TopHat-Fusion is an enhanced version of TopHat, an efficient program that aligns RNA-seq reads without relying on existing annotation. Because it is independent of gene annotation, TopHat-Fusion can discover fusion products deriving from known genes, unknown genes and unannotated splice variants of known genes. Using RNA-seq data from breast and prostate cancer cell lines, we detected both previously reported and novel fusions with solid supporting evidence. TopHat-Fusion is available at  To do this, we aligned each end of every mate pair separately, allowing them to be aligned to at most two places, and then combined and converted them to the input format required by FusionSeq.Because it is also a standalone fusion detection system, we ran FusionSeq (0.7.0)  on one oRPS6KB1-TMEM49) because it was supported by only one mate pair. In contrast, TopHat-Fusion found this fusion because it was supported by three mate pairs from TopHat-Fusion's alignment algorithm: one mate pair contains a read that spans a splice junction, and the other contains a read that spans a fusion point. These spliced alignments are not found by Bowtie or ELAND. With this spliced mapping capability, TopHat-Fusion will be expected to have higher sensitivity than those based on non-gapped aligners. When the minimum number of mate pairs is reduced to 1, FusionSeq found all three known fusions at the expense of increased running time (9 hours versus just over 2 hours) and a large increase in the number of candidate fusions reported .When we required at least two supporting mate pairs for a fusion , FusionSeq missed one true fusion  that would otherwise eliminate two of the true fusions. FusionSeq reported 14,510 gene fusions , BCAS4-BCAS3 , and RPS6KB1-TMEM49 . First, we used SPER of 0.161181 and DASPER of 0.133692 to find the minimum set of fusion candidates that include the three known gene fusions. This reduced the number of candidates from 14,510 to 11,774. Second, we used the SPER and DASPER values from ARFGEF2-SULF2 and BCAS4-BCAS3, which resulted in 1,269 and 512 predicted fusions, respectively.FusionSeq reports three scores for each transcript: SPER , DASPER (difference between observed and expected SPER), and RESPER . Because RESPER is proportional to SPER in the same data, we used SPER and DASPER to control the number of fusion candidates: We next compared TopHat-Fusion with deFuse (0.4.2) . deFuse CSE1L-ENSG00000236127, was not considered because ENSG00000236127 has been removed from the recent Ensembl database). Both programs missed two fusion transcripts: DHX35-ITCH and NFS1-PREX1. However, TopHat-Fusion had far fewer false positives: it predicted 42 fusions in total, while deFuse predicted 1,670 , one of the most powerful features in TopHat-Fusion is its ability to map reads across introns, indels, and fusion points in an efficient way and report the alignments in a modified SAM (Sequence Alignment/Map) format .Unlike previous approaches based on discordantly mapping paired reads and known gene annotations, TopHat-Fusion can find either individual or paired reads that span gene fusions, and it runs independently of known genes. These capabilities increase its sensitivity and allow it to find fusions that include novel genes and novel splice variants of known genes. In experiments using multiple cell lines from previous studies, TopHat-Fusion identified 34 of 38 previously known fusions. It also found 61 fusion genes not previously reported in those data, each of which had solid support from multiple reads or pairs of reads.The first step in analysis of an RNA-seq data set is to align (map) the reads to the genome, which is complicated by the presence of introns. Because introns can be very long, particularly in mammalian genomes, the alignment program must be capable of aligning a read in two or more pieces that can be widely separated on a chromosome. The size of RNA-seq data sets, numbering in the tens of millions or even hundreds of millions of reads, demands that spliced alignment programs also be very efficient. The TopHat program achieves efficiency primarily through the use of the Bowtie aligner , an extrTopHat-Fusion implements several major changes to the original TopHat algorithm, all designed to enable discovery of fusion transcripts Figure . After iThe algorithm then uses Bowtie to map the 25-bp segments to the genome. For normal transcripts, the TopHat algorithm requires that segments must align in a pattern consistent with introns; that is, the segments may be separated by a user-defined maximum intron length, and they must align in the same orientation along the same chromosome. For fusion transcripts, TopHat-Fusion relaxes both these constraints, allowing it to detect fusions across chromosomes as well as fusions caused by inversions.et al. [HOMEZ-MYH6 and KIAA1267-ARL17A were supported by 2 and 11 multi-mapped read pairs, respectively. Therefore, instead of eliminating all multi-mapped reads, we impose an upper bound M (default M = 2) on the number of mappings per read. If a read or a pair of reads has M or fewer multi-mappings, then all mappings for that read are considered. Reads with > M mappings are discarded.Following the mapping step, we filter out candidate fusion events involving multi-copy genes or other repetitive sequences, on the assumption that these sequences cause mapping artifacts. However, some multi-mapped reads  might correspond to genuine fusions: for example, in Kinsella et al. , the knoTo further reduce the likelihood of false positives, we require that each read mapping across a fusion point have at least 13 bases matching on both sides of the fusion, with no more than two mismatches. We consider alignments to be fusion candidates when the two 'sides' of the event either (a) reside on different chromosomes or (b) reside on the same chromosome and are separated by at least 100,000 bp. The latter are the results of intra-chromosomal rearrangements or possibly read-through transcription events. We chose the 100,000-bp minimum distance as a compromise that allows TopHat-Fusion to detect intra-chromosomal rearrangements while excluding most but not all read-through transcripts. Intra-chromosomal fusions may also include inversions.As shown in Figure i with no gap, (2) chromosome j where it spans an intron, and (3) a fusion contig formed between chromosome m and chromosome n. Our scoring function prefers (1), followed by (2), and by (3). For IUM read 2  an alignment to chromosome k with a small deletion. These two alignments both incur the same penalty, but we select (1) because it has fewer mismatches.After stitching together the segment mappings to produce full alignments, we collect those reads that have at least one alignment spanning the entire read. We then choose the best alignment for each read using a heuristic scoring function, defined below. We assign penalties for alignments that span introns (-2), indels (-4), or fusions (-4). For each potential fusion, we require that spanning reads have at least 13 bp aligned on both sides of the fusion point.  After applying the penalties, if a read has more than one alignment with the same minimum penalty score, then the read with the fewest mismatches is selected. For example, in Figure We imposed further filters for each data set: (1) in the breast cancer cell lines , we required two supporting pairs and the sum of spanning reads and supporting pairs to be at least 5; (2) in the VCaP paired-end reads, we required the sum of spanning reads and supporting pairs to be at least 10; (3) in the UHR paired-end reads, we required (i) three spanning reads and two supporting pairs or (ii) the sum of spanning reads and supporting pairs to be at least 10; and (4) in the UHR single-end reads, we required two spanning reads. These numbers were determined empirically using known fusions as a quality control. All candidates that fail to satisfy these filters were eliminated.5 to just a few hundred.In order to remove false positive fusions caused by repeats, we extract the two 23-base sequences spanning each fusion point and then map them against the entire human genome. We convert the resulting alignments into a list of pairs . For each 23-mer adjacent to a fusion point, we test to determine if the other 23-mer occurs within 100,000 bp on the same chromosome. If so, then it is likely a repeat and we eliminate the fusion candidate. We further require that at least one side of a fusion contains an annotated gene (based on known genes from RefSeq), otherwise the fusion is filtered out. These steps alone reduced the number of fusion candidates in our experiments from 10et al. [As reported in Edgren et al. , true fuEven with strict parameters for the initial alignment, many of the segments will map to multiple locations, which can make it appear that a read spans two chromosomes. Thus the algorithm may find large numbers of false positives, primarily due to the presence of millions of repetitive sequences in the human genome. Even after filtering to choose the best alignment per read, the experiments reported here yielded initial sets of about 400,000 and 135,000 fusion gene candidates from the breast cancer  and prostate cancer (VCaP) cell lines, respectively. The additional filtering steps eliminated the vast majority of these false positives, reducing the output to 76 and 19 fusion candidates, respectively, all of which have strong supporting evidence Tables  and 3.The scoring function used to rank fusion candidates uses the number of paired reads in which the reads map on either side of the fusion point in a consistent orientation Figure  as well The overall fusion score is computed as:where lcount is the number of bases covered in a 300-bp window on the left Figure , lavg iswhere lwindow is the size of the left window (300 bp).TopHat-Fusion outputs alignments of singleton reads and paired-end reads mapped across fusion points in SAM format , enablinbp: base pair; DASPER: difference between observed and expected SPER; IUM: initially unmapped; RESPER: ratio of observed SPER to the average of all SPERs; SAM: Sequence Alignment/Map; SPER: supportive paired-end reads; UHR: universal human reference.DK developed the TopHat-Fusion algorithms, performed the analysis and discussed the results, implemented TopHat-Fusion and wrote the manuscript. SLS developed the TopHat-Fusion algorithms, performed the analysis and discussed the results, and wrote the manuscript. All authors have read and approved the manuscript for publication.Table S1 - 76 candidate fusions including multiple fusion points in the breast cancer cell lines. Additional details for the 76 fusions detected by TopHat-Fusion in the breast cancer cell lines . Some of the genes contain multiple fusion points, presumably due to alternative splicing.Click here for fileTable S2 - 19 candidate fusions including multiple fusion points in the prostate cancer cell line. Nineteen fusion genes detected by TopHat-Fusion in a prostate cancer cell line (VCaP), including several with multiple fusion points due to alternative splicing.Click here for fileBCR-ABL1 fusion for single-end and paired-end readsFigure S1 - read distributions around . This figure shows read distributions around the BCR-ABL1 fusion gene in Universal Human Reference (UHR) data. (a) The read distribution for single-end reads (100 bp or less). (b) Read distribution for paired-end reads (50 bp) from 300-bp fragments. Coverage was similar with either data set.Click here for fileTable S3 - the top 20 fusion candidates reported by TopHat-Fusion in the UHR data. The top 20 fusion genes from the Universal Human Reference (UHR) data found by TopHat-Fusion, sorted by the scoring scheme described in Figure Click here for fileTable S4 - 45 fusion candidates reported by TopHat-Fusion in Illumina Body Map 2.0 data. Using two samples (testes and thyroid) from Illumina Body Map 2.0 data, TopHat-Fusion reports 45 fusions.Click here for fileList of 14,510 fusion candidates reported by FusionSeq for MCF7 sample data.Click here for fileTable S5 - 42 fusion candidates reported by TopHat-Fusion in SKBR3 and MCF7 cell lines. Twenty-eight and fourteen candidate fusions are reported in SKBR3 and MCF7 samples, respectively, when the filtering parameters are changed to one spanning read and two supporting mate pairs.Click here for fileList of 275 fusion candidates reported by deFuse in MCF7 sample data.Click here for fileList of 1,395 fusion candidates reported by deFuse in SKBR3 sample data.Click here for fileSupplementary methods.Click here for fileFigure S2 - Finding fusions using two segments and partner reads in paired-end reads. (a) TopHat allows one to three mismatches when mapping segments using Bowtie, which enables segments to be mapped even if a few bases cross a fusion point . These two segments, mapped to two different chromosomes, are used to identify a fusion point. (b) For paired-end reads, the mapped position of the partner read is used to narrow down the range of a fusion point. The second segment (shown in green) cannot be mapped because it spans a fusion point. Here, its partner read is mapped and the fusion point is likely to be located within the inner mate distance ± standard deviation of the left genomic coordinate of the partner read. TopHat-Fusion is able to use this relatively small range to efficiently map the right part of the second segment to the right side of a fusion (case 2). The left part of the second segment is aligned to the right side of the mapped first segment (case 3).Click here for fileFigure S3 - stitching segments to produce a full read alignment. (a) The segment in the third row for segment 1 and the one in the first row for segment 2 are connected because they are on the same chromosome (i) in the forward direction and with adjacent coordinates. These are then matched to the second row in segment 3 and glued together, producing the full-length read alignment at the bottom. (b) TopHat-Fusion tries to connect the segment in the second row for segment 1 with segments in the first and second rows for segment 2, but neither succeeds. Case 1 would require two fusion points in the same read, and case 2 cannot be fused with consistent coordinates. (c) Attempts to connect the segment in the second row for segment 2 with the one in the first row in segment 3: in case 3, there is no intron available, there is no fusion in case 4, and case 5 would require more than one fusion.Click here for file


We analyzed the levels of selected micro-RNAs in normal prostate tissue to assess their potential to indicate tumor foci elsewhere in the prostate. Histologically normal prostate tissue samples from 31 prostate cancer patients and two cancer negative control groups with either unsuspicious or elevated prostate specific antigen (PSA) levels  were analyzed. Based on the expression analysis of 157 microRNAs in a pool of prostate tissue samples and information from data bases/literature, we selected eight microRNAs for quantification by real-time polymerase chain reactions (RT-PCRs). Selected miRNAs were analyzed in histologically tumor-free biopsy samples from patients and healthy controls. We identified seven microRNAs , which displayed significant differential expression in normal prostate tissue from men with prostate cancer compared to both cancer negative control groups. Four microRNAs  remained to significantly discriminate normal tissues from prostate cancer patients from those of the cancer negative control group with elevated PSA levels. The transcript levels of these microRNAs were highly indicative for the presence of cancer in the prostates, independently of the PSA level. Our results suggest a microRNA-pattern in histologically normal prostate tissue, indicating prostate cancer elsewhere in the organ. Understanding the biology of early tumors is of eminent strategic importance, due to its potential impact on the diagnosis, treatment and prognosis of prostate carcinoma. To date, knowledge about early prostate cancer development, however, is still limited. Nevertheless, it is clear that morphologically detectable malignant changes are preceded by aberrations at the molecular-biological level. The volume of such molecularly altered areas should be substantially larger than the histologically manifest cancer or even the prostatic intraepithelial neoplasia (PIN) area therein . These gThe identification of such molecular alterations in unsuspicious prostate tissue could lead to an interesting application in clinical routine: the assessment of prostate cancer risks by evaluation of apparently tumor-free biopsy specimens. To date, the definitive diagnosis of prostate cancer is exclusively dependent on prostate biopsies, which are taken from patients with elevated PSA levels  and examined histopathologically. The absence of malignant cells precludes the diagnosis of cancer. Now, small tumors are often missed by prostate biopsies. In such apparently negative cases, histologically normal biopsy specimens possessing molecular signatures, which suggest the presence of adjacent cancer or imminent progression towards malignancy , could rOnly one decade ago, microRNAs—small RNA molecules of 20–22 nucleotides length—have been identified to be important regulators of the human transcriptome associated with several disorders— especially cancer ,5. Each Here, we conducted a pilot study on the expression of well defined microRNAs in normal prostate tissue to test their potential to indicate occult prostate cancer.As for transcripts in our previous study , this stp-values in the graphs in Indeed, we found a downregulation of these microRNAs in the tumor positive group. As demonstrated by explorative univariate statistics on three groups , the differences between these collectives were significant  compared to both age-matched cancer negative groups (TN-nPSA group and TN-hPSA group). This could indicate that the shutdown of these microRNAs is involved in early malignant transformation (see below). If this is true, it would fit with observations of an extensive micro-transcriptome repression in “mature” cancer tissues ,8, indicp-values in the graphs in An attempt to find a robust marker set indicating occult prostate cancer, however, should regard the clinical everyday situation. There, usually only individuals with high PSA values are submitted to intensive diagnostic procedures, such as biopsy-taking. Therefore, we tested whether the observed differential microRNA transcription pattern discriminates the TP group from the TN-hPSA group alone  and stromal (COL1A1) tissue resulted in comparable gene expression rates in the TP group and the TN-hPSA group , suggesting that the proportions of different tissue types in the biopsies were similar.The quantification of gene markers for epithelial  in one principal cluster, dividing it from a second principal cluster composed of individuals from both TN groups .A discriminant function assigned almost 90% of the TP group individuals correctly to suffer from PCa, while 70% of the TN-hPSA group was correctly “diagnosed” to be tumor free. Notably, 100% of the TN-nPSA group , was correctly assigned to be tumor-free. Overall, 83.3% of all individuals were correctly classified .vs. TN-hPSA group) was examined by a ROC curve ]. It also has been shown that let-7a downregulates the oncogenes, E2F2 and cyclin D2 [let-7a, let-7b, miR-16 and miR-185 are known to regulate tumorigenic processes in general or in prostate cancer. Let-7a has been shown to downregulate the transcript of the ma cells  and in lma cells . Elevatemalities  and resumalities . PreviouTP group )—a causayclin D2 . Let-7b yclin D2 . miR-16 yclin D2 . Also, myclin D2 , togetheyclin D2 . These syclin D2 –23.To test principal miRNAs expression in prostate tissue, we prepared an RNA pool of tumor negative prostate biopsies from 17 healthy donors  to assesAre the microRNAs associated with prostate cancer (according to MiRó data)? If yes, then:Are (prostate-) cancer-related target transcripts of the microRNAs experimentally validated or else consistently predicted by three independent prediction algorithms ? If yes, then:Are there reports on a role in human early cancer formation, malignant transformation or field cancerization in scientific literature (PubMed search results up to the year 2008)?The following microRNAs fitted best to these requirements and were chosen for further analyses in the main experiment: miR-124a, miR-146a, miR-146b, miR-185, miR-16, let-7a and let-7b.miR-187—a microRNA expressed in our prostate tissue pool, but not fitting to the selection criteria described above—was also included to the main experiment analysis for control purposes (see below).For the main experiment, we defined three collectives of tumor-free prostate biopsies taken by diagnostic ultrasound-guided transrectal biopsy probing :the tumor-positive group (TP group) composed of patients with proven PCa,the tumor negative/high PSA group (TN-hPSA group), composed of patients with an elevated PSA level, but a high likelihood of being cancer-free (as confirmed by multiple repeated negative biopsies ),the tumor negative/normal PSA group (TN-nPSA group), comprising healthy men with normal serum PSA (<1 ng/mL).The approval for the study was obtained from the local ethical committee. All patients gave written permission to additional tissue sampling for scientific purposes.Tissue specimens from all individuals were collected as previously described . Shortly20 ng of microRNA fractions were reversely transcribed to cDNA in volumes of 20 μL using the cDNA archive kit, according to the manufacturer’s protocol , using specific loop-RT-primers for all investigated mature microRNAs (see below). RT-PCRs were performed on the Gene-analyzer 7900 (Applied Biosystems) using microRNA assays on demand  to amplify up to 157 microRNAs  in dupliFor initial screens in the healthy prostate tissue pool, RT-PCR runs were examined for principal microRNA expression by assessment of dCt values and regular sigmoid shaped curves. For the main experiment, relative expression differences of selected microRNAs between the analyzed collectives were calculated using the ΔΔCt method . Small nARG, GUSB) and stromal (COL1A1) cells were analyzed on all groups by RT-PCRs, as previously published [Levels of tissue type-specific transcripts for epithelial  and an analysis for the creation of a discriminant function on the TP group and the TN-hPSA group, implying all individuals with complete quantification results of the included microRNAs. Sensitivities and specificities were compared by ROC curves. Statistic evaluation was performed using Microsoft Excel and SPSS.Due to non-Gaussian distribution patterns of the results, non-parametric tests  were applied to test for significant microRNA level differences of the compared groups. Transcript level differences were considered to be significant when means of the RNU44/48-double-normalized RQ sets displayed a In our previous study, we found indications for the existence of a gene transcription phenotype in histologically normal prostate tissue, indicating neighboring tumors . Here, tHowever, our study has some limitations. First, the selection procedure explained in the experimental section missed some miRNAs with a high consensus in newer prostate cancer research, as miR-96, miR-221/222 or miR-205 . HenThe clear, significant microRNA level differences found between the compared groups nevertheless give reason to further test our microRNA signature on larger and independent collectives for their applicability as diagnostic surrogate markers in biopsies from patients with elevated PSA serum levels.


Brassicaceae. Although the gene orf138 has been isolated as a determinant of Ogura-type CMS, no homologous sequence to orf138 has been found in public databases. Therefore, how orf138 sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene orf138.Plant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS) is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF) created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in atp9. As for tRNA, normal-type contained 17 tRNAs, while Ogura-type contained 17 tRNAs and one additional trnfM. The gene orf138 was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions . It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions  that are non-syntenic to normal-type genome, and the gene orf138 was found to be located at the edge of the largest unique region. Blast analysis performed to assign the unique regions showed that about 80% of the region was covered by short homologous sequences to the mitochondrial sequences of normal-type radish or other reported Brassicaceae species, although no homology was found for the remaining 20% of sequences.Ogura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional Brassicaceae mitochondrial sequences. This suggests that the regions unique to the Ogura-type genome were generated by integration and shuffling of pre-existing mitochondrial sequences during the evolution of Brassicaceae, and novel genes such as orf138 could have been created by the shuffling process of mitochondrial genome.Ogura-type mitochondrial genome was highly rearranged compared with the normal-type genome by recombination through one large repeat and multiple short repeats. The rearrangement has produced four unique regions in Ogura-type mitochondrial genome, and most of the unique regions are composed of known  Brassica hirta to 11.3 Mb in Silene conica[, due to the expansion of non-coding sequence and duplication of a large segment. It is also well known that plant mitochondrial genome evolves rapidly in structure and slowly in sequence. Plant mitochondria contain a number of repeats. The recombination via repeat sequences is believed to be responsible for extensive structural change. Incorporation of foreign DNA fragments that originated from plastid and nuclear genomes also can be found in plant mitochondrial genome. Because of frequent recombination and incorporation of foreign DNA, extensive genome reorganization and gene-order shuffling may occur. These changes in mitochondrial genome can produce novel open reading frames, some of which result in cytoplasmic male sterility (CMS) [Compared with animal mitochondrial genomes that are compact and relatively uniform in size, plant mitochondrial genomes are extremely complex and have characteristic features, including large genome size, frequent reorganization and incorporation of foreign DNA . Plant mne conica,3, due tty (CMS) .1 hybrid seed production in many crops. CMS has been widely observed in higher plants, and many reports show that CMS is caused by alteration of the mitochondrial genome [CMS is a maternally inherited trait in which a plant is unable to produce functional pollen. CMS is an economically important trait for Fl genome . These aBrassicaceae[1 seed production of European, Chinese and Japanese radishes. The cytoplasm was introduced into Brassica crops by intergeneric hybridization and somatic cell fusion, and has been utilized for F1 seed production in Brassica crops such as rapeseed worldwide [The Ogura male-sterile cytoplasm, which was originally found in an unknown variety of Japanese radish, is the most widely studied cytoplasm in ssicaceae. This cyorldwide ,8.orf138, is responsible for Ogura male sterility and specifically present in the mitochondrial genome of various radishes with Ogura-type cytoplasm [orf138 itself is also interesting; there is no evidence that orf138 is a chimeric gene consisting of native mitochondrial genes or sequence. No sequence homologous to orf138 is found in public databases; therefore, the origin of orf138 is a mystery. In addition, it has been shown that the mitochondrial genome of Ogura-type cytoplasm is highly rearranged compared with that of normal cytoplasm in radish. These rearrangements occurred in the loci atp1atp6cox1 and most notably orf138-atp8[orf138 is specifically located in the 5’ upstream region of the gene atp8 in Ogura radish, whereas the region is occupied by the gene cox1 in normal radish [Raphanus, how and when these extensive rearrangements occurred between two types of mitochondrial genome are not known.A mitochondrial gene, ytoplasm . The accytoplasm . Althougytoplasm , the molf138-atp8-17. The l radish . In the orf138 and Ogura-type mitochondrial genome in radish, we determined the complete nucleotide sequences of two mitochondrial genomes of radish, that is, Ogura- and normal-type mitochondrial genomes, and compared their structures. Gene contents and unique regions for each genome were analyzed in detail at the sequence level. Furthermore, we focused on short repeated sequences that may be involved in the recombination, leading to extensive rearrangements of the two genomes and to generation of the gene orf138.In this study, in order to obtain a better understanding of the origin of Brassicaceae. About 7% of Ogura and 3% of normal sequences are unique to each mitochondrial genome, and 80% of Ogura and 84% of normal sequences show high similarity to the sequence of rapeseed (Brassica napus L.) mitochondrial genome (GenBank: AP006444.1) [The Ogura and normal mitochondrial genomes are assembled as 258,426-bp and 244,036-bp circular molecules, respectively Figure . The ove06444.1) .atp9 (atp9-2). Gene content is identically conserved between the two radish mitochondrial genomes and it is also identical to that of rapeseed, except atp9-2 in Ogura-type genome. Normal-type genome contains similar sequence to atp9-2, but 3’ part of this sequence lacks similarity to atp9-2. The atp9-2 like sequence in normal-type genome encodes 70 amino acid protein, which is lacking atp9 domain partially. Eighteen of the conserved 33 protein-coding genes produce components of the electron transport chain and ATP synthase: nine subunits of complex I(nad1nad2nad3nad4nad4Lnad5nad6nad7 and nad9), apocytochrome b (cob) of complex III, three subunits of complex IV (cox1cox2 and cox3) and five subunits of complex V (atp1atp4atp6atp8 and atp9). Five additional proteins are involved in the biogenesis of cytochrome c (ccmBccmCccmFN1/ccmFN2 and ccmFC). Another eight genes encode ribosomal proteins . The two remaining genes encode maturase (matR) and orfX. The gene ccmFN is divided into two reading frames in mitochondrial genomes of radish like in those of rapeseed and Arabidopsis[rrn5rrn18 and rrn26) are highly conserved between the two mitochondrial genomes of radish.Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, while Ogura-type genomes contained same genes and one additional abidopsis,19. Threatp6 is highly diverged between the two radishes because of recombination that occurred in the locus [orfX also lacks similarity to that of normal-type. This is caused by a 48-bp repeated sequence that is regarded as a minisatellite. Eleven SNPs were identified in the nine genes: four were synonymous and seven were non-synonymous (Table trnfM (trnfM-2). The trnfM-2, which is located close to the gene orf138  . Seventy-two percent of unique region I  and 100% of unique region II  were occupied by five mitochondrial sequences of Brassicaceae , even though orf138 sequence itself had no similarity. Homologous sequences detected by bl2seq were plotted on unique region IV in Ogura-type mitochondrial genome  LD-CMS line has a smaller mitochondrial genome than fertile line, while CW-CMS derived from wild rice (O. rufipogon) has a larger genome than fertile line [Oryza genus is also attributable to large repeats. It should be noted that the size difference between two radish mitochondrial genomes mainly arises from a novel sequence of 15,255 bp, not from a large repeated sequence.Ogura-type mitochondrial genome was 14 kb larger than that of normal type. Region IV that is unique to Ogura-type mitochondrial genome mainly contributes to this size difference. Comparative analysis has been performed between mitochondrial genomes of CMS and normal lines in other higher plant species. In sugar beet, Owen-type CMS line has a significantly larger mitochondrial genome than fertile line because the Owen-type mitochondrial genome contains one 86-kb large repeat . In wheaile line . The difBrassicaceae species including Brassica and Arabidopsis have small mitochondrial genomes among higher plants [Brassicaceae mitochondrial genomes reported so far do not contain much nuclear sequence. The number of large repeats (>1 kb) is less in reported Brassicaceae (Arabidopsis and Brassica) than in other plants species [Brassica and Raphanus have only two large repeats. In addition, sequences derived from nuclear or plastid genomes were rarely observed. This is one of the reasons why mitochondrial genomes of Brassica and Raphanus are small. Because of the small genome size and few large repeats, we can consider Brassica and Raphanus as species that have the “simple” mitochondrial genomes in higher plants. An understanding of the remarkable reorganization events that occurred in the simple radish genome will provide us with a clear picture of mitochondrial genome evolution, such as insertion of a 15-kb large novel sequence in Ogura-type.The reported r plants . It is a species . While matp6 in Ogura-type mitochondrial genome is, however, different from that of normal-type. Recombination had occurred at the site of syntenic region 4 and atp6, which produced a new initiation codon for atp6 in Ogura-type genome. According to the genome sequence, Ogura-type atp6 encodes a different polypeptide from that of normal-type atp6. However, the N-terminal of ATP6 polypeptide of Ogura-type is processed to produce a protein identical to normal-type ATP6 [orfX also lacks similarity to the sequence of normal-type. This is caused by a 48-bp repeated sequence regarded as a minisatellite. This minisatellite will be a useful marker to detect polymorphism among radish mitochondrial genomes including Ogura and normal types [trnfM (trnfM-2). This additional trnfM-2 is located near orf138, and it is interesting to note that this trnfM-2 has a unique SNP that has not been found in other reported trnfMs in Brassicaceae. As for unique ORFs, Ogura-type has six unique ORFs including orf138. These ORFs can be related to unique biological characteristics in Ogura-type cytoplasm. In particular, transcription of four ORFs (orf122orf154orf117 and orf102) that are specific to Ogura-type genome should be investigated in future study.As expected, gene contents and their sequences were highly conserved between the two mitochondrial genomes in radish. The 5’ sequence of the ype ATP6 . The 3’ al types . A totalal types . As for T-urf13 in maize [orf522 in sunflower [pcf in petunia [orf456 in pepper [orf107 in sorghum [orf79 in rice [Brassicaceae crops, in addition to orf138 in radish, three CMS-related ORFs have been reported: orf224 and orf222 in rapeseed [orf267 in Brassica tournefortii[orf138, all these CMS-related ORFs have a chimeric structure that contains the sequence of known mitochondrial genes. The orf138, however, does not contain any conserved mitochondrial sequence. The sequence of orf138 is totally novel, and is not present in normal-type mitochondrial genome of radish. It is quite interesting to reveal how the orf138 sequence was created during the evolution of radish mitochondrial genome. However, it now appears difficult to infer the origin of the gene orf138, since no sequence homologous to the main part of orf138 is present in either Ogura- or normal-type mitochondrial genome.CMS-related ORFs have been isolated in various plant species, such as in maize , orf522 unflower , pcf in  petunia , orf456 n pepper , orf107  sorghum  and orf7 in rice . In Brasrapeseed , and orfrnefortii. With thorf138 is located in this region. Blast analysis using the sequence in region IV as a query showed that 80% of this region has similarity to the mitochondrial sequences in other Brassicaceae plants, including rapeseed and Arabidopsis. The analysis also showed that the homologous sequences are scattered throughout unique region IV  in Ogura-type mitochondrial genome. The gene V Figure . TherefoThis composition of novel sequence in radish contrasts to that reported for sugar beet. In sugar beet mitochondrial genome, 7.6% of the unique regions showed significant homology to previously determined mitochondrial sequences, 17.9% to nuclear DNA, 4.6% to mitochondrial episome and 0.1% to plastid DNA .Brassicaceae mitochondrial genome. The next question relates to how this unique region IV was generated or integrated into the radish mitochondrial genome. We searched for repeated sequences around this region. Ogura-type unique region IV was shown to be sandwiched between syntenic regions 7 and 11  is almost conserved at the edge of syntenic region 11 of normal radish. The position of the promoter exactly matched the size of atp8 transcript (ca. 600 bp in size) [atp8. Actually, in Ogura-type genome, atp8 is co-transcribed with orf138 and trnfM, while in normal-type, atp8 is transcribed from its own promoter.Previously, we conducted sequence analysis on the radishes . While pf radish . The pren Figure . In Arabtermined . A promoin size) . The recpol -type and nap-type, co-exist among many rapeseed cultivars, and SSS can explain the emergence of these CMS [orf138 was reported to be present in substoichiometric molecules with low copy number by genomic PCR [orf138 in various normal radishes. The fact that no sequence homologous to orf138 was found in the current pyrosequencing data  from a normal radish suggests that substoichiometric molecules containing orf138 are absent in normal radish. The discrepancy between our results and a previous study [orf138 regarded as subliminal fragments in various radishes with Ogura-type cytoplasm. This indicates that, if there were orf138 sequences in normal radish, they would be at quite a low level, and the contribution of SSS as a possible mechanism to explain generation of the Ogura-type mitochondrial genome from normal-type genome remains to be elucidated.Another possibility to explain the emergence of Ogura-type mitochondrial genome is substoichiometric shift (SSS). SSS is a phenomenon by which copy number of the substoichiometric molecule dramatically increases over generations . Researchese CMS . In radiomic PCR . Howeverus study  can be aus study ,44 vs. 4us study ). Howeveorf138 in 107 Japanese wild radishes, 29 cultivated radishes and seven R. raphanistrum[orf138 variants, we inferred the mechanism behind the differentiation of Ogura-type cytoplasm. These studies showed that the original Ogura-type radish is derived from Japanese wild radish and that orf138 sequence of Japanese wild radish was introduced from R. raphanistrum[Raphanus and molecular evolutionary studies of the data should provide clues to resolve this issue.Previously, we conducted a large-scale sequence analysis of hanistrum. On the hanistrum-46. HoweBrassicaceae mitochondrial genomes. Insertions of sequences derived from plastid or nuclear genome were not identified. This suggested that the unique regions were generated by integration and shuffling of pre-existing mitochondrial sequences during the evolution of Brassicaceae, and novel genes such as orf138 could have been created by the shuffling process.The complete nucleotide sequence of the mitochondrial genome from Ogura male-sterile radish reveals that the genome has been highly rearranged compared with that of normal radish. The radish mitochondrial genome consists of 11 syntenic regions. The reorganization of the genome occurred via a pair of large repeats and multiple pairs of short repeats, and produced four regions unique to the Ogura-type mitochondrial genome. Most of these unique regions consist of sequences homologous to known Raphanus sativus var. hortensis) with a long white root. UC-G and MS-G have normal cytoplasm and Ogura-type cytoplasm, respectively. The MS-G was developed by introducing Ogura-type male-sterile cytoplasm into UC-G and maintained by repeated backcrosses.A cultivar 'Uchiki-Gensuke' (UC-G) and its male-sterile line 'MS-Gensuke' (MS-G) were used in this study. These two lines are in the category of Japanese cultivated radish  and de novo assembly were conducted by Hokkaido System Science . A nucleotide sequence of 20,646,046 bp in total was obtained. Sequences were assembled to 105 contigs . On the basis of the physical map for the normal-type mitochondrial genome, we connected contigs to develop a master circle. If contigs had similarity to plant plastid genome and their depth value was low, we ignored them. All linkages between contigs were confirmed by genomic PCR  by Takara-Bio . A nucleotide sequence of 12,059,770 bp in total for Ogura-type was obtained. Sequences were assembled to 147 contigs , using GS De Novo Assembler version 2.0 (Roche). The average sequence depth, which was defined as the total nucleotide number used for assembly divided by the total length of contigs, was 36. On the basis of linkage information of the contigs and the physical map of radish mitochondria, we connected contigs to develop a master circle by a parsimonious method, so that each contig appeared at least once to construct the smallest genome . If the http://blast.ncbi.nlm.nih.gov/Blast.cgi). A tRNA gene search was conducted with the tRNA scan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/) [http://emboss.dbcls.jp/). ORFs predicted to encode proteins longer than 100 amino acids were picked up in the current analysis.The genes encoding known mitochondrial proteins and those for rRNAs, as well as repeated sequences, were identified using the Basic Local Alignment Search Tool (can-SE/) . ORFs enCMS = Cytoplasmic male sterility; mtDNA = Mitochondrial DNA; ORF = Open reading frame; SNP = Single nucleotide polymorphism; SSS = Substoichiometric shifting.The authors declare that they have no competing interests.YT carried out the sequence data analysis, and drafted the manuscript. MT isolated mitocnodrial DNA from radish. KY participated in the sequence analysis. HY prepared plant materials. TT supervised the work and edited the manuscript. All authors read and approved the final manuscript.List of repeated sequences in normal-type genome.Repeated sequences in normal-type genome were listed.Click here for fileList of repeated sequences in Ogura-type genome.Repeated sequences in Ogura-type genome were listed.Click here for fileThe sequences homologous to Brassicaceae mitochondrial genome plotted on Ogura-type specific region I -III. A black line indicates the region specific to the Ogura-type mitochondrial genome. Black boxes indicate the sequence homologous to radish and/or other Brassicaceae mitochondrial genomes.Click here for fileValidation of contig linkage by PCR analysis. The primer information used for this PCR analysis is described in Additional file 5.Click here for fileThe primers used for the PCR analysis to validate the linkage between contigs of normal-type genome. Information on the primers used for the PCR analysis in Additional file 4.Click here for fileThe primers used for PCR analysis to validate the short repeats. Information on the primers used for PCR and sequencing, in order to confirm short repeats in Ogura-type genome.Click here for file


Multiple investigators have established the feasibility of using buccal brush samples to genotype single nucleotide polymorphisms (SNPs) with high-density genome-wide microarrays, but there is currently no consensus on the accuracy of copy number variants (CNVs) inferred from these data. Regardless of the source of DNA, it is more difficult to detect CNVs than to genotype SNPs using these microarrays, and it therefore remains an open question whether buccal brush samples provide enough high-quality DNA for this purpose.To demonstrate the quality of CNV calls generated from DNA extracted from buccal samples, compared to calls generated from blood samples, we evaluated the concordance of calls from individuals who provided both sample types. The Illumina Human660W-Quad BeadChip was used to determine SNPs and CNVs of 39 Arkansas participants in the National Birth Defects Prevention Study (NBDPS), including 16 mother-infant dyads, who provided both whole blood and buccal brush DNA samples.We observed a 99.9% concordance rate of SNP calls in the 39 blood–buccal pairs. From the same dataset, we performed a similar analysis of CNVs. Each of the 78 samples was independently segmented into regions of like copy number using the Optimal Segmentation algorithm of Golden Helix SNP & Variation Suite 7.R ratios had an average correlation of 0.899 between blood-buccal pairs of samples from the same individual, while the average correlation between all possible blood-buccal pairs of samples from unrelated individuals was 0.318. An independent analysis using the QuantiSNP algorithm produced average correlations of 0.943 between blood-buccal pairs from the same individual versus 0.332 between samples from unrelated individuals.Across 640,663 loci on 22 autosomal chromosomes, segment-mean log R ratios had an average correlation of 0.539 between mother-offspring dyads of buccal samples, which was not statistically significantly different than the average correlation of 0.526 between mother-offspring dyads of blood samples (p=0.302).Segment-mean log We observed performance from the subject-collected mail-in buccal brush samples comparable to that of blood. These results show that such DNA samples can be used for genome-wide scans of both SNPs and CNVs, and that high rates of CNV concordance were achieved whether using a change-point-based algorithm or one based on a hidden Markov model (HMM). Multiple investigators have established the feasibility of using buccal brush samples to genotype single nucleotide polymorphisms (SNPs) with high-density genome-wide microarrays -3, but tMany studies, including the National Birth Defects Prevention Study (NBDPS) , rely onTo demonstrate the quality of CNVs generated from these DNA samples and genotyping instruments, we evaluated the concordance of CNV calls from DNA extracted from blood to DNA extracted from subject-collected mail-in buccal brushes (i.e. cheek swabs). The Illumina Human660W-Quad BeadChip was used to determine SNPs and CNVs of 39 Arkansas participants in the NBDPS who had provided both whole blood and buccal brush DNA samples.Genotype calls across 561,490 SNPs were generated using Illumina’s GenomeStudio software under default settings; genotypes with a GenCall score [R ratios, which is defined as the base-2 log-ratio of the sample’s direct intensity (R) at the given locus, versus a reference sample [For our analysis of CNVs, we restricted data to the 22 autosomal chromosomes, because part of our analysis was a comparison of maternal and infant CNVs, and there were both male and female infants in the study sample. In addition to 547,937 SNPs, an additional set of 92,726 non-polymorphic, but copy-number-informative, loci were included in the analysis, for a total of 640,663 loci on 22 autosomal chromosomes. Using the Optimal Segmentation algorithm of SNP & Variation Suite 7 , each of the 78 samples was independently segmented into regions of common mean. This segmentation was performed on the 78 sequences (one per sample) of 640,663 log R ratios. We judged concordance, therefore, by comparing the 78 sequences of segment-mean log R ratios, in which individual log R ratios are replaced by their corresponding segment mean. Among the 39 blood-buccal pairs of DNA samples from the same individual, segment-mean log R ratios had an average Pearson’s correlation coefficient of 0.899, while the average correlation between all possible blood-buccal pairs of unrelated individuals was 0.318.Optimal Segmentation does not explicitly assign copy number state, but instead produces a parsimonious segmentation of the genome in which each locus in a segment shares the same underlying distribution of log As an illustration, Figure R ratios between the 39 blood-buccal pairs of samples from the same individual was 0.613, while the average correlation of segment-mean log R ratios from the same pairs of samples was 0.899 . Similarly, correlations averaged 0.323 between all possible unrelated pairs of buccal samples, compared to 0.321 (p=0.177) from blood samples. Buccal and blood DNA samples, therefore, performed comparably well in detecting CNVs.The correlation between mothers and offspring allows us to compare the performance of buccal and blood samples. We would expect higher quality DNA to yield higher correlations between mothers and offspring, because there would be less measurement error in the resulting data. In our data, segment-mean log QuantiSNP  is a BayR ratio is largely insensitive to the choice of cutoff. Table p=0.025). Using an LBF cutoff of 50, however, reduces the average number of CNVs as well as the difference between brush and blood .An independent analysis using QuantiSNP produced average correlations of 0.943 between blood-buccal pairs versus 0.332 between unrelated pairs, using an LBF cutoff of 30. Table R ratios generated by QuantiSNP and SVS Optimal Segmentation. The first panel shows that QuantiSNP yields uniformly higher self-to-self correlations, while the second and third panels show higher average mother-offspring correlations and correlations between unrelated pairs of samples. This may be due to QuantiSNP’s use of B allele frequency data, in addition to log R ratio, to infer CNVs, which allows loss of heterozygosity (LOH), for example, to inform CNV calls.Figure The use of subject-collected mail-in DNA samples exposes a study to an additional risk of non-compliance, namely, the possibility of poor sample collection or handling by study subjects. Out of our initial collection of 46 buccal brush samples that were run on non-defective microarrays, one had a low genotyping call rate that was likely caused by poor DNA quality, and one had a high call rate but was genetically inconsistent with the three other members of its blood/buccal/mother/offspring quartet. This left 44 buccal samples (96%) that had high call rates and the expected genetic relatedness to the rest of their respective quartets. The NBDPS collects DNA from mothers, fathers, and infants, which allows a consistency check of genetic identity. In the buccal brush samples used in this study, total DNA yield ranged from 390 to 11,004 ng and averaged 2,474 ng  Center for Birth Defects and Developmental Disabilities. All study subjects gave informed written consent. For minors, informed written consent was obtained from their legal guardian.In 1997, eight CDC-supported centers for birth defects research were established and began participation in the NBDPS ,17,18. AInformation regarding multiple maternal exposures and lifestyles hypothesized to impact the developing embryo are obtained from all case and control mothers who participate in the NBDPS by a phone interview. Once a mother has completed the interview, a DNA sample-collection kit  is mailed to her.In Arkansas, there is one pediatric tertiary care stand-alone children's hospital – Arkansas Children's Hospital. A subset of Arkansas residents who completed the NBDPS were also included in another study at Arkansas Children's Hospital Research Institute; thus, some Arkansas families who were eligible for both studies provided both blood and buccal cell samples.All participants in the NBDPS are asked to submit maternal, paternal, and infant DNA samples using mail-in CytoSoft CYB-1 buccal brush kits . In ordeOut of the 96 DNA samples, all but six had SNP call rates of 99.1% or higher, with a mean call rate of 99.8%. Four of the six low call rates were caused by a defective BeadChip, which was determined using diagnostic data plots and subsequently confirmed by Illumina. We cannot definitively determine the cause of the remaining two low call rates, which came from one blood and one buccal sample of unrelated individuals on two different BeadChips. Out of the remaining 42 subjects with both high blood and high buccal call rates, 39 had blood-buccal SNP concordance rates greater than 99.9%. Of the three subjects (two infants and one mother) with high call rates and low concordance rates, we were able to determine whether the blood or buccal sample was more likely to contain incorrect DNA, by comparing genotypes to those from the related subject  and assuming Mendelian inheritance.Based on these comparisons, the three likely mislabeled or miscollected DNA samples were one maternal blood, one infant blood, and one infant buccal sample. Out of the 46 buccal samples that were hybridized to non-defective BeadChips, therefore, one had a low call rate (83%) and one appeared to be either mislabeled or miscollected, based on a high call rate but low concordance with its corresponding blood sample and with the two maternal DNA samples. The other 44 buccal samples (96%) exhibited high call rates and high concordance with their corresponding blood samples, as well as Mendelian consistency with their related samples. There were 39 subjects with high SNP call rates for both blood and buccal, and high SNP concordance rates between the two, and these subjects form the basis for our analysis of CNV. Out of these 39, 32 were comprised of 16 mother-infant dyads.Methods for the collection and processing of blood and buccal cells are well established using approved IRB protocols for the DNA Bank for Congenital Malformations and the NBDPS . SamplesGenotype calls across 561,490 SNPs were generated using Illumina’s GenomeStudio software under default settings; genotypes with a GenCall score of 0.15 or lower were considered unreliable and set to no-calls .R ratios under three different LBF thresholds: 10, 30, and 50, representing a range of thresholds that might be used in practice.The QuantiSNP manual recommends filtering out CNVs with LBF less than 10 to prevent large numbers of false positive calls, while setting a threshold at 30 or more is recommended to obtain low false positive rates. We therefore computed correlations of segment-mean log SNP: Single nucleotide polymorphism; CNVs: Copy number variants; NBDPS: National Birth Defects Prevention Study; IRB: Institutional Review Board; HMM: Hidden Markov model; LBF: Log Bayes factor; LOH: Loss of heterozygosity; CDC: Centers for Disease Control and Prevention.The authors declare that they have no competing interests.SWE conducted statistical analysis and drafted the manuscript. SLM conducted DNA extraction and genotyping and assisted with the manuscript. CAH participated in the study design and manuscript development. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/13/51/prepubTable S1.DNA Yields and Concentrations.Click here for file


Pokemon is an oncogenic transcription factor involved in cell growth, differentiation and oncogenesis, but little is known about its role in human breast cancer. In this study, we aimed to reveal the role of Pokemon in breast cancer progression and patient survival and to understand its underlying mechanisms.Pokemon expression and its correlation with the progression and prognosis of the disease. DNA microarray analysis of MCF-7 cells that overexpress Pokemon was used to identify Pokemon target genes. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis were utilized to determine how Pokemon regulates survivin expression, a target gene.Tissue microarray analysis of breast cancer tissues from patients with complete clinicopathological data and more than 20 years of follow-up were used to evaluate P = 0.0148) and lymph node metastasis (P = 0.0014). Pokemon expression led to worse overall  and disease-related  patient survival. DNA microarray analyses revealed that in MCF-7 breast cancer cells, Pokemon regulates the expression of at least 121 genes involved in several signaling and metabolic pathways, including anti-apoptotic survivin. In clinical specimens, Pokemon and survivin expression were highly correlated . ChIP and site-directed mutagenesis indicated that Pokemon induces survivin expression by binding to the GT boxes in its promoter.Pokemon was found to be overexpressed in 158 (86.8%) of 182 breast cancer tissues, and its expression was correlated with tumor size (survivin expression and thus may be a potential target for the treatment of this malignancy.Pokemon promotes breast cancer progression by upregulating  ZBTB7 gene .Human cDNA microarrays covering 22 kb cDNA spots  were used. In brief, total RNA was extracted from MCF-7 cells with ectopic Pokemon expression in MDA-MD-231 breast cancer cells was knocked down using chemically synthesized small interfering RNA (siRNA) siRNA#1 , siRNA#2  [ Utt-3')  and siRN Utt-3') , and pro Utt-3') . SolublePokemon on survivin promoter activity in MDA-MB-231 and MCF-7 cells as described previously [5 cells were mixed with survivin promoter constructs and Pokemon expression vector or Pokemon siRNA#1. At 48 hours after transfection, cell extracts were prepared with 1 × lysis buffer, and a 10-μl aliquot of the supernatant was mixed with 50 μl of luciferase assay reagent (Promega) and analyzed with a Microplate Luminometer . Luciferase activity was normalized by using a Renilla luciferase internal control.Transient gene delivery was carried out to assess the effect of eviously ,36. Briesurvivin promoter.MCF-7 and MDA-MB-231 cells were fixed by the addition of 1% formaldehyde to the medium for 10 minutes. Formaldehyde was quenched by the addition of 1 × glycine for 5 minutes at room temperature. A chromatin immunoprecipitation (ChIP) assay was performed as described previously  with 3 μPokemon and survivin expression, the Wilcoxon rank-sum test or the Kruskal-Wallis test were utilized with categorical variables and Kaplan-Meier survival curves were produced to examine the relationship between Pokemon expression and mortality. Results were considered statistically significant at P < 0.05.Spearman's rank-correlation coefficients were used to assess the relationship between Pokemon expression. This array consists of 246 breast cancer cases and normal controls, among which 182 malignant tissues were histologically interpretable. The results showed that Pokemon was undetectable in normal breast lobules, but overexpressed in 158 (86.8%) of 182 cancerous tissues. The level of Pokemon expression was assessed according to the intensity of immunostaining and assigned the score of 0 to 3 (negative to most intensive)  and lymph node metastasis (P = 0.0014), but not with patient age, tumor type and nuclear grade. Pokemon was also found to be overexpressed in 28 (80%) of 35 interpretable recurrent breast tumors in another YTMA-89 microarray.The YTMA-23 tissue microarray, provided by the Yale Cancer Center Critical Technology Group, was evaluated for ) Figure . This YTPokemon expression on patient survival was further analyzed using Kaplan-Meier plots. As shown in Figure Pokemon expression was negatively correlated with overall survival  and in particular disease-related survival  of breast cancer patients, indicating that Pokemon is a negative prognostic indicator.Tumor size and lymph node metastasis are well-known prognostic factors for breast cancer, and therefore the effect of HER2) genes are well-established biomarkers and therapeutic targets for breast cancer. The correlation of Pokemon expression with these three molecular markers was assessed using Kruskal-Wallis tests, and the results showed that Pokemon expression did not correlate with ER, PR or HER2 alone or with any combination of the three genes.The estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 , a negative prognostic factor for breast cancer [survivin. MCF-7 cells were transfected with Pokemon expression vector to increase the level of Pokemon, while MDA-MD-231 cells were transfected with Pokemon siRNA to knock down its expression. As shown in Figure Pokemon mRNA successfully knocked down its expression and in turn led to a decrease of survivin, proving the cDNA microarray data. A time-course study showed that significant silencing of Pokemon by siRNA was observed at 48 hours after transfection. Interestingly, Pokemon silencing in MDA-MD-231 led to upregulation of p14ARF, but had no effect on Bcl-2 expression. In addition, transient expression of Pokemon in MCF-7 cells induced survivin upregulation in a parallel manner to Pokemon levels . In normal breast tissues, the expression levels of both Pokemon and survivin were low (data not shown).To confirm that the induction of Pokemon regulates the expression of survivin. The promoter region of survivin, containing 2,080 bp upstream of the transcription start site, was cloned and used to drive the expression of luciferase reporter in MCF-7 and MDA-MB-231 breast cancer cells. Progressive deletions of the 5' end of the survivin promoter revealed that the -441-bp fragment possesses full promoter activity  from ogenesis . In thisPokemon was overexpressed in 86.8% of breast cancer tissue, but not in normal beast tissue, indicating its tumor-specific expression. More importantly, Pokemon expression correlated positively with tumor size and lymph node metastasis, both of which are indicators of unfavorable clinical outcome. Indeed, the level of Pokemon expression was negatively correlated with survival rate. These findings indicate that Pokemon may be a novel prognostic marker for breast cancer and a potential therapeutic target. We therefore attempted to elucidate the underlying mechanism of its tumor-promoting function.Using tissue microarray technology, we have shown that Survivin, an antiapoptotic protein and a negative prognostic indicator of breast cancer [survivin and Pokemon were highly correlated , suggesting that Pokemon serves as an upstream inducer of survivin in breast cancers.Previous studies have shown that Pokemon functions as an oncoprotein by inhibiting the ARF/p53 pathway . Using ct cancer ,39, is osurvivin expression and that loss of p53 function would lead to the induction of survivin, resulting in cancer growth and resistance to chemotherapeutic agents [survivin indirectly by suppressing p53. However, in this study, we have demonstrated that Pokemon can directly induce the expression of survivin by binding to the GC boxes in its promoter. This finding reveals a new signaling pathway of Pokemon-mediated oncogenesis and advances understanding of survivin regulatory mechanisms -23 contained 246 breast cancer cases with clinical records. The descriptive statistical data are summarized below.Click here for filePokemonMCF-7 cell clones with stable overexpression of . MCF-7 cells were transfected with pcDNA3.1/Pokemon or the empty vector, and the transfected cells maintained with neomycin were subjected to Western blot analysis for the detection of Pokemon expression.Click here for file


Accumulating evidence indicates that motor imagery and motor execution share common neural networks. Accordingly, mental practices in the form of motor imagery have been implemented in rehabilitation regimes of stroke patients with favorable results. Because direct monitoring of motor imagery is difficult, feedback of cortical activities related to motor imagery (neurofeedback) could help to enhance efficacy of mental practice with motor imagery. To determine the feasibility and efficacy of a real-time neurofeedback system mediated by near-infrared spectroscopy (NIRS), two separate experiments were performed. Experiment 1 was used in five subjects to evaluate whether real-time cortical oxygenated hemoglobin signal feedback during a motor execution task correlated with reference hemoglobin signals computed off-line. Results demonstrated that the NIRS-mediated neurofeedback system reliably detected oxygenated hemoglobin signal changes in real-time. In Experiment 2, 21 subjects performed motor imagery of finger movements with feedback from relevant cortical signals and irrelevant sham signals. Real neurofeedback induced significantly greater activation of the contralateral premotor cortex and greater self-assessment scores for kinesthetic motor imagery compared with sham feedback. These findings suggested the feasibility and potential effectiveness of a NIRS-mediated real-time neurofeedback system on performance of kinesthetic motor imagery. However, these results warrant further clinical trials to determine whether this system could enhance the effects of mental practice in stroke patients. Subsequently, whether neurofeedback could enhance cortical activation associated with motor imagery was analyzed. Sequential finger movements from the right hand were utilized for the motor imagery task; hemoglobin signal changes from the left motor cortex were evaluated as “real” feedback, and sham information irrelevant to cortical signals served as control or “sham” feedback. A small feasibility study was initially conducted using actual finger movements for the task. The main purpose of this initial experiment was to confirm that the real-time signal analysis method was consistent with off-line analyses, as well as to determine which hemoglobin oxygenation parameters – Oxy- or Deoxy-Hb signals – would be suitable for the feedback signal. Based on findings from the first experiment, a second experiment was conducted in which self-assessment scales of motor imagery and cortical activation mapping were compared between the two feedback conditions to determine whether real feedback significantly affected motor imagery quality and related cortical activation.In accordance with the Declaration of Helsinki, written informed consent was obtained from each subject, who participated in the present study. The study was approved by the Ethics Committee of Morinomiya Hospital .A total of five healthy, right-handed subjects were recruited to test consistency of the neurofeedback system. Handedness of the subjects was measured by the self-report of the side of their hand used in writing and eating, and no subjects had history of correction of handedness. The mean (± SD) age of participants was 36.8 (±13.5) years, with one female subject. Written informed consent was obtained from each subject.Self-paced, sequential movements of the right fingers were used for the motor task. Participants were asked to sequentially fold their right fingers from the thumb to the little finger, and then to unfold them from the little finger to the thumb. They repeated these movements during the 5-s task period. The experiment consisted of 15 executions of the motor task, with randomized inter-task rest periods ranging from 8–15 s . The totThe NIRS-mediated neurofeedback system consisted of the NIRS system, a data-processing computer, and a monitor to display feedback information. A schematic overview of this system is illustrated in It was assumed that NIRS detects hemoglobin signal changes derived from local vascular reactions coupled with neuronal activation at the cortical surface http://www.MRIcro.com), together with the Brodmann's area (BA) image and Automated Anatomical Labeling (AAL) image According to the fiber arrangement shown in via a local area network cable. In the data-processing computer , transferred data were buffered and processed using a general linear model (GLM) and least-square estimation, which can be suitable for using shorter inter-task intervals NIRS measures task-related changes in OxyHb and DeoxyHb signals on the cortical surface. In the present study, the OxyHb signal was primarily utilized as a cortical activation marker and feedback signal source. Previous studies demonstrated that OxyHb signals exhibit superior sensitivity in task-related signal changes and a greater correlation with blood oxygen level–dependent signals in functional magnetic resonance imaging (fMRI) t-test against zero, and the calculated t-value was used as a marker of cortical activation at each channel. Because it took 50 ms to calculate t-values for 80 points of data from 50 NIRS channels, it was possible to calculate t-values for each data acquisition point. As shown in t-value for these three channels is displayed as the height and color of the vertical bar to provide feedback for the subjects , the maximum height of the vertical bar was set to 8.0 for better visibility. The displayed vertical bar color, which ranged from blue (zero) to red (8.0), also varied according to the t-value.A two-parameter gamma hemodynamic response function (HRF), which was utilized in fMRI data analysis, served as the predictor for task-related hemoglobin signal changes subjects . If all t-values were calculated for each task/channel, and maximal t-values in channels 4, 9, and 17 were used as reference standards for cortical signals for each task (TRef).As a reference standard for cortical activation of each task, an off-line task-by-task GLM analysis was performed using all data points for the dataset. In this analysis, the time series was divided by task, and each individual task was represented by three box-car functions together with three basis sets for HRF. The design matrix consisted of 52 columns: a constant for collecting offsets, 3×15 columns (3 basis functions×15 repetitions of the task) for task data, and 6 columns for discrete cosine transform functions as high-pass filters with a cut-off frequency of 0.0125 Hz to remove baseline drifts . The t-vt-values were calculated from the sliding-window GLM analyses and were averaged from onset of one task to the next. The averaged value (TSWA) served as the feedback signal for each task. Although OxyHb signal-based analysis was most often utilized, a similar correlation analysis was performed using DeoxyHb signals. Pearson's correlation coefficient was used to measure correlations between the two cortical signals from each subject. In addition to the level of significance (P<0.05), r-values were calculated for effect size and represented small (0.1<), medium (0.3<), or large (0.5<) correlations To analyze real-time assessment, the referenced cortical signals calculated from the task-by-task analysis were compared with feedback signals calculated from the sliding-window GLM analysis. By comparing reference signals, A total of 24 healthy, right-handed subjects were recruited, with no history of neurological or psychological disease. As Experiment 1, handedness of the subjects was measured by the self-report of the side of their hand used in writing and eating, and no subjects had history of correction of handedness. Written informed consent was obtained from each subject. Only two subjects, who participated in Experiment 1, were included.e.g., feel the movements as they physically perform the task) Participants were asked to perform two sessions of the motor imagery task. Each session consisted of 15 sets in which participants performed imagery of right-finger movements, without physical movement, for 5 s. Environmental settings were similar to those in Experiment 1. Subjects were asked to imagine self-paced and sequential folding of the right fingers similar to the movements in Experiment 1. The subjects were also asked to kinesthetically imagine movements rather than visually  motor cortex  were displayed as vertical bars. Based on results from Experiment 1, OxyHb signal changes served as measurements of cortical activation for feedback. In the sham feedback session, normalized random values irrelevant to cortical activation were used for feedback, similar to a previously described neurofeedback study using functional MRI randn. The generated values were truncated if values were <0. The subjects were aware that only one of the two experimental sessions was real and the other was a sham condition. However, the order of real and sham feedback sessions was randomized and this information was not provided to the subjects.During the motor imagery task, participants were instructed to watch the monitor where feedback information was displayed as vertical bar height and color . In one After each session, the participants were asked to evaluate a self-assessment scale of motor imagery quality. They were asked to image finger-folding and to score how well they kinesthetically imagined the finger movements. If the subject experienced a vivid kinesthetic feeling that he/she had performed the task physically, then the 11-point scale scored a performance of 10, while the worst performance was scored as zero.t-test. To exclude the possibility that the order of motor imagery feedback conditions affected motor imagery quality, self-assessment scales were compared between first and second sessions in order. In addition, the average height of the presented feedback bar was compared between the two conditions, because it could possibly influence the self-assessment scores. Pearson's correlation analysis was used to compare self-assessment scores and presented height of feedback bar. The significance level was set to P<0.05.To estimate the effect of neurofeedback on motor imagery quality, self-assessment scale scores under real and sham feedback conditions were compared using a two-tailed paired vs. baseline, sham feedback vs. baseline, sham vs. real, and real vs. sham.As a first-level analysis, the effect of neurofeedback on cortical activation maps associated with motor imagery was analyzed, and the contrast between motor imagery task and baseline in real and sham feedback conditions was estimated. In addition, intra-subject contrasts between the two conditions were evaluated. Accordingly, three beta-values were calculated from four different contrasts, including real feedback t-test distinct from zero was performed for the contrast between task and baseline, and two-tailed one-sample t-test was performed for the contrast between two feedback conditions. The significance level was set to P<0.01 (uncorrected).Oxy- and DeoxyHb signal changes were analyzed. For each contrast, a positive beta-value indicated an increase, and a negative beta value indicated a decrease in hemoglobin signals in the former condition compared to the latter condition. In the design matrix, discrete cosine transform functions were included as high-pass filters with a cut-off frequency of 0.0125 Hz to remove baseline drifts. Averaged signal changes from 50 channels were included for eliminating global effects, such as autonomic responses relevant to motor imagery. To adjust for the auto-correlated error term, an autoregressive model of order 1 was used In addition to statistical analysis using GLM, a timeline analysis of OxyHb and DeoxyHb signals from the left lateral premotor cortex (channels 3), left sensorimotor cortex (channel 4), and left parietal association cortex (channel 25) was also performed. In each channel, averaged data from 315 trials  under real feedback and sham feedback conditions were plotted from 1 s before to 12 s after task onset to validate GLM analysis.SWA) and results from conventional task-by-task GLM analyses (TRef) in five subjects are shown in In the feasibility study, which utilized NIRS-mediated real-time neurofeedback system data from five healthy right-handed subjects, all subjects completed the right-finger folding task without obvious head motion. Correlation analyses between real-time feedback signals derived from sliding-window GLM analyses , but the order of conditions did not affect the score . The mean (± SD) heights of presented feedback bars under real and sham feedback conditions were 1.7 (±0.6) and 1.2 (±0.03), respectively, and there was a significant difference between the conditions . However, the correlation between presented feedback bar height and self-assessment scores was small and non-significant . Most subjects were not aware of the order of the two conditions.Although 24 healthy, right-handed subjects were recruited to analyze the effects of neurofeedback on motor imagery task-related cortical activation, data from three subjects were excluded due to overt finger movements during the motor imagery task. Subsequent analyses comprised data from the remaining 21 subjects. The mean (± SD) age of the 21 subjects was 34.3 (±10.3), with 4 female subjects. The average (± SD) self-assessment scale scores for kinesthetic motor imagery, which were assessed after real- and sham-feedback conditions, were 5.0 (±1.6) and 4.1 (±1.8), respectively . The scoCortical activation mapping with OxyHb signals revealed significantly increased motor imagery-related signals in the left sensorimotor and bilateral prefrontal cortex under real feedback conditions . Under s20 = 1.13, P = 0.14). However, in the left lateral premotor cortex, task-related OxyHb signals were more evident under real feedback conditions compared with sham feedback conditions . In contrast, the bilateral parietal association cortex exhibited significantly increased OxyHb signals under sham feedback conditions compared with real feedback conditions . The task-related DeoxyHb signal did not significantly change in the left lateral premotor and sensorimotor cortex , but decreased DeoxyHb signals in the left parietal association cortex were more evident under sham feedback conditions than real feedback conditions . R from time-line analyses were consistent with findings from cortical mapping analyses, which suggested that neurofeedback induced enhanced contralateral premotor activation and reduced parietal association cortex activation.Results from the present study demonstrated that the NIRS system can be used to detect real-time task-related hemoglobin signal changes, and this system can be reliably used as a neurofeedback tool. During motor learning processes, correct information feedback about performance (“knowledge of result”) is known to be effective in healthy subjects and in stroke patients Results from Experiment 1 revealed that hemoglobin signal changes were detected by the sliding-window GLM analysis with a hemodynamic delay of several seconds. Results from real-time processing in this system represented task-related cortical hemoglobin signal changes. Compared with other neuroimaging modalities, the NIRS-mediated neurofeedback system exhibits several advantages for clinical application, including relative robustness of subject motion, shorter attachment time, and less subject constraint. However, one main technical flaw of the NIRS measurements is the delay of several seconds between neuronal activation and hemoglobin signal changes. Simultaneous measurement of NIRS and EEG, which measures direct neuronal activation and has greater temporal resolution, could be a possible solution for methodological limitations The present results demonstrated that OxyHb was more robust under real-time assessment conditions for task-related cortical activation. However, it remains to be determined which hemoglobin parameters are more suitable for measuring cortical activation. Although the current theoretical framework for blood level-dependent (BOLD) signals in fMRI suggests that decreased DeoxyHb concentrations correlate with greater BOLD signals Results from Experiment 2 demonstrated that NIRS-mediated neurofeedback enhanced motor imagery-related cortical activation in the contralateral premotor cortex. These findings were consistent with previous findings from a fMRI-mediated neurofeedback study, which showed rostral enlargement of motor imagery-related motor cortical activation Results from the present study also demonstrated significantly greater cortical activation in the parietal association cortex under sham feedback conditions. Previous studies have suggested that the medial parietal association cortex is involved in memory-related visual imagery Bilateral prefrontal cortex and right premotor cortex were activated by the motor imagery task, regardless of type of provided feedback . Because motor imagery requires much attention and concentration, prefrontal activation could be related to cognitive processes involved in motor imagery. Indeed, previous reports have consistently documented greater cortical activation in the bilateral premotor cortex and prefrontal cortex during motor imagery tasks compared with motor execution tasks via the autonomic nervous system, which was evoked by motor imagery, could have affected the task-related hemoglobin signal changes. However, the focal activation patterns were different between feedback conditions and were unlikely to be the result of extracerebral contamination. Previous studies have introduced techniques to eliminate the effect of systemic vascular changes on NIRS signals Because cognitive processes, including planning, inhibition, and motor imagery, could evoke changes in heart and respiratory rate Sham information served as feedback information for the control condition. In previous neurofeedback studies, several kinds of signals, including background signals with random fMRI noise There were several limitations in this study. First, because the study included only healthy subjects, the effect of neurofeedback on stroke patients remains unclear. Although motor imagery in stroke patients is generally not impaired In conclusion, results from the present study demonstrated the feasibility of a NIRS-mediated neurofeedback system and revealed the modulative effect of this system on motor imagery-related cortical activation. Results suggested that neurofeedback could facilitate individual skills for kinesthetic motor imagery. The NIRS-mediated neurofeedback system could be a promising tool, which could be applied in widespread areas, including neurorehabilitation. However, further clinical trials are needed to determine whether this system could enhance mental practice in stroke patients.


The evidence that individual dietary and lifestyle factors influence a person’s weight and waist circumference is well established; however their combined impact is less well documented. Therefore, we investigated the combined effect of physical activity, nutrition and smoking status on prospective gain in body weight and waist circumference.We used data of the prospective EPIC-PANACEA study. Between 1992 and 2000, 325,537 participants  were recruited from nine European countries. Participants were categorised into two groups  for each of the following being physically active, adherent to a healthy  diet, and never-smoking for a total score ranging from zero to three. Anthropometric measures were taken at baseline and were mainly self-reported after a medium follow-up time of 5 years.Mixed-effects linear regression models adjusted for age, educational level, alcohol consumption, baseline body mass index and follow-up time showed that men and women who reported to be physically active, never-smoking and adherent to the Mediterranean diet gained over a 5-year period 537  and 200  gram less weight and 0.95  and 0.99  cm less waist circumference, respectively, compared to participants with zero healthy behaviours.The combination of positive health behaviours was associated with significantly lower weight and waist circumference gain. General and abdominal adiposity are related to mortality and many adverse health-related outcomes, such as diabetes, cardiovascular diseases, and several types of cancer Given these associations observed with individual health behaviours, we were interested in investigating the combined impact of these behaviours on subsequent changes in general and abdominal obesity. Studying the combined effect will provide insight on the potential benefits of adopting not just one but a range of healthy lifestyle habits. It has been shown that the combined impact of health behaviours was associated with a reduced risk of major chronic diseases including myocardial infarction, stroke, diabetes mellitus and cancer and was also inversely related to mortality The aim of the present study was to investigate the combined effect of physical activity, smoking status, and diet on future weight and waist circumference gain by combining these behaviours into a simple health behaviour score in the large EPIC-PANACEA study.From 1992–2000, in the EPIC study more than 500,000 individuals aged between 25 and 70 years were recruited from 23 centres in 10 countries  2 at follow-up (n = 140)). Information on smoking, physical activity or dietary variables was not available for 48,279 participants. Physical activity was mainly missing because centres in Norway and Umea (Sweden) used different questionnaires to assess physical activity. Therefore, the final study population comprised 325,537 participants from nine countries.Of the total cohort of 519,931 participants, we excluded 22,196 individuals because of missing dietary and non-dietary questionnaires, extreme energy intake to energy expenditure ratio, pregnant women, and missing baseline weight or extreme anthropometry. Furthermore, we excluded participants with missing follow-up weight  and parts of cohorts from Norway and Naples ) or extreme anthropometry at follow-up  or BMI<16 kg/mA total of 88,972 participants from Florence , Potsdam (Germany), Doetinchem (Netherlands) and Cambridge (United Kingdom) also provided data on waist circumference at baseline and follow-up.Baseline body weight (kg) and height (cm) were measured according to standardised procedures without shoes 2). For descriptive purposes, the accuracy of self-reported anthropometric measures was improved with the use of prediction equations derived from subjects with both measured and self-reported values  At follow-up, weight and waist circumference were self-reported in all centres except in Cambridge (UK) and Doetinchem (Netherlands) where it was measured according to the baseline protocol. BMI was calculated as body weight (kg) divided by height squared (mAs follow-up times between first and second anthropometry assessment differed by centre (2–11 years), our main outcome was the 5-year change (i.e. ((follow-up weight or waist circumference -baseline weight or waist circumference)/years of follow-up)*5).We investigated the combined effect of three lifestyle factors, that is physical activity, diet  at recruitment was assessed with standard questionnaires. For the current purpose, participants were grouped in two categories: ever smokers (current and former) and never smokers. In a subpopulation of 288,167 participants also information on smoking status at follow-up was available We constructed a simple pragmatic health behaviour score. Participants could get one point for each health behaviour, i.e., physically active, high adherence to the mMDS and never-smoking for a total score ranging from 0–3. Comparable scores were previously used and were associated with reduced risk of major chronic diseases Total amount of daily consumed alcohol over the last 12 months was assessed by country-specific validated dietary questionnaires Baseline characteristics of the study population are presented by health behaviour score and gender by using mean and standard deviation for continuous variables and percentages for categorical variables.2, continuous), education , total energy intake , and alcohol intake . The model with 5-year waist circumference change as the outcome was additionally adjusted for baseline waist circumference  and in a second model also for BMI change (continuous) for estimation of the relationship between the health behaviour score and waist circumference change independently of changes of general obesity.Associations between the health behaviour score and 5-year weight and waist circumference changes were modelled using sex-specific multilevel random mixed-effects linear regression, taking clustering of participants within countries and centres into account. Analyses were adjusted for age , follow-up time , baseline BMI (kg/m2), and education were explored by adding a product term. Stratified analyses were conducted when the product term was significant.Plausible effect modification by age (<60 and ≥60 years of age), baseline BMI  and current (0 point) in the health score.Results were computed using SAS 9.2. STATA 11 was used for the meta-analyses.In the current population, mean (standard deviation) 5-year weight gain was 0.71 (5.14) kg; when we applied the Oxford correction equations to improve the accuracy of the self-reported measurements, 5-year weight gain was 2.08 (5.07) kg. Five-year increase in waist circumference was 3.80 (6.42) cm (uncorrected) and 6.17 (6.48) cm (corrected). A total of 11.2% of men and 19.3% of women were scored to have all three health behaviours at study recruitment . These mFor changes of weight and waist circumference, significant interactions were found between the health behaviour score and baseline BMI, except in women when waist circumference change was the outcome. Stratified analyses showed that the relationship between the score and weight change was stronger for people with normal weight (BMI<25) and less obvious for obese people (BMI≥30) . The rel2 = 60.7% (p = 0.002)) waist circumference change (I2 = 62.0% (p = 0.033)). In most countries, the associations between the health behaviour score and anthropometry changes were inverse, with the exception for Cambridge and Oxford-Health Conscious (United Kingdom), and Greece where non-significant positive associations with weight change were observed.Heterogeneity in the association between the health behaviour score and anthropometry changes between countries was assessed using the meta-analysis approach . For men, no heterogeneity was present (p for heterogeneity>0.05). For women a significant degree of heterogeneity was found for the association with body weight and (IExcluding participants with chronic diseases at baseline did not change the results (data not shown).In 288,167 participants information on smoking habits at follow-up was available. Twenty-two percent of baseline smokers quit smoking during follow-up. Excluding these quitters from the analyses resulted in an attenuation of the effect of the health behaviour score on weight in men  and a null effect in women  . ResultsSensitivity analyses including smoking as non-current (1 point) and current (0 point) in the health score yielded results comparable to our main analyses: Men and women who reported to be physically active, non-smoking and adherent to the Mediterranean Diet gained lesser weight and waist  and women: −331 grams  and men: −0.93 cm  and women: −1.18 cm , respectively) compared to participants with no healthy behaviours.In the present large prospective study, the combination of positive health behaviours was associated with significantly lower weight gain and smaller increase in waist circumference in a dose-response manner. Men and women who reported to be physically active, never-smoking and adherent to the Mediterranean Diet (mMDS) gained over a 5-year period on average 537 and 200 grams less weight and 0.95 and 0.99 cm less waist circumference, respectively, as compared to men and women who reported to be physically inactive, ever smokers and not adherent to the mMDS.Our results confirm findings from studies investigating individual relationships between physical activity, smoking and diet and changes in general and abdominal adiposity showing that each individual behaviour is related with changes in adiposity In contrast to others Combining the health behaviours in one score led to stronger associations compared to the associations of the individual behaviours with weight and waist circumference changes we reported earlier Recently in controlled intervention studies, it was reported that physical activity did not further affect weight loss in addition to a diet Strengths of the present study are the prospective design, the large sample of participants from 9 European countries and the use of validated and standardized questionnaires.A limitation of our study was that in most of the centres weight and waist circumference at second assessment were self-reported and were therefore possibly underreported Another limitation is that lifestyle assessment for creating the score was only done at baseline and possible behavioural changes during follow-up were not taken into account. In participants for whom we had information on smoking status during follow-up, quitting smoking was associated with weight gain compared to stable smokers We constructed a pragmatic simple health behaviour score by using dichotomous variables where 1 point was given for the presence of each of the positive health behaviours without weighting the individual strength of the relationship with anthropometry. A weighted approach might improve the estimation of the effects of the individual score, but the combined effect of reporting all three healthy behaviours would be similar. Nechuta et al. We developed a score including health behaviours that are presumable easy to achieve in the general population. We, for example, dichotomized physical activity into inactive  and active (any category with activity levels above the latter). Therefore, a relatively high proportion of participants scored one or more points in our health behaviour score.The choice of the components of our score was based on prior findings from EPIC-PANACEA. Therefore, we used the mMDS, which was associated with weight change Our results suggest that public health programs aiming at reducing the burden of obesity could benefit from targeting a cluster of behaviours. We show that combining health behaviours was associated to a lower gain in waist circumference – a valid marker of central adiposity In conclusion, the combination of three positive health behaviours was associated with significantly lower 5-year weight and waist circumference gain compared with participants who scored to be inactive, smoking and low adherent to the Mediterranean Diet.Figure S1Country/Centre-specific association between the health behaviour score  and 5-year weight change (kg) in men. Country or centre specific estimates were calculated using general linear models in centres and countries with one centre only, or multilevel mixed-effects linear regression models in countries with more than one centre, and were adjusted age, total energy intake, baseline body mass index, education, alcohol intake and follow-up time. The overall estimate was calculated using random effect meta-analyses. Because of differences in assessment of follow-up weight, and/or different follow-up times, the centres in Germany , United Kingdom  and the Netherlands  were treated as separate cohorts.(TIF)Click here for additional data file.Figure S2Country/Centre-specific association between the health behaviour score  and 5-year weight change (kg) in women. Country or centre specific estimates were calculated using general linear models in centres and countries with one centre only, or multilevel mixed-effects linear regression models in countries with more than one centre, and were adjusted age, total energy intake, baseline body mass index, education, alcohol intake and follow-up time. The overall estimate was calculated using random effect meta-analyses. Because of differences in assessment of follow-up weight, and/or different follow-up times, the centres in Germany , United Kingdom  and the Netherlands  were treated as separate cohorts.(TIF)Click here for additional data file.Figure S3Country/Centre-specific association between the health behaviour score  and 5-year waist circumference change (cm) in men. Country or centre specific estimates were calculated using general linear models in centres and countries with one centre only, or multilevel mixed-effects linear regression models in countries with more than one centre, and were adjusted age, total energy intake, baseline body mass index, baseline waist circumference, education, alcohol intake and follow-up time. The overall estimate was calculated using random effect meta-analyses.(TIF)Click here for additional data file.Figure S4Country/Centre-specific association between the health behaviour score  and 5-year waist circumference change (cm) in women. Country or centre specific estimates were calculated using general linear models in centres and countries with one centre only, or multilevel mixed-effects linear regression models in countries with more than one centre, and were adjusted age, total energy intake, baseline body mass index, baseline waist circumference, education, alcohol intake and follow-up time. The overall estimate was calculated using random effect meta-analyses.(TIF)Click here for additional data file.