene expression. One report has observed very high (up to R = 0.98) correlations between cDNA sequencing and qPCR, but selected just 34 genes spanning a 6000-fold range of expression . AnotherFor 9 genes that were significantly differentially expressed in both technologies but where the technologies disagreed on the direction of the change, the qPCR results were inconclusive. All 9 genes were measured with fewer than 300 reads. The majority, six out of 9, showed no differential expression by qPCR. They were likely false positives for both sequencing and arrays or false negatives for qPCR. Two of the qPCR assayed genes agreed with the array prediction, while the third agreed with the sequencing data. These numbers were too small to draw definitive conclusions.-2. cDNA sequencing shows great promise for generating a coding SNP map in any genome for which a reference sequence is available.Our analysis, both in our comparison to arrays and qPCR, focused on the differential gene expression within the RM11-1a strain across two environmental conditions (RMg and RMe). This represents a realistic situation in which the strain of interest has several polymorphisms compared to a reference sequence. Moreover, gene expression differs greatly between these conditions . Calculain silico data set, generated by combining reads from RMg and BYg, we are able to detect 9,848 known sites of heterozygosity while falsely calling 654 sites . This performance was notably worse than detecting SNPs from haploid cDNA. Heterozygous site detection requires more reads than homozygous SNP detection because bases from each allele must be present to provide sufficient evidence for the existence of the site. This is further complicated by the presence of strong allele specific expression (ASE) for many genes, resulting in fewer reads from the less expressed allele. Consequently, our results with an actual BY4716/RM11-1a heterozygous diploid indicated that at high specificity only a smaller percentage, approximately 15%, of heterozygous sites could be detected. The discrepancy between the ability to detect heterozygosity for the in silico diploid and the actual BYg/RMg diploid can also be partially attributed to the fact that the in silico diploid was modeled by combining data from two sequencing runs and so had twice the data generated for the actual diploid.In our assessment of heterozygous site detection in a diploid, the total number of reads available limited predictive power. In the in silico diploid showed significantly higher correlation with gene expression . Because the in silico diploid contained the number of reads obtained from two lanes of a sequencer, this result suggests that the total number of reads was the limiting factor.Similarly, in our assessment of the quantitative measurement of ASE in a diploid, the total number of reads available limited the accuracy of the results. Correlation of ASE as determined by sequencing with ASE as measured by RT-PCR was weak . In contrast, ASE calculated for the Our analysis of next generation sequencing was motivated by the rapidly converging cost of microarray analysis with a single lane of data from an Illumina 1 G sequencer. In summary, our results indicate that, at least for the present, microarrays remain a highly competitive technology for quantitative measurement of differential gene expression. Arrays have an advantage in measuring low-abundance transcripts. Also, the current throughput of array experiments is higher than the 8 lanes present on an Illumina 1 G instrument. It is routine to process 24–96 arrays in a single day, while the Illumina instrument is limited to 8 lanes that require approximately 48 hours to run. Multiplexing approaches can increase the number of samples per lane of the instrument, but our analysis suggests that accurate analysis of gene expression in yeast by full-length cDNA sequencing requires at least the entire output of a single lane. More lanes may be necessary in other species with more complex mRNA pools.For BY9e, BY9g, RM9e, and RM9g, samples were identical to those described in . RM-11a/S. cerevisiae genome downloaded from SGD on April 1, 2008, allowing for up to 2-edits . DNA bases called by the Illumina IG decrease in accuracy toward the 3' end of the read, so up to two bases were trimmed from the 3' end. We used the method of Baeza-Yates and Perleberg [Reads were aligned to the S288C erleberg  to match1. Index the genome using a suffix array. We used the implementation of Doring et al.2. Search the genome exactly for the read, If the read did not match uniquely or in multiple locations exactly, allow 1-mismatch for the read by searching for regions of the genome that match either half of the read. Once such a region is found, use the Myers bitvector algorithm  to quick3. For all matches within 2-edits, use the Smith-Waterman algorithm to recover an alignment.4. For reads that did not match up to two edits, trim one base at a time from the 3' end, up to two bases, and align again using the same strategy allowing for up to 0, 1, and 2-edits.1. Index the intervals covered by the reads in a balanced dynamic priority search tree .2. Use stabbing and interval queries to obtain reads that overlap a gene's open reading frame (ORF).3. Compute expression value for that gene as the number of reads returned by the stabbing and interval queries by gene length. If a read matched multiple locations within a single ORF, it contributed only one count for the read.The following quality criteria were applied to genes in the array data: intensities well above background, no non-uniformity outliers, and no missing data. A single quality criterion was used for the sequencing data: the ORFs of genes the genes do not overlap. Statistical difference in expression for cDNA sequencing data was evaluated using Fisher's exact test on a 2 × 2 contingency table on a per gene basis in which the two columns of the table were separated by reads that overlap the gene's ORF and reads that do not overlap the ORF and the two rows were separated by the two compared samples RMg and RMe. Fisher's exact test on the contingency table corrected for overall differences in the number of filtered reads obtained for each sample. For the microarray data, statistical significance was evaluated using the Mann-Whitney test on n = 6 biological replicates; the sequenced cDNA originated from one of these 6 samples. We further corrected for multiple testing using a q-value approach. Significant difference was determined using Fisher's exact test for the cDNA sequencing data, and the Mann-Whitney test for the array data with the additional criteria of a FDR less than 1% and a 1.5 fold or greater change between the two conditions. All correlation confidence intervals reported were calculated in R using 10,000 re-samplings.We began with the ratio of median RMe and median RMg values measured in the array experiment. We randomly perturbed the log ratio of the array data with Gaussian noise with a variance of 0.5 to model unknown sources of variance for each gene. Next, we calculated the sum of the read counts from RMe and RMg from the observed Illumina data for each gene. For each gene read counts were redistributed between the strains such that the ratio of the read counts equaled the noise-perturbed ratio from the array data. The sum of the read counts for each gene remained unchanged. For each gene a probability between 0 and 1 was generated by dividing this simulated read count by the total read counts across all genes for that strain. For each strain and each gene we simulated sequencing data by randomly sampling once from a binomial distribution defined by this probability and the total counts for each strain.192 genes were selected by pooling a list of genes corresponding to those differentially expressed by arrays only, sequencing only, or both and randomly sampling once from this list. In addition, we included all 9 genes that were significantly differentially expressed in both technologies but where the technologies disagreed on the direction of the change. Primers for quantitative PCR were designed using BatchPrimer3 1.0 , an implWe identified SNPs for the haploid strains and heterozygous sites for the diploid strains by calculating a likelihood ratio statistic. The likelihood that the reads for a base comes from a binomial distribution with a 2% or smaller error in sequencing any base was compared to the likelihood that comes from a binomial distribution where the probability that reads differ from the reference base is greater than the sequencing error rate. The choice of the error rate is a conservative estimate. A LOD score for all bases with at least at least one discrepancy between an Illumina base-call and the reference sequence was determined as follows:n = total number of reads aligning to a basey = total number of reads aligning to a base that match the referencep = probability that reads match the reference baseBases with LOD>3 were called SNPs or sites of heterozygosity.JSB and ZK analyzed the data and drafted the manuscript. AAC performed the experiments and drafted the manuscript. MS, LK designed the study and helped draft the manuscript.Table S1 – Read Utilization. Table S2 – Unique Read Origin.Table S3 – Multiple Read Origin. Table S4 – Correlations of Differential Gene Expression Between Illumina 1 G and Agilent Arrays. Tables of Illumina sequencing read utilization and read origin. Correlations of differential gene expression, with and without thresholding on read coverage.Click here for file


Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration.In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear's neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene,  phoenix mutation is in a new gene family class that is specifically required for the supporting cells to enter into mitosis after hair cell damage. What is particularly unusual about this mutation is that it appears not to affect the normal development and differentiation pathways, but only seems to affect the cells' post-differentiation regeneration.By screening for regeneration deficient zebrafish mutations, we identified a zebrafish mutant line deficient in a highly specific regeneration process, the renewal of hair cells in the lateral line. Although this organ is specific to fish and amphibians, it contains essentially the same mechanosensory cells (the hair cells) that function in the ear for sound and balance detection in all vertebrates. Mammals are unusual vertebrates in that they have lost the ability to regenerate functional hair cells after damage by sound or chemical exposure. All other vertebrates retain their ability to regenerate their hair cells after damage, but this process is not well understood at the molecular level. The retroviral insertion linked to the  During development of the vertebrate inner ear, a subset of neuroepithelial cells specialize to give rise to hair cells and supporting cells Fish and amphibians have an additional organ related to the inner ear called the lateral line, which is used to detect water currents in vivo assay using both copper and neomycin to follow regeneration of hair cells in neuromasts of 6dpf to 8dpf larvae. Using this assay, we characterized two allelic mutant lines generated by retroviral insertion phoenix (pho). Homozygous mutants did not display obvious morphological or behavioral phenotypes and developed a functional lateral line as ascertained by FM1-43 incorporation. However, when phoenix larvae were treated with either copper or neomycin, they showed a strongly reduced regeneration of the hair cells. In parallel, we demonstrated that another form of regeneration, the growth of the tail after amputation, was not affected in the phoenix mutants, arguing that the regeneration defect was specific to hair cells.We utilized an phoenix mutant larvae. Furthermore, we monitored cell death over the time course of our assay in treated wild-type and mutant neuromasts and did not find significant differences in supporting cell death. Strikingly, we found that the proliferation rate in this progenitor cell population was strongly reduced in mutants. Thus, impaired proliferation is tightly correlated to the observed deficit of regenerated hair cells in the phoenix mutant.We show that, the number of supporting cells was not significantly different before or after copper and neomycin treatments in neuromasts of wild-type versus Phoenix is the first documented member of a novel gene family, which has an important role in the regeneration of hair cells in the lateral line.We characterized the gene carrying the retroviral integration linked to the phenotype, and find that it is a novel gene, with no previously described homologs. Expression at 3-4dpf is upregulated in the supporting cells of the neuromasts. Thus, phoenix. Homozygotes displayed no behavioral defects  or visible morphological abnormalities until 5dpf, when the swim bladder failed to inflate. Later, around 7 to 8 dpf, mutants display necrosis in the liver, and death ensues at approximately 14 dpf. Because the gene was expressed in the lateral line neuromasts (see below), we performed a detailed assessment of this organ in 2 to 12 dpf larvae  were indistinguishable from wild-type larvae (upper panels). Camera lucida drawings . Again, we did not find a significant difference between the absorption of the dye in wild-type (top) and mutant (bottom) hair cells, nor did we see any significant observable structural differences. Therefore, although the gene is expressed in the lateral line, its initial development appears unaffected in mutant larvae.We have utilized an assay to monitor hair cell regeneration in the lateral line of 5dpf zebrafish larvae, using transient exposure to copper sulphate (10mM) or neomycin (200mM) dissolved in the water. Our intention was to test for defective hair cell regeneration from a collection of mutant lines previously identified in a retroviral integration screen f larvae . Semi-thf larvae  of phoendrawings  of each drawings  did not drawings  show, lidrawings . As showhi43) of the phoenix mutant line was generated by retroviral mutagenesis as previously reported phoenix (hi43 (black triangle) landed in the first exon and was adjacent to the first splice donor site.The first allele . This mutant line carried a retroviral insertion in the first exon (75bp downstream of the ATG) of the phoenix gene  we were able to identify the syntenic region for four other fish species: Takifugu rubripes (fugu), Tetraodon nigroviridis (tetraodon), Oryzias latipes (medaka), and Gasterosteus aculeatus (stickleback). The genes flanking phoenix in zebrafish are aspa and c11orf54 homologs. In all fish species examined, this synteny was maintained, but a synteny break occurred at this location in other vertebrate genomes. Located between aspa and c11orf54 in all four fish species was a large predicted ORF. The corresponding predicted genes  had very weak but noticeable identity with the zebrafish phoenix gene. The overall identity of the fish homologues was still not significant . The main feature of all proteins was a low complexity sequence with a large number of proline and lysine residues. Using ClustalW2 , we showed that the only significant identity in these predicted transcripts was in the amino-terminal end of the proteins . It contained an abATPase signature, PSVHSPPSDS (P-[SAP]-[LIV]-[DNH]-X(3)-SXS), encoded in the first exon with the last serine overlapping the splicing site  on embryos from 8 hours post fertilization (hpf) through 5 dpf. The expression of the phoenix gene was first detected in the anterior and posterior lateral line system  and mutant (right) cristae  larvae. At most, one remaining hair cell could be seen, but its irregular shape indicating that it was probably a dying cell  and mutant (lower panel) larvae and followed them to 9dpf (phoenix (pink bar) larvae was identical. Thus, we conclude that the phoenix mutation affects hair cell but not tail regeneration, indicating a defect specific to the lateral line.To determine if phoenix has an important role in the regeneration process of hair cells in the lateral line.Taken together, these observations strongly suggest that phoenix versus wild-type larvae could be due to a difference in susceptibility of the progenitor cells to the ototoxic agents used in our assays. There is substantial evidence pointing to supporting cells as the source of new hair cells in neuromast regeneration. The new hair cells are likely to arise from a population of dividing progenitor cells phoenix mutant background in order to obtain mutant transgenic individuals. We evaluated the effect of either copper or neomycin treatment on the number of supporting cells in wild-type and mutant treated larvae after copper or neomycin exposure. We confirmed that there were no significant differences in the appearance of the supporting cells in the mutant transgenic larvae by performing the regeneration assay, followed by immuno-staining against GFP to detect supporting cells and anti-Myosin VI antibody to detect hair cells. In phoenix mutant (second column) neuromasts, hair cells were present and centrally located as visualized by anti-myosin VI antibody staining . The supporting cells, as expected, surrounded them as visualized with an anti-GFP antibody staining . After copper . We counted the supporting cells in 4 different neuromasts in the head  and ten supporting cells visible (nuclei outlined in blue). In the mutant neuromast, three hair cells and ten supporting cells were visible. Four hours after neomycin treatment hair cells were completely destroyed in the wild-type and in the mutant neuromast (phoenix mutant neuromasts. Ten nuclei in the wild-type and ten in the mutant neuromast were visible (as outlined in the camera lucida in blue). Thus, at all observed stages, we did not notice a significantly higher cell death rate among the supporting cell population in phoenix mutant larvae.Another possible explanation for the observed phenotype is that mutant progenitor cells are dividing but that the newly formed precursor cells do not survive because they are hyper-sensitive or unstable after the chemical exposure. To detect dying cells in the supporting cell layer in the mutant neuromasts, we analyzed semi-thin sections  larvae showed a striking difference in the number of BrdU positive cells. On average, we found eight BrdU positive cells in the transgenic wild-type neuromasts, in mutant neuromasts we typically found no positive cells while at most we found two. Thus the number of BrdU positive cells in the progenitor cells population in the mutant neuromasts is drastically reduced, most likely explaining the decrease in the number of newly regenerated hair cells in phoenix mutant embryos.Since the number of supporting cells in mutant neuromasts is not unlike that of wild type neuromasts, we turned our attention to differences in cell division to explain the regeneration phenotype observed in y red in  and neomphoenix mutant (yellow line) neuromasts per larva. BrdU was added to the medium 6h before fixation allowing us to determine rates of DNA synthesis in equivalent time-windows during the 3 days regeneration period. Since recovery to a normal number of hair cells is reached at around 72 hours after treatment, this time point represents the rate of BrdU incorporation in undamaged neuromasts, or the BrdU baseline. At +24h of recovery, we had an eight-fold difference in the number of BrdU positive cells between wild-type and mutant neuromasts. This difference was still two-fold at +48h of recovery and both returned to the base line frequency at +72h. We performed similar tests after neomycin treatment  used.We further show that the regeneration deficiency is specific to the lateral line, as we did not see a similar effect during tail regeneration. In combination with the restricted expression of the gene in supporting cells of the lateral line, this strongly suggests that, zygotic It is intriguing that while the two treatments trigger similar responses, they are significantly different in the amplitude of the elicited effect. The reason there is a nearly 2.5 times greater inability of the supporting cells to respond properly after copper compared to neomycin treatments, is not clear. However, it is to be noted that the wild-type regeneration is also significantly slower (about two times) after the copper treatments when compared to neomycin treatments, as we found and as it has been described previously phoenix is upstream of the differential response between neomycin and copper, as the differences in the rates of response in mutant larvae remain similar to the differences seen in wild-type larvae.Additionally, it was recently shown in a screen to identify genes that modify hair cell resistance in the lateral line, that different ototoxic agents can elicit different pathways for protection and survival of hair cells in treated neuromasts phoenix mRNA is expressed in all supporting cells, therefore the most likely explanation is that the regenerative potential of all supporting cells is severely impaired.We showed that the death rate of supporting cells was unaltered in mutant neuromasts as observed by three different approaches . Furthermore, making use of the MSC1 transgenic line that expresses GFP exclusively in the supporting cells, we demonstrated no loss in the number of those cells in mutant neuromasts. It is to be noted that the counting of cells in the neuromast is technically challenging. Although we did not see obvious differences, we cannot exclude a possible subtle reduction in the number of supporting cells. If a key subset of cells critical to the initiation of regeneration is missing, even a subtle difference in number might have a drastic effect on regeneration, which we cannot exclude at our level of analysis. However, it was previously shown that the regeneration process is happening in many supporting cells simultaneously, encompassing most of the neuromast phoenix mutant larvae, this regenerative process is happening in a significantly reduced number of supporting cells. Further work is required to address the timing of the blockage occurring in the supporting cells in mutant neuromasts, but it is clearly occurring before DNA synthesis initiates. This invaluable information will help to elucidate the mechanism of action of the novel phoenix gene in the regeneration process of hair cells in the lateral line.To assess proliferation of the progenitor cells, we follow the incorporation of BrdU during the S-phase of the cell cycle in neuromasts. The incorporation of BrdU is detected in cells that have exited the cell cycle and have differentiated. Therefore, it provides no information on the post-mitotic state of the BrdU positive cells. However, as the number of regenerated hair cells correlates closely to the number of BrdU labeled cells and as we do not see additional cell death in either wild-type or mutant neuromasts, this would suggest that the progenitor cells that actually enter S-phase proceed through the entire cell cycle and into differentiation. In the All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the appropriate committee (NHGRI protocol # G-01-3).hi43 allele of phoenix was recovered in a retroviral screen performed at MIT GGAGATCGACAGCGCCCTGAAG and AAACTGCTGAGGGCTGCTGGACCGCATC. The zm allele (ZM_00003486) was purchased  and carriers were genotyped using the primers CGAGACCCCGCCGCCTGATGTT and GACGCAGGCGCATAAAATCAGTC. The MSC1 transgenic line was a gift from B. Weinstein and carriers were identified by detecting specific expression of GFP in the lateral line. The cldnB::GFP line pou4f3::GFP line ET20::GFP line Fish care and husbandry were performed according to All active agents were added to system water at 28°C. During the staining/treatment, larvae were kept in cell strainers  in 6 well plates ) at a maximum of 35 larvae/well. This allowed rapid transfer of the larvae during the post-treatment rinsing. All larvae were rinsed 6×3 minutes with system water and then transferred into large Petri dishes with abundant fresh system water. After the various treatments, only healthy looking larvae displaying no additional morphological or behavioral defects were kept for subsequent analysis over the following 72 hours. The number of hair cells and the AO positive cells were counted in 10 head and all the trunk and tail neuromasts, in each larva.YoPro-1 (Molecular probes) for 15 minutes at 2mM, as described in FM1-43 (Molecular probes) for 1minute at 2mM, as described Acridine orange (AO) hemi  for 5 minutes at 2mg/ml, as described Copper (Copper(II) sulfate, Sigma) was added for 2h at 10mM as described in Neomycin  for 1h at 200mM as described in We added Five day old larvae were allowed to recover for an hour after copper or neomycin treatment. Subsequently, larvae were anesthetized with 0.005% MS-222 (Sigma) and placed on a clean glass slide and a portion of their tail was amputated with a scalpel. We cut not just the fin, but also a portion of the actual tail including the neural tube, the notochord, and the somites, to evaluate the regeneration of other tissues in addition to the fin itself. Three day old untreated larvae were similarly processed. To follow and image the tail growth over 3 to 6 days post amputation, we mounted anesthetized larvae on cover slips in 2% methylcellulose. Measurements were made on pictures taken from live larvae on the inverted Zeiss AXIOVERT200M, using the Axiovision software from Zeiss. The region of amputation was usually easily identifiable, as it presented healing scars, allowing a reasonably accurate measurement of the newly grown tail.BrdU (Sigma) was diluted to 10mM in system water and larvae were exposed for 6h before fixation at the chosen time points  o/n with 4% formaldehyde (Electron Microscopy Sciences) in 1× PBS . Double staining was done as described Untreated and copper treated larvae were fixed o/n with 4% formaldehyde (Electron Microscopy Sciences) in 1× PBS , at various stages of interest and subsequently stored in 100% methanol. After progressive rehydration ), larvae were treated with acetone for 7mn at −20°C. Subsequently, we rehydrated and rinsed them 3×5mn in PTW. Next we digested them with 1mg/ml collagenase (Sigma) in PTB (PTW + 10% goat serum +10% BSA) for 35mn. After 5×5mn rinses with PBT, we pre-incubated the larvae 4 hours in PBT. Larvae were incubated o/n with the polyclonal rabbit primary antibody (1/200) against Myosin VI  and a fluorescently labeled monoclonal mouse primary antibody (1/200) against GFP (Abcam). The next day we rinsed the larvae 6×10 mn in PTW and preincubated them again for 4 hours in PBT. The fluorescently labeled secondary anti-rabbit antibody (1/500) was added o/n. The next day 6×10 mn rinses were performed. Larvae were mounted on slides in Aquapolymount  for imaging on an upright confocal microscope (Zeiss AXIOVERT).Larvae were fixed overnight at in 2.5% glutaraldehyde (Sigma) and 4% paraformaldehyde prepared from paraformaldehyde (Sigma) in 0.1M sodium cacodylate buffer (Sigma). Larvae were then rinsed and post-fixed 1h at room temperature in reduced osmium (1∶1 mixture of 2% aqueous potassium ferrocyanide) as described previously TCAACTGATGTATTTCCTGGGC and GTTTTGCTCCACTATCTGACCTTT. The probe was hybridized at 62°C. Larvae were mounted on slides in Aquapolymount  for imaging on an upright confocal microscope (Zeiss AXIOVERT).Performed as described previously AGATCTTGAGATTGCCGAATGT and CATCTCTCTCACCTTCTTCAGTGAC. The BAC was sequenced, by shotgun assembly and brought to finished quality (≤1 error per 50kb) at the National Intramural Sequencing Center (NISC). Sequences were aligned to the zebrafish genome (UCSC Genome Browser http://genome.ucsc.edu/) and syntenic regions identified using genomic chain comparisons in Takifugu rubripes (fugu), Tetraodon nigroviridis (tetraodon), Oryzias latipes (medaka), and Gasterosteus aculeatus (stickleback). Genescan or N-SCAN http://www.ebi.ac.uk/Tools/clustalw2/index.html). The BLOSUM protein matrix We screened the zebrafish BAC library  with a probe generated from total RNA (5 dpf larva), with the primers CCATATCAAAACAGAGCTGTGCTAC and GTCAAGGCAGAGTAAGCAAGTGACACTG, both located in the 5′UTR, respectively starting 142bp and 121bp upstream of the start codon. For the cloning of transcripts 2, 3 and 6, they were respectively coupled with the reverse primers GTTCTGTTTTCTTTTCCTTGTCAACGCC and CCTTCTCTCCCTGATAAAGTCTGGCAACC both found in the 5′end of exon 7. For the cloning of the transcript 1, we coupled them respectively with the reverse primers CATTCATTCATTCATTCATTTTCCT and TTTCCTTTTGCTTAGTCCCTTATTT both found in the 3′end of exon 2. For the cloning of the transcript 4 we coupled them respectively with the reverse primers TTTTACATCTACAGAATCGTGAAAAA and AATTGTGATTCTCATTTTAGCCAGA, both found in the 3′ end of exon 6. For the cloning of the transcript 5 we coupled them respectively with the reverse primers TTTTTCTTTCATAGCGAACACAAAG and TTTAAACTGTCAGCTCTTGATGC, both found in 3′end of exon 5. To verify the quality of the Total RNA from different stages, which we used for the RT-PCR, we amplified a 159bp fragment of b-actin using the following primers, GACCCAGACATCAGGGAGTGATGG and AGGTGTGATGCCAGATCTTCTCCAGT. All sub-cloning for subsequent sequencing were done using the TOPO TA cloning Kit (Invitrogen), following the manufacturer protocol. Sequencing was performed by ACGT, Inc. The analysis of the sequences was done using the Sequencher (Gene Codes Corp.) program, with the genomic sequence of the phoenix gene (as previously isolated in the BAC) as a reference.We prepared total RNA extracts from embryos and Pac2 cells  with two samples of equal variance and considered significant when p was <0.05. Error bars in the different graphs represent the standard deviation, or standard error, depending on the number of samples (n) analyzed.Stacks of images collected on the inverted microscope were treated with the Axiovision software from Zeiss. Stacks of images collected on the confocal microscope were treated with the LSM software from Zeiss. All images or stack of images were exported as tiff files, which were subsequently processed with Photoshop.Figure S1phoenix background show no differences from wild-type larvae. A. We crossed the phoenix mutant line into the cldnB::GFP transgenic background, which expressed GFP (green in first and third panels) in all cells of the neuromast. We stained untreated 5 dpf larvae with FM1-43 (red in second and third a panel). Shown are images of live wild-type (top panels) and phoenix (bottom panels) neuromasts. A camera lucida drawing of each merged picture is highlighting the GFP positive cells contours (green) and the FM1-43 positive cells (red) in the fourth panel. B. We crossed the phoenix mutant line into the pou4f3::GFP transgenic background, which expressed GFP in all of the neuromast's hair cells. Dorsal views (left panels) and lateral views (right panels) of wild-type (top panels) and pho/pho (bottom panels) are shown. A camera lucida drawing of each panel is outlining the GFP positive hair cells of each respective 41 panel. C. We crossed the phoenix mutant into the ET20::GFP transgenic background, which expressed GFP in a subset of supporting cells known as the mantle cells. Two representative examples of wild-type (top panels) and of phoenix mutant (bottom panels) neuromasts are shown. A camera lucida drawing of each panel is outlining the GFP + cells of each respective panel. - 10 microns in all panels.Live imaging of transgenic lines crossed into the (6.66 MB TIF)Click here for additional data file.Figure S2phoenix mutant ear. Wild-type expression of the phoenix mRNA and protein. A. Live images in pou4f3;;GFP transgenic animals of hair cells in a crista of a wild-type (left panel) and a phoenix mutant (right panel) ear. B. In situ hybridization with an antisense probe against phoenix in the inner ear of a 4dpf old embryo (top panel). A camera lucida drawing (lower panel) shows the positions of the two otoliths (red), of the cristae's (light green) and the maculae's (dark green) hair cells. The AP/DV ventral orientation is indicated. C. Immunofluorescence on cultured zebrafish cells (Pac2) using EX1 (green in the top and bottom left panels) or EX7 (green in the top and bottom right panels) antibodies. To highlight the ER, we co-stained with an antibody against PDI . DAPI was used to counterstain nuclei (blue in the two bottom left and right panels). Merged images  showed a cytoplasmic staining of phoenix, which was more concentrated in the perinuclear region. It appeared excluded from the ER, as there was no co-localization with DPI. Note how phoenix protein was upregulated in cells preparing to enter division (white arrow in the top right panel). D. Western blot on PAC2 cell extracts with two rabbit polyclonal antibodies, raised against an epitope encoded by exon 1 (EX1) or by exon 7 (EX7). A common band in the range of 95KDa was found with both antibodies (black arrow). - 10 microns in A, 50 microns in B and 3 microns in C.Hair cells seem unaffected in the (3.51 MB TIF)Click here for additional data file.


Air pollutants may induce airway inflammation and sensitization due to generation of reactive oxygen species. The genetic background to these mechanisms could be important effect modifiers.2-adrenergic receptor (ADRB2), glutathione S-transferase P1 (GSTP1), and tumor necrosis factor (TNF) genes for development of childhood allergic disease.Our goal was to assess interactions between exposure to air pollution and single nucleotide polymorphisms (SNPs) in the βx) as an indicator based on emission databases and dispersion modeling and estimated individual exposure through geocoding of home addresses. We measured peak expiratory flow rates and specific IgE for inhalant and food allergens at 4 years of age, and selected children with asthma symptoms up to 4 years of age (n = 542) and controls (n = 542) for genotyping.In a birth cohort originally of 4,089 children, we assessed air pollution from local traffic using nitrogen oxides . Children with Ile105Val/Val105Val genotypes were at increased risk of sensitization to any allergen when exposed to elevated levels of traffic NOx . In children with TNF-308 GA/AA genotypes, the GSTP1–NOx interaction effect was even more pronounced. We observed no conclusive interaction effects for ADRB2.Interaction effects on allergic sensitization were indicated between several GSTP1 and TNF variants, supporting a role of genes controlling the antioxidative system and inflammatory response in allergy.The effect of air pollution from traffic on childhood allergy appears to be modified by  We evaluated genes that have previously been identified as target genes for allergic diseases and with a potential for interaction with air pollutants in order to select suitable candidates from among genes that are involved in the inflammatory response and antioxidative pathway, or as airway receptors  . The objeceptors . Thus, bBetween 1994 and 1996, 4,089 newborn infants were recruited in the BAMSE study, and questionnaire data on parental allergic diseases and residential characteristics were obtained . The catTNF, ADRB2, and GSTP1 single nucleotide polymorphisms (SNPs), but we subsequently excluded 40 controls and 33 wheezers because of > 50% missing data from genotype failures, indicating poor DNA quality. Thus, we included 982 children  in the analyses.The study design for the genetics part of BAMSE has been presented elsewhere in detail , 2005. Fn = 635) is smaller than the full genetics study sample (n = 982), and other phenotypes may be enriched among the wheezers, so if bias is not suspected to be an issue, analysis using the full sample size may instead be advantageous.The subcohort mirrors the full BAMSE cohort well in terms of sociodemographic factors, wheeze, and sensitization prevalence . The subThe study was approved by the ethical committee at Karolinska Institutet, and parental consent to genetic analyses was obtained for all participating children.a) transient wheezing: three or more episodes of wheezing between 3 months and 2 years of age, but no episode in the preceding 12 months at 4 years; b) persistent wheezing: one or more episodes of wheezing between 3 months and 2 years of age and one or more episodes in the preceding 12 months at 4 years; and c) late-onset wheezing: no episode of wheezing between 3 months and 2 years of age, but one or more episodes in the preceding 12 months at 4 years. We defined current asthma as reported physician-diagnosed asthma up to 4 years of age and one or more episodes of wheezing in the preceding 12 months at 4 years (nonwheezing children without asthma diagnosis at 4 years served as controls).We subdivided children with history of wheezing into three groups according to reported episodes during the early (3 months to 2 years) and recent (last 12 months at 4 years) age periods , using tA/L)] with Phadiatop : a mixture of cat, dog, horse, birch, timothy, mugwort, Dermatophagoides pteronyssinus, and Cladosporium allergens and fx5 (Phadia AB): a mixture of milk, egg white, soy bean, peanut, fish, and wheat allergens. Twenty-nine percent of the wheezing cases were sensitized, versus 25% of the controls , in total providing 266 sensitized cases. To investigate whether the degree of sensitization might influence the results, we also used a higher cutoff for serum IgE antibodies (≥ 3.5 kUA/L) in some analyses. Of the 982 children with genotype information, 860 performed acceptable PEF tests (88%), and we used PEF as a continuous variable. In the subcohort of 635 children, 175 were sensitized to inhalant or food allergens (28%), and 557 (88%) had acceptable PEF measurements.We measured sensitization to either inhalant or food allergens , and one was out of Hardy-Weinberg equilibrium (HWE) , leaving three SNPs for complete analysis . Of seven potential SNPs in the TNF gene , one was monomorphic (rs3093665), and one had a success rate below 85% (rs1800630/–863), leaving five SNPs for complete analysis . Out of seven selected GSTP1  SNPs, one we excluded because of success rate below 85% (rs1871041) and included the remaining six SNPs in the study .We extracted DNA from peripheral blood leukocytes using a standard nonenzymatic method or the Puregene kit . We selected a number of registered SNPs [preferably with proven or suggested functional effects based on the existing literature or according to the National Center for Biotechnology Information (NCBI) database] in each of the n = 982). We designed primers for multiplexing PCR and extension reactions by the SpectroDesigner software  and performed PCR and extension reactions according to standard protocols (detailed information is available from the authors on request). We performed the SNP analysis by matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry (Sequenom), which has an automatic quality control program that checks all genotypes with regard to consistency of genotypes, peak intensity, signal-to-noise ratio, and the like. In addition to using this program, we also manually double-checked all genotypes as an extra quality control step.We then analyzed the included SNPs in all wheezing cases and controls (p > 0.01) and had a genotyping success rate of 93–99%.All SNPs included for analyses  were in r2) and linkage disequilibrium (LD) coefficient (D′) using Haploview 3.2  frequencies between cases and controls using the chi-square test and used a linear regression model to test for associations with mean PEF values. For the primary interaction test analyses, we used model-free (unconstrained) genotype coding with indicator (dummy) variables for heterozygote and rare homozygote genotypes to investigate the pattern of effects across different genotypes, except for SNPs where the rare homozygotes were too few, in which case we used a dominant genetic model. We then used multiple regression modeling (linear regression for PEF) to test for gene–environment interactions between genotypes and exposure to traffic NOx by adding an interaction term between the genotype and exposure of interest. We performed a likelihood-ratio test between the models with and without interaction terms to test for genotype–exposure interaction. Thus, the obtained p-value indicates a departure from a multiplicative interaction model on the odds ratio (OR) scale and indicates whether the effect of a certain exposure is significantly altered by presence of a genotype  during the first year of life  to assess a p-value cutoff that corresponds to a nominal value of < 0.05 after correction for multiple tests.We evaluated deviations of observed genotype frequencies from HWE by the chi-square test. To check the haplotype inheritance of the markers we estimated the correlation , 1.4–3.3] and between TNF-308 and sensitization to inhalant and/or food allergens , with p-values < 0.05 after correction for multiple tests. The GSTP1 114Val allele also had a suggestive association with increased risk of persistent wheezing, and TNF-308A with asthma and persistent wheezing (p < 0.05 unadjusted). We saw no clear main genetic effects for ADRB2. Estimation of the correlation and LD between the SNPs indicated relatively high correlation in the GSTP1 gene but generally low correlation in the TNF gene [http://www.ehponline.org/members/2008/11117/suppl.pdf)]. Based on the LD pattern, we defined one haplotype block in each gene . We also saw the association between TNF and sensitization in haplo-type analyses of the four SNPs in the block , whereas haplotype associations in the ADRB2 and GSTP1 genes were not significant after correction for multiple tests (data not shown).We performed screening for overall main genetic effect using allelic association tests . We obsex during the first year of life as the environmental exposure . Interaction effects were suggestive between all tested SNPs in the GSTP1 gene  and exposure to traffic NOx during the first year of life for sensitization at 4 years of age (A/L) when exposed to elevated levels of traffic NOx during the first year of life , whereas we observed no increased risk in children homozygous for Ile/Ile. For comparison, GSTP1 genotypes and for exposure to traffic NOx during the first year of life, respectively. Using a higher IgE cutoff level for sensitization (≥ 3.5 kUA/L), we found a similar association to traffic NOx in the Ile105Val/Val105Val group . Interaction with respect to PEF values was also suggestive for two GSTP1 SNPs, including Ile105Val . However, the interaction p-value for PEF analyses was not significant after correction for multiple tests. The other coding GSTP1 SNP, Ala114Val, showed an interaction pattern similar to that for Ile105Val for sensitization . In general, the increased risk seemed to be largely driven by the heterozygote individuals . However, the number of individuals in such subgroup analyses is very small , and this aspect of our results should be interpreted with caution.We next assessed gene–environment interaction effects using exposure to traffic NOs of age . Correctle105Val . Childretization , and we TNF and ADRB2 genes and exposure to traffic NOx with regard to respiratory symptoms, PEF, or sensitization. However, TNF and GSTP1 SNPs have been suggested to have joint effects with respect to air pollution exposures  than in children with the nonrisk genotype (GG)  .We observed no significant interactions between variants in the xposures , and we ctively) . We obseAs a sensitivity analysis, we also performed association and interaction tests using sensitization and PEF as outcomes in the representative subcohort of 635 children. The results and interpretations were very similar to from analyses including all children (both wheezing cases and controls), which supports the general significance of the findings (data for subcohort only not shown).GSTP1, and long-term exposure to ambient air pollution from traffic with respect to sensitization to common allergens in children. TNF and ADRB2 polymorphisms did not seem to directly influence the effect of air pollution from traffic on the development of asthmatic symptoms and sensitization, but in combination with GSTP1, TNF substantially modified the effect of traffic-related exposure.We present results that support a role of interactions between a natural antioxidant enzyme, The GSTP1 enzyme is of particular interest in relation to the respiratory system because it has been reported to provide more than 90% of the GST activity in the lung . The GSTConflicting results on allergy and asthma have been presented for individuals homozygous for the Val105 variant. Although some studies have reported a protective effect for childhood respiratory illness , others GSTP1 variants modifying the effect of exposure to air pollutants and tobacco smoke with respect to asthma and allergy. In an experimental study, 19 ragweed-sensitized adults were challenged intranasally with allergen alone and with allergen plus diesel exhaust particles (x and SO2 levels) (Previous interaction studies support the role of articles . Patientarticles . A study levels) . The Val levels) .GSTP1 SNPs implies that we cannot directly identify the SNP responsible for the observed interaction pattern. Most other studies have focused on the functional Ile105Val rather than Ala114Val, because of the low allele frequency and unknown functional effects of the latter. In the interactions seen between Ile105Val and traffic NOx with respect to sensitization, and the weaker effect but with a similar pattern with respect to PEF values, carriers of the 105Val allele  consistently showed the highest risk estimates in relation to air pollution levels. These results contrast with previous studies, where Ile105 homozygotes seem to have the greatest IgE response after air pollution or tobacco smoke challenge  were at the highest asthma risk, especially if they lived < 75 m from a major road  seem to be related to asthma risk predominantly in subjects from nonsmoking families or children in low-ozone-exposure communities  seems to potentiate the risk associated with exposure, and we speculate that increased TNF levels may in part explain these observations, based on previous results on increased expression associated with the A allele  have been reported to affect not only the long-term response to ADRB2 agonists but also the risk of asthma in relation to active and passive smoking, and we specifically selected ADRB2 in this study based on previous interaction effects  (ADRB2 interactions with air pollutants other than smoking.Polymorphisms in the  effects . Unfortu = 0.38) . To our x in this study increases the power to detect interactions, but the number of individuals with different genotypes who are cases or controls is still limited. Therefore, we cannot rule out the possibility that we obtained false-negative results because of low power. In the whole BAMSE cohort, NOx exposure was associated with sensitization, especially to pollen  (TNF-308 SNP) and interaction effects  were somewhat stronger for inhalant allergens than for food allergens, but the differences were small, and the directions of association were similar (data not shown).Different gene–environment effects in different settings may occur for several reasons. The pattern of exposure in observational studies can differ substantially between countries or areas, and the genotype frequencies also show large global differences . Differe10–2.53) . In thisThere are many practical issues to consider when attempting examining lung function in a large group of 4-year-olds. To ensure good-quality measurements, we used a reproducibility criterion in addition to the test leader’s opinion of the child’s effort. The usefulness of the PEF measurement could be criticized, because it may not correlate well with other measures used in the diagnostic procedure of asthma. However, we compared the effect on airflow between groups of children and did not attempt to use the PEF measurements as a tool for diagnosis or diagnostic criterion. Further, in a previous publication on the same cohort, we were able to show that PEF is significantly reduced in groups of children with asthma compared with healthy children .GSTP1–NOx interaction is likely not to be a false positive. There is, however, a lack of functional data from experimental studies to directly support our findings of gene–environment interactions. Because we found a high correlation between exposure to traffic NOx and traffic PM10 in this data set, we cannot disentangle effects between NOx and PM10 or, for that matter, other traffic-related pollutants such as ultrafine particles. Thus, exposure to source-specific NOx in this study should rather be viewed as a proxy for traffic-related air pollutants.The possibility of false-positive findings also needs to be considered, and we have addressed the multiple testing problems with false discovery rate analyses, which suggest that the observed GSTP1 and TNF genes seemed to modify the effect of early long-term exposure to ambient air pollution from traffic with respect to sensitization to common allergens in children. These results support the role of genes controlling the antioxidative system and inflammatory response in the pathogenesis of allergy and asthma.In conclusion, our data indicate gene–environment interaction effects on the development of allergic diseases in childhood. Specifically, variants in the


In the present study we report a systematic Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Streptococcus agalactiae and Enterococcus faecalis. The inhibition of adenovirus and lentivirus infection of MDCK cells was also tested. A homologous series of cationic surfactants, alkyl-N,N,N-trimethylammonium bromides (CnTAB), with varying alkyl chains were shown to be bactericidal and fungicidal at doses that were related to the surfactant critical micelle concentrations (CMC), all of them at concentrations significantly below the CMC. In general, bacteria were more susceptible to this surfactant group than C. albicans and this organism, in turn, was more susceptible than MDCK cells. This suggests that the CnTAB may be useful as vaginal disinfectants only in so far as bacterial and fungal infections are concerned. None of the surfactants examined, including those that have been used in pre-clinical studies, showed inhibition of adenovirus or lentivirus infection of MDCK cells or spermicidal activity at doses that were sub-toxic to MDCK cells.Toxicity was evaluated in mammalian columnar epithelial (MDCK) cells, human sperm cells, The results of this study lead us to propose that systematic analysis of surfactant toxicity, such as we report in the present work, be made a mandatory pre-condition for the use of these substances in pre-clinical animal and/or human studies. On the other hand, the world population continues to grow at an alarming rate with a high incidence of unwanted pregnancies. Condoms, when used correctly and consistently, provide a high level of protection against STDs (including HIV) and pregnancies, but many women lack the social and/or economic power to persuade their partners to use them. These facts have highlighted the importance of efforts to develop other approaches to prevent STDs and unplanned pregnancies, especially women-targeted methodologies such as vaginal prophylactic products, especially microbicides nTAB are also known as microbicides and antifungal agents The answers to these questions require a multi-step, systematic investigation on the effect of different types of surfactants on fungal and bacterial growth, viral infection and the viability of mammalian cells  and human sperm cells before any possible application is proposed. The results have also to be related to the physico-chemical properties of the surfactants since these are known to condition the effect of surfactants on lipid bilayer membranes nTAB were bactericidal and fungicidal at concentrations that were not toxic to mammalian columnar epithelial cells grown to confluence. At concentrations that were sub-toxic to the mammalian epithelial cells all the surfactants examined were neither spermicidal nor did they prevent viral infection of the epithelial cells.We show here that only the CEscherichia coli, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Streptococcus agalactiae, and Enterococcus faecalis as bacterial models. The effect of four surfactant classes with similar apolar parts; namely, cationic (C12TAB), anionic (SDS), zwitterionic (DDPS), and nonionic (Triton X-100) were first tested for their effects on the growth curves of E. coli at concentrations corresponding to various fractions and/or multiples of their respective CMCs. The CMC of the surfactants was used as a reference concentration for the following reason: The formation of surfactant micelles in aqueous media may be considered, under certain conditions, to be similar to a phase separation between an aqueous solution of surfactant monomers and a micellar phase. The surfactant concentration at which this phase separation occurs is the CMC. Within a given homologous series of surfactants the CMC is linearly proportional to the free energy of partitioning of the apolar part of the surfactant between an apolar environment such as a micelle or the lipid bilayer of a cell membrane and the aqueous phase We chose 12TAB began inhibiting the growth of E. coli at a concentration of CMC/25 and was totally inhibitory at all higher concentrations examined. DDPS showed some inhibition of bacterial growth only at CMC/1 and SDS at a concentration of CMC/2. The non-ionic surfactant, Triton X-100, did not inhibit the bacterial growth even at concentrations as high as CMC×100 (not shown). For convenience the CMC of these detergents is listed in the legend to As shown in 12TAB, we examined the effect of some homologues of this surfactant, namely C10TAB and C14TAB, on the growth curves of E. coli. The results are also shown in nTAB homologues albeit at different concentrations relative to their respective CMC. The toxicity of C10TAB began to be noticeable at CMC/100, that of C12TAB at CMC/25 and that of C14TAB at CMC/5. The bromide anion was not the determinant in the toxicity of these surfactants since no differences were found between the toxicities of the CnTAB and their chloride analogues (results not shown).Given the results obtained with CnTAB surfactants was also tested on E. coli cultures growing at log-phase. The results are shown in 12TAB and C14TAB show no or only a very small bactericidal effect at their respective CMC/100 concentrations. In contrast, C10TAB was still effective at its CMC/100 concentration and significant bactericidal activity was seen at 60 and 120 min of exposure at the concentration of CMC/100  infections we examined the toxicity of the same surfactants towards alactiae  the surffaecalis  was, simCandida albicans as a fungal model to test for toxicity of the surfactants examined in this work. This organism is responsible for vulvovaginal candidiasis, a common mucosal infection in women of childbearing age. Furthermore, fungal infections have recently emerged as a growing threat to human health in seriously debilitated and immunocompromised diseases, such as AIDS and cancer We chose the yeast C. albicans. Although here, as in the case of E. coli, the toxic ranking of the surfactants examined, using the concentration criterion mentioned above, is C10TAB≈C12TAB>C14TAB>DDPS>SDS>Triton X-100, there are subtle differences that merit attention: C. albicans was more susceptible to the cytotoxic effects of SDS and DDPS than was E. coli. These differences suggest that surfactant toxicity is not just dependent upon the chemical structure of the surfactants but also upon the nature of the cell membrane, the C. albicans membrane being different both in chemical composition and physical properties from the membrane(s) of bacteria. Unlike the case of the bacteria, the toxicity of the CnTABs toward C. albicans is not related to the chain length.in vitro epithelial cell system. The reason for this choice was that: 1) This cell line is derived from a columnar epithelium and the vaginal columnar epithelium was shown to be the primary site of damage in studies of surfactant use in animal models We chose Madine-Derby Canine Kidney (MDCK) cells as a model nTAB, C14TAB was the least toxic, the toxicity beginning to be noticeable at a concentration higher than CMC/5. In contrast C10TAB and C12TAB were noticeably toxic already at concentrations above CMC/25 (result not shown). SDS was toxic to the polarized MDCK cells at concentrations of CMC/5. The results with the MTT test for viability  and lentivirus (an enveloped retrovirus) were chosen as viral models since they are easily and relatively safely handled in the laboratory (both viruses are not able to replicate in humans). The lentivirus used here is derived from HIV-1 and pseudotyped with the envelope G-protein from the Vesicular Stomatitis virus. This confers higher physical stability in vitro toxicity to a number of species of bacteria and viruses, including HIV, 10TAB and C12TAB were the most toxic. The toxicity ranking of surfactants studied towards human sperm cells, using the same concentration criterion as used before, was: C10TAB≈C12TAB>SDS>C14TAB>DDPS>Triton X-100. The results indicated again that the toxic effects of surfactants depend on the target cell. Cytotoxicity studies were also performed with N-9 and revealed that this spermicide exhibited toxicity towards human sperm at concentrations of CMC/1 (results not shown).The effect of these surfactants on sperm viability, on sperm-membrane integrity as monitored by the Hypo-Osmotic Swelling (HOS) test, and as potential spermicides using the Sander-Cramer criteria were also assayed in human samples . N-9, a 14TAB and N-9 were able to affect the integrity of the plasma membrane of the sperm cells  tested by us are neither spermicidal nor bactericidal, nor protective against viral infection of polarized epithelial cells, below concentrations at which they are toxic to the epithelial cells themselves. This result was somehow unexpected for SDS (the anionic surfactant tested by us), which is considered an attractive candidate microbicide due to its lower cytotoxicity In this work we have demonstrated an easy and fast 10TAB does not show cytotoxicity towards mammalian cells at concentrations between CMC/25 and CMC/100 but is toxic to all the bacteria (with the exception of P. aeruginosa) and to C. albicans at concentrations of CMC/25 or lower. It also has a rapid microbicidal action (<30 min). 2) The anti-bacterial and anti-fungal activities were related to the surfactant CMC. The chain length dependence (C10TAB>C12TAB>C14TAB) may be related to the fact that the absolute concentrations are also an important determinant in their microbicidal action since the CMC of C10TAB is about 10-fold higher than that of C12TAB and about 100-fold higher than that of C14TAB. Similar results have been reported for amphiphilic betaine esters with a chain length of C10–C1412TAB is more toxic than C10TAB or C14TAB.We may highlight two interesting observations in the present study: 1) There appears to be some selectivity in the action of the cationic quaternary ammonium compounds on bacteria and yeast on the one hand and mammalian epithelial cells and spermatozoa on the other. CE. coli. Once in the cytoplasm the surfactant may interact with several cytoplasmic components and alter their activity. In particular, the cationic surfactants may be expected to interact with negatively charged polymers such as DNA and RNA for which they are known to have very high affinities and with which they form compact structures Though the disinfectant properties of the cationic surfactants have been extensively reported, their mechanism of action is not fully understood. It has, plausibly, been proposed to involve changes in the surface electrostatic potential of the membranes into which the hydrophobic portion of these surfactants is inserted as a consequence of the hydrophobic effect in vitro experiments have the advantage of convenience, flexibility, speed, and low cost. The results obtained from the present systematic study provide insights on the applicability and the safety of using surfactants as contraceptive, anti-viral, and anti-microbial agents. On the basis of these results, none of the surfactants tested by us could qualify for testing with animal, and much less with human, subjects.In conclusion, although animal models and pre-clinical studies offer more complete experimental conditions, 12TAB), and tetradecyltrimethylammonium bromide (C14TAB) were obtained from Sigma. Decyltrimethylammonium bromide (C10TAB), was from Fluka. Stock solutions of surfactants were prepared as multiples of their CMC MDCK cell culture media were from Invitrogen or PAO Laboratories. All chemicals were of the highest commercially available purity and were used as received. 3--2,5-diphenyltetrazolium bromide (MTT), the nonionic surfactant Triton X-100 (TX-100), the anionic sodium dodecyl sulfate (SDS), the zwitterionic  and the cationic surfactants: dodecyltrimethylammonium bromide  and Neisseria gonorrhoeae (49226) were from the ATCC. E. coli, P. aeruginosa, S. agalactiae and E. faecalis were grown overnight in Luria broth medium at 37°C and shaking at 200 rpm. Bacterial and yeast growth was assessed by measuring the absorbance at 600 nm as a function of time, in presence or absence of detergents.Wild type Bactericide activity was quantified by plating bacteria that had been previously incubated with surfactants on LB/agar plates after separation from the added surfactants by centrifugation. The LB/agar plates were then incubated at 37°C for 16–18 h and microbicide efficiency was determined by counting the number of colonies formed.N. gonorrhoeae was grown on Chocolate Agar PolyViteX® plates (BioMerieux), and GCB plus Kellogg's supplement 2 atmosphere for 1 h. The samples were then diluted 100-fold and 100 µL were inoculated on Chocolate Agar plates. The plates were incubated at 37°C in a 5% CO2 atmosphere for 24 h and scored for colony growth. Counts from bacteria treated with surfactants were compared with those from the controls which contained no surfactant.C. albicans was grown in conical flasks in YEPD medium  on an orbital shaker at 37°C and shaking at 200 rpm.MDCK cells were grown on glass-coverslips or on 12-mm Transwell polycarbonate filters  for 4 days in MEM with 10% FCS. On the third day, the cells were infected with adenoviruses or lentiviruses for 1 hour and the surfactants were added 10 min before the infection. After incubation for 1 h the viruses and surfactant were removed and fresh medium was added to the cells. Only in the experiments with lentivirus the infection was done in the presence of 4 µg/mL polybrene (Sigma). At 16–18 h (adenovirus) or 48 h (lentivirus) post-infection, the cells were fixed with PFA, permeabilized with Triton X-100 and incubated with rhodamine-phalloidin. The glass-coverslips or the polycarbonate filters were then mounted and observed under a Zeiss Axioskop 2 plus or a Zeiss confocal microscope.The generation of the plasmid (GFP-GL-GPI) and adenovirus used were described previously Viable cells were measured by their ability to reduce the tetrazolium salt, 3--2,5-diphenyltetrasodiumbromide (MTT), to a formazan dye detectable by spectrophotometric analysis in a microplate reader.+), at 340 nm.The activity of the cytoplasmic enzyme LDH in the extracellular medium, which evaluates plasma membrane integrity was measured spectrophotometrically according to the method described by Bergmeyer and Bernt The effect of surfactants on human sperm was quantified using three distinct assays for sperm viability, sperm integrity and spermicidal activity of added substances.Human sperm was obtained from the Human Reproduction Laboratory, Department of Maternal-Fetal Medicine, Genetics and Human Reproduction , from healthy males undergoing routine semen analysis or fertility treatment. All donors signed informed consent forms, and all human material was used in accordance with the appropriate ethical guidelines provided by the Coimbra University Hospitals and its Internal Review Board (IRB). Fresh semen samples were obtained by masturbation after 3–5 days of sexual abstinence, and routine seminal analysis was performed according to the World Health Organization guidelines . The effect of surfactants on sperm viability was determined microscopically after nuclear staining with SYBR-14 and propidium iodide (PI), both prepared in DMSO The Hypo-Osmotic Swelling (HOS) test was carried out 1 h after the addition of surfactants. Briefly, after treatment with surfactants human sperm was incubated in a 150 mOsm hypo-osmotic saline solution at 37°C during 60 min Activity of surfactants on sperm motility was determined by the Sander–Cramer assay p value of <0.05 was considered to be statistically significant.Results were expressed as the means±standard deviations (S.D.). Statistical significance was assessed by the ANOVA test. A


Child with mild scoliosis is always a subject of interest for most orthopaedic surgeons regarding progression. Literature described Hueter-Volkmann theory regarding disc and vertebral wedging, and muscular imbalance for the progression of adolescent idiopathic scoliosis. However, many authors reported spontaneous resolution of curves also without any reason for that and the rate of resolution reported is almost 25%. Purpose of this study was to question the role of paraspinal muscle tuning/balancing mechanism, especially in patients with idiopathic scoliosis with early mild curve, for spontaneous regression or progression as well as changing pattern of curves.An observational study of serial radiograms in 169 idiopathic scoliosis children (with minimum follow-up one year) was carried. All children with Cobb angle < 25° and who were diagnosed for the first time were selected. As a sign of immaturity at the time of diagnosis, all children had Risser sign 0. No treatment was given to entire study group. Children were divided in three groups at final follow-up: Group A, B and C as children with regression, no change and progression of their curves, respectively. Additionally changes in the pattern of curve were also noted.Average age was 9.2 years at first visit and 10.11 years at final follow-up with an average follow-up of 21 months. 32.5% (55/169), 41.4% (70/169) and 26% (44/169) children exhibited regression, no change and progression in their curves, respectively. 46.1% of children (78/169) showed changing pattern of their curves during the follow-up visits before it settled down to final curve. Comparing final fate of curve with side of curve and number of curves it did not show any relationship (p > 0.05) in our study population.Possible reason for changing patterns could be better explained by the tuning/balancing mechanism of spinal column that makes an effort to balance the spine and result into spontaneous regression or prevent further progression of curve. If this which we called as "tuning/balancing mechanism" fails, curve will ultimately progress. A major concern of orthopaedic surgeons in managing children with idiopathic scoliosis with a minor curvature is identifying how many and which curve will progress to severe deformities that requires treatment -9. AccurSoucacos et al reported 27.4% spontaneous improvement of at least 5° in the curve and 9.5% out of them had complete resolution . Brooks In adolescent idiopathic scoliosis, a larger back muscle volume has been reported at the apex on the convex side of the spine ,13. LiteIn present study, we reviewed the curve pattern of immature idiopathic scoliosis patients; and observed different pattern of curve during progression, regression or resolution of curve. Based on our observation we think that tuning or balancing mechanism of spinal column may be an important factor in progression, stabilization or regression of curve, especially in mild degree early curves. We tried to answer our question that in growing spinal muscles or ligaments try to balance the whole spinal column as they get matured and, due to this balancing mechanism mild scoliosis curve shows regression or resolution . Those pA retrospective observational study was carried out in 169 idiopathic scoliosis patients  who had regular visits in our outpatient clinic. Average age of patients was 9.2 ± 2.1 years  at the first visit and 10.11 ± 2.8 years  at final follow-up. Patients who had idiopathic scoliosis, Risser sign 0 at first visit, at least one year regular follow-up, initial curve between 11 and 25 degree and no prior treatment either in form of bracing or manipulation were selected for this study.We retrospectively observed serial radiograms for all patients and compared their initial Cobb angle with follow-up and at last follow-up Cobb angle. All children underwent for standing anteroposterior and lateral radiogram of whole spine including both hip joints and full length both lower limb radiograms by a single radiologist. All radiograms were taken on a single X-ray machine to avoid any error. By clinical as well as radiological examination, leg length discrepancy was ruled out in the subject group. Based on our observation, we divided the entire study population into three groups: group A who had regression or resolution of curve for 5° or more at final follow-up; group B who had no change (or difference less than 5°) in Cobb angle at final follow-up; and group C who had progression of curve for more than 5° at final follow-up. We found out the percentage of children in each group from our observation to have an idea of incidence of regression, progression or stabilization of curve. We also tried to find out any change in the curve pattern during each follow-up that might be responsible for regression or progression of curve. Criteria for considering children in changing pattern were the change in side of curve during follow-up or/and wavy pattern of Cobb angle  Figure  and 2. FWe also analyzed side of primary curve, number of curves (single or double) and gender of patients in each group to find out any relationship with regression, stabilization or progression of curve. We used Chi-square test to analyze the statistical significance between side of curve, number of curve and gender with curve regression or progression. P value < 0.05 was considered for significance for all tests.Average follow-up was 21 ± 9  months. Average follow-up in each group with their average age at initial and final follow-up are shown in table There were 26 boys and 29 girls in group A; 16 boys and 54 girls in group B; and 8 boys and 36 girls in group C. Comparing fate of curves according to gender it showed significant relationship  between gender of patient and curve regression, stability or progression which suggested that boys have greater tendency to stabilize curves while girls have higher tendency for the progression. 38, 56 and 28 curves were right sided and 17, 14 and 16 curves were left sided in group A, B and C, respectively. Comparing the side of curves in each group with final outcome it did not show any relationship  between side of curve and regression, stabilization or progression of curve. Similarly there were 33, 35 and 19 children had single curve  and 22, 35 and 25 children had double curves (major thoracic and minor lumbar or major lumbar and minor thoracic) in group A, B and C, respectively. Comparing number of curves in each group it also did not exhibit any relationship  between number of curves and regression, stabilization or progression of curves.Results of our study showed that in immature patients who were detected with mild scoliosis for the first time, changing pattern in their curves before displaying regression, stabilization or progression of curves was frequently observed. The incidence rate of changing pattern was noted 46.1%. The possible reason for these changing patterns could be better explained by the tuning/balancing mechanism of paraspinal muscles which try to balance the spine, and result into spontaneous regression or stabilization of curve. If this mechanism which we call as "tuning/balancing mechanism" fails, the curve will ultimately show progression.Only a subset of curves detected through screening are destined to progress to a point of potential clinical significance. The probability that curves will progress more than 5° can vary from 5% to 90%, depending on the patient's age, sex, and skeletal maturity, and the pattern and magnitude of the curve ,26,27. PThe aetiology of all but idiopathic is self-evident and the progression of deformity is popularly believed to be linked to the mechanical modulation of growth theory ,30. It iIn another experimental study by Schwartzmann and Miles showed that selective muscle imbalance can be produced without muscle excision by the use of inert material to prevent muscle reattachment which will produce lateral curvature . Muscle The possible criticism for this study might be angle of curve to consider as scoliotic angle. We were concerned about role of spinal column in the developing (immature) curve and that's why we considered those children who had curve more than 10° as per definition of scoliosis. Additionally initial curves of all three groups did not show any significant difference p = 0.15, ANOVA) in our study. Second criticism might be average follow-up period which is comparatively less with an average of 21 months (minimum 12 months). We would like to clear that actual follow-up period for these patients are longer than that; however, to keep uniform follow-up in all three groups we considered final follow-up when the curves started showing uniform pattern during three follow-up which did not fall in changing pattern category by us. And we did not observe any changes later on as described by us in subsequent follow-up. Cheung et al , ANOVA i can't bePresent study shows the possible role of spinal column tuning mechanism in skeletally immature children with mild scoliosis curve for regression, stabilization or progression. If rehabilitation or physical therapy program is applied during this period of immaturity, scoliosis curve might regress with increased activation.The authors declare that they have no competing interests. Each author certifies that he has no commercial associations  that might pose a conflict of interest in connection with the submitted article.HNM has contributed in conception and design and acquisition of data, analysis and interpretation of data, drafting the manuscript and revising it critically, SWS has contributed in conception and design of data, drafting the manuscript and given the final approval of manuscript, JHY has contributed in acquisition of data, revising the manuscript critically and given the final approval, JYH has contributed in acquisition of data and analysis and interpretation of data; and KPV and NM have contributed in revising the manuscript critically.All authors read and approved the final manuscript.