Igf2/IGF2) is a robustly imprinted gene, important for fetal growth in both mice and humans. In utero IGF2 exhibits paternal expression, which is controlled by several mechanisms, including the maternally expressing untranslated H19 gene. In the study by Soubry et al., a correlation is drawn between the IGF2 methylation status in fetal cord blood leucocytes, and the obesity status of the father from whom the active IGF2 allele is derived through his sperm. These data imply that paternal obesity affects the normal IGF2 methylation in the sperm and this in turn alters the expression of IGF2 in the baby.The relationship between the parental genomes in terms of the future growth and development of their offspring is not critical. For the majority of the genome the tissue-specific gene expression and epigenetic status is shared between the parents equally, with both alleles contributing without parental bias. For a very small number of genes the rules change and control of expression is restricted to a specific, parentally derived allele, a phenomenon known as genomic imprinting. The insulin-like growth factor 2 ( Igf2) [Igf2r), which show reciprocal imprinting effects and associated growth patterns [IGF2 is an important human fetal growth promoter that is regulated by the nearby H19 gene, which is also oppositely imprinted in humans [IGF2, their transcription is potentially more vulnerable. Alterations in their regulation through either mutation or disturbed epigenetic processes may lead to functional abnormalities and disease states.The "parental conflict hypothesis" is the most widely accepted explanation for genomic imprinting . This hyIgf2)  and its patterns . IGF2 isn humans ,6. For aIGF2 and H19 methylation are mechanistically implicated and are considered to be functionally important [Good examples of epigenetic syndromes are the rare imprinted human growth disorders Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS), where mportant . It now mportant . DNA metIGF2 and its receptors on fetal growth. Given the limitations of this type of study it is not surprising that they are usually confined to samples obtained at the time of birth. Unfortunately, many of these studies seem to report conflicting results. For example, in one study no correlation was reported between IGF2 expression and birth weight [IGF2 expression is either increased [There have been many studies investigating the effect of human placental expression of h weight , whereasncreased , or decrncreased , includincreased . Neverthet al. [IGF2 and H19 gene DMRs in the leucocytes from cord blood of their newborns (n = 79). They find an association between the father's obesity status and the loss of methylation at the DMR linked to IGF2 but not the H19 DMR. The main caveats of this study are the small numbers and the mixture of ethnicities. In addition, it has not been shown in this study whether a small change in methylation  at a DMR has any truly functional significance at the phenotype level. The authors point to their previous finding that a 5% loss of methylation could lead to a 10% increase in serum concentration of IGF2 [et al. [IGF2 is influenced by increased paternal BMI and the study makes a compelling case for a larger analysis to be performed. It is surprising and counter intuitive to note that a similar effect of hypomethylation on the IGF2 DMR is seen both for paternal obesity and maternal preconceptual famine [In the study by Soubry et al. , the aut of IGF2 . While i [et al.  study, fl famine . One migin utero is complex and will involve multiple gene pathways, many of which will not be haploinsufficient or driven by one parental allele. It is important and of particular human interest to allude to the differing roles of the parents in the developmental outcome of the future offspring. Without even considering imprinted genes, it is obvious that the two parents must have different physiological roles in pregnancy. We must consider the whole genome/epigenome/metabolome in an unbiased way, involving all key genes and networks. It is also important to critically assess environmental players. It is tempting to over-emphasize the role of a small number of parent-of-origin expressing genes and to speculate about the effects of modest variation in methylation, but we must not be too hasty to blame either parent for their offspring's health outcomes without being certain that these effects are consequentially robust.Fetal growth and development BMI: Body Mass Index; BWS: Beckwith-Wiedemann syndrome; DMR: Differentially methylated region; Igf2r: Insulin-like growth factor-2 receptor; Igf2/IGF2: Mouse/Human Insulin-like growth factor-2; SGA: Small for Gestational Age; SRS: Silver-Russell syndromeThe authors declare that they have no competing interests.GEM and PS both contributed to drafting and editing of the manuscript and both read and approved the final manuscript.http://www.ucl.ac.uk/babybiobank).GEM and PS have a specialist interest in human fetal growth and development with a view to understanding pregnancy complications. GEM was awarded an Honorary Fellowship of the Royal College of Paediatrics and Child Health in 2012 and is the Co-Director the Baby Bio Bank (The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/11/30/prepub


By focusing on the genes that correlate with the expression of miRNAs without necessarily being direct targets of miRNAs, we have clustered miRNAs into different functional groups. This has resulted in the identification of three novel miRNAs that are linked to the epithelial-to-mesenchymal transition (EMT) in addition to the known EMT regulators of the miR-200 miRNA family. In addition, an analysis of gene signatures associated with EMT, c-MYC activity, and ribosomal protein gene expression allowed us to assign different activities to each of the functional clusters of miRNAs. All correlation data are available via a web interface that allows investigators to identify genes whose expression correlates with the expression of single miRNAs or entire miRNA families. miRConnect.org will aid in identifying pathways regulated by miRNAs without requiring specific knowledge of miRNA targets.micro(mi)RNAs are small non-coding RNAs that negatively regulate expression of most mRNAs. They are powerful regulators of various differentiation stages, and the expression of genes that either negatively or positively correlate with expressed miRNAs is expected to hold information on the biological state of the cell and, hence, of the function of the expressed miRNAs. We have compared the large amount of available gene array data on the steady state system of the NCI60 cell lines to two different data sets containing information on the expression of 583 individual miRNAs. In addition, we have generated custom data sets containing expression information of 54 miRNA families sharing the same seed match. We have developed a novel strategy for correlating miRNAs with individual genes based on a summed Pearson Correlation Coefficient (sPCC) that mimics an  Micro(mi)RNAs are small, 19–22 nucleotide long, non-coding RNAs that regulate gene expression mostly by targeting the 3′UTR of mRNAs resulting in reduced translation of proteins or degradation of the mRNAs. miRNAs are fundamental regulators of cell differentiation and developmental processes. They have also been recognized to be highly relevant in cancer formation and progression miRNAs contain at their 5′ end a short stretch of 6–8 nucleotides complementary to the seed match in the target mRNA. This complementarity is accessible to computational analysis and multiple algorithms have been developed to predict miRNA targets HMGA2 and IMP1 as targets of the let-7 family of miRNAs miR-200 family and validated two E-box binding transcription factors, ZEB1 and ZEB2 as targets FAP1 as a miR-200 target let-7 and miR-200 in the NCI60 cells also allowed prediction and confirmation of the biological function of these miRNAs. Let-7 was found to be a marker for differentiated cancer cells miR-200 was identified as a powerful marker and regulator of the epithelial-to-mesenchymal transition (EMT) We have recently used the NCI60 cells Most of our findings were made by comparing miRNA and mRNA expression levels, which at the time was surprising, considering that miRNAs in mammalian cells were believed to mostly act by translational silencing without affecting mRNA expression levels. However, it had also been demonstrated that mRNA abundance of the majority of the targeted genes was somewhat affected by miRNAs per se, but are biological effectors relevant to the function of the miRNA. Especially when detected with the steady state system of the NCI60 cells, these biological effector genes may hold important information regarding the endogenous function of miRNAs. This was most obvious for miR-200. A small change in miR-200 expression (about 2 fold) resulted in a moderate change in mRNA levels of its targets ZEB1 and ZEB2 (about 5–7 fold), which resulted in a massive change in the E-cadherin/Vimentin ratio (over 8 orders of magnitude) miR-200 and the E-cadherin/Vimentin ratio allowed us to assign the function of “epithelial regulator” to the miR-200 family even before we had identified ZEB1 and ZEB2 as targets. Encouraged by this analysis, we have now used the NCI60 system to identify secondary correlators of miRNAs : 1) the expression profile of 208 human miRNAs quantified by real-time PCR (the “Q” data set) The most common and well-defined strategy to explore miRNA-gene associations is the Pearson's Correlation Coefficient (PCC) In this method we performed a standard COMPARE analysis In this method  from highest to lowest . Then inHOX) genes that have well characterized links to the expression of certain miRNAs.In addition to the theoretical level, it was necessary to test the ability of the sPCC method to identify miRNA/gene connections that have been established in known biological systems. We therefore made use of both host genes and homeobox (Many miRNAs are encoded within co-located genes (host genes) and share promoters with them. Expression of these miRNAs is driven by the promoters of their host genes, and positive correlations between expression of miRNAs and their host genes have been reported HOX genes as a unique system of four gene clusters, each containing at least one intergenic miRNA. HOX genes regulate embryonic development and in mammals they are grouped into 4 clusters (HOXA-D) including 9 to 11 genes HOXA, HOXC, and HOXD clusters harbor one miRNA gene each and the HOXB contains two , which are encoded within the HOX clusters, and all human genes. We observed that in the sPCC method, in 3 of 4 of the clusters a HOX gene adjacent to the co-localized miRNAs had the highest positive correlation with these miRNAs out of ∼18,000 human genes  (data not shown). For each of the 136 miRNAs, we calculated sPCC values with genes in the 4 HOX gene clusters. The sPCCs of all HOX genes within a cluster were added up and the miRNAs were ranked according to the cumulative sPCC for each HOX cluster  used for characterizing our approach. The sPCC method was, therefore, used in the subsequent analyses.In summary, these data demonstrate that the steady state system of the NCI60 mRNA and miRNA data is useful in detecting biologically meaningful connections between miRNAs and their host genes. The sPCC method, which was designed to mimic a miRNA titration experiment, was superior to the dPCC method in two assays (negative correlation with expressed mRNAs and miRConnect.org (or miRConnect.net)As introduced above, in addition to actual target genes, genes that are not predicted to be miRNA targets as well as the large number of both positively and negatively correlating genes may hold important information with respect to the status of miRNA cellular expression levels, which may provide insight into the biological activities of miRNAs. All negatively and positively correlating genes for the 136 miRNA and the 25 miRNA families determined with both the dPCC and the sPCC method in the Q data set, as well as the information on how many of them are predicted targets by TargetScan 5.0, can be found under miRConnect-Q at a searchable web interface: E-cadherin and Vimentin, which positively and negatively correlate with the expression of the miR-200 family, respectively . This phenomenon suggested that the grouping of miRNAs was partly based on, but not restricted to miRNA seed sequences.Interestingly, all 5 members of the miR-106b/93/25, miR-17∼92, miR-194-2/192, miR-183∼182, miR-99b/let-7e/125a, miR-206/133b.The clustering of miRNAs in this analysis was partially due to the fact that some family members are part of the same transcriptional unit. miRNAs that share chromosomal co-localization and also found in the same functional clusters included the transcriptional units of miR-141/200a and miR-200bc/429 seed families were clustered together although they comprise two gene clusters on separate chromosomes  and XI (including the miR-30 family) contained most of the miRNAs that are enriched in colon cancer cells  or downregulated (211 genes) by c-MYC (obtained from http://www.myc-cancer-gene.org/) and determined how many of them were either positively or negatively correlated with the expression of each of the 136 miRNAs. The result is visualized in c-MYC induced or c-MYC repressed genes. Examples are the miRNAs in cluster V which positively correlated with c-MYC induced genes and negatively correlated with c-MYC repressed genes  before and after a 4-day treatment with TGF-βTGF-β induced EMT While all 5 miR-200 family segregated in the cluster including the most epithelial miRNAs together with miR-7, miR-203, and miR-375  to more than 81% (for miR-200c). The miR-200 miRNAs were closely followed by miR-203 (55%), miR-7 (48%) and miR-375 (45%). miR-425* showed a much lower degree of identity in correlating genes (22%). However, for the 7 most mesenchymal miRNAs, virtually no genes were found to overlap with the genes that correlated with expression of miR-200 . In addition, these two miRNAs also did not cocluster with the other epithelial miRNAs in the Principal Component analysis  While the ability to predict miRNA targets represented a great advancement in the field and has helped to identify many miRNA targets, the false positive rate of all of the algorithms is high. This hampers accurate prediction of miRNA function based solely on the list of predicted targets. While it is well established that the co-expression of miRNAs which are part of the same transcriptional unit is often indicative of a common biological function, it has not been consistently so, and also has not permitted accurate grouping of miRNAs according to their function. We now present a new method to group miRNAs, which relies on identification of co-expressed effector genes for which a large amount of functional data is available.E-cadherin for epithelial cells or early carcinomas). 2) A substantial change in the expression of these marker genes is brought about by a relatively small change in the expression of genes that are actual miRNA targets (i.e. ZEB1/2). MiRNAs therefore are part of amplification loops. 3) A cancer-relevant differentiation stage is characterized as much by expression of positively correlating marker genes (i.e. E-cadherin) as by negatively correlating ones (i.e. Vimentin) which are not direct targets of the correlating miRNA (i.e. miR-200 does not target Vimentin). Our method is complementary to existing methods and, in conjunction with prediction algorithms and improved methods for confirmation of actual in vivo targeting events Our analysis is based on three premises: 1) Cancer-relevant miRNAs act in pathways that regulate cellular differentiation stages which can be recognized by the signature of expression of a number of stage-specific marker genes .In one early work, a computational strategy to predict miRNA regulatory modules (MRMs) was developed using bipartite graphs to model miRNA-mRNA binding structures ZEB1 and the miR-200 family). 2) The learning procedure of a Bayesian Network is computationally exhaustive Two approaches A recent approach was based on the statistical enrichment of miRNA targeting signatures in annotated gene sets. It allowed prediction of a number of novel targets for various miRNAs and prediction of disease relevant pathways in silico titration. We argued that, in order to detect mRNAs whose expression was repressed by miRNAs, the miRNA had to be expressed. We predicted that very high expression of miRNAs would suppress expression of targets representing certain biological processes. In contrast, in cells completely lacking expression of miRNAs, targeted genes would either be expressed or not expressed as a consequence of other regulatory mechanisms. In other words the “pressure” of a miRNA on a gene should be best detectable by comparing cells with high versus low expression of a miRNA. In order to reflect this pressure, different weights were assigned to each of the cell lines based on their miRNA expression levels. One could argue that the classical PCC method might also be modified in this way. However, in practical terms, it would be arbitrary to add weights directly to each cell line. First, the PCC calculation is not a linear process, and hence any change within the PCC function may have uncontrollable consequences. Second, it would not be clear how much weight to assign to each cell line. To solve this problem, we adopted the “titration” method sPCC, with a gradient weighting approach for cell lines sorted according to miRNA expression. We decided to pick 30 cell lines as a threshold, for the following reasons: 1) the model adds weight at the level of cell line selection, so it is linear and additive for PCCs with different cell line selections (e.g. from pattern 30 to pattern 59); 2) the model fully considers the biological importance of the 30 cell lines with the highest expression of each miRNA, as every pattern includes them; and 3) the minimal number of 30 cell lines ensured the methodological stability, as PCC is highly unstable and easy to be interfered by background noise when its sample size is too small (e.g. <10). We intentionally reduced the importance of the rest of the 29 cell lines, rather than simply neglecting them. If we had performed the dPCC analysis by only using the 30 highest expressing cell lines, it would be problematic since different sets of cell lines would be selected for each miRNA. In order to test the stability of our threshold selection, we used a randomized sPCC (rsPCC) method as an internal negative control  when compared to the dPCC method. Interestingly, only the sPCC was good enough to group miRNAs for exploring their potential biological functions. Regardless of whether positively or negatively correlating genes were used, the dPCC method failed to group some of the most biologically relevant miRNAs, such as members of the miR-200 and miR-17 families (data not shown). As an important control the sPCC method performed far better than the randomized rsPCC method. Hence, this method is robust enough to explore biological functions of miRNAs. We decided to let the user decide whether to use the dPCC or sPCC analysis on miRConnect.org. In summary, our data demonstrate that across all NCI60 cells the sPCC method performed best at both the theoretical and biological level, permitting detection of general connections between miRNAs and their downstream effectors.The performance advantage of the sPCC was obvious when the methods were tested for the ability to detect the conserved miRNA targets with the highest total TargetScan context score in the human genome. Only with the sPCC method the correlation increased with the more strongly predicted targets . We chosHOX gene clusters, which provided us with a unique system in which each cluster of multiple genes contained at least one miRNA gene. For both the host gene and the HOX gene cluster case we found a highly significant correlation between the expression of the gene(s) and the miRNAs that are linked to their co-transcription. Of the 73 host gene/miRNA pairs in our analysis of the Q data set, 37 showed a positive sPCC, but negative sPCCs were only found in 5 cases and they were of low significance , and few HOX genes negatively correlated with these miRNAs. This analysis in particular illustrates the strength of the expression correlation that can be found using the NCI60 data analysis. The sPCC method achieved the highest positive correlations between the four miRNAs and their adjacent HOX genes. Out of ∼18,000 human genes the following HOX genes had the highest positive sPCC for the miRNAs that are coded within a HOX gene cluster: HOXA9 and miR-196b, HOXB7 and miR-10a, HOXC10 and miR-196a and HOXD8 and miR-10b. Interestingly, our data allow us to predict that miR-196a-1, which is part of the HOXB gene cluster, is not significantly expressed in cancer cells, since neither of the two methods of analysis detected any HOXB genes that were co-expressed with this miRNA , a known regulator of EMT miR-7 expression . miR-203 was recently linked to EMT regulation and stem cells miR-203 was a potent inducer of E-cadherin upregulation. Predicted targets include the EMT regulator Snail2 (Slug) which also had a low negative sPCC of −8.01 (miRConnect.org). Finally miR-375 was recently reported to target 3-phosphoinositide-dependent protein kinase-1 (PDK1) miR-155, which we had already identified as most highly expressed in mesenchymal cancer cells Induction of EMT induces changes in expression of thousands of genes in the human genome. Many of these changes involve sharp on-off regulation of genes. Examples of genes that are regulated in a switch-like manner are miRConnect.org site we have made data sets available based on two different miRNA expression data sets. In a recent analysis the level of concordance between 4 different miRNA expression data sets obtained with the NCI60 cells was moderate let-7, miR-200 or the miR-17∼93 family were more consistent (data not shown). Regardless of the nature of the differences they must not necessarily be due to fluctuation between the analyses. The two platforms that generated the Q and the L data sets are very different and each has both advantages and caveats. The LNA-based arrays that underlie the L data set cannot discriminate between mature miRNAs and their pre-miRs but are more robust in detecting different isomiRs. In contrast, the Q data set relies on real-time PCR involving loop primers. While they are highly selective for the mature miRNAs they cannot distinguish between certain isomiRs. This could be relevant because in a recent study it was demonstrated that miRNAs exit as multiple isomiRs and this varies among species and tissues miRConnect.org.On the c-MYC and let-7, and ZEB1/ZEB2 and miR-200miR-200 to EMT or miR-10/miR-196 to HOX gene expression) without the need to validate specific mRNAs as miRNA targets.Expression levels of miRNAs and mRNAs have been correlated previously in attempts to establish causality Based on our data, we propose to focus more on individual pathways when assigning cancer relevant functions to miRNAs and less on global activities. We believe that the functional grouping of miRNAs using miRConnect will significantly aid miRNA researchers and will provide a novel resource to the field that complements the study of miRNA function based on their seed sequences. We propose to group miRNAs families according to seed families, gene clusters, and functional correlations.http://dtp.nci.nih.gov/mtargets/download.html as WEB_DATA_ISRAEL_MIR.ZIP. For each miRNA the number of cell lines with an expression level above the limit of detection was determined. The real time PCR data set for miR-429, which was not part of the original data set, was provided by Dr. Arti Gaur . Expression of the miRNA was determined by normalizing the data set as previously described TargetScan.org. To develop the sPCC method, the integration of miRNA IDs and measurements was done using a customized Java platform (Netbeans java console project MicroRNAparser). A few miRNAs were not explicitly included in the TargetScan family assignments and a few used different nomenclatures than that used in TargetScan. In these instances miR_family assignments were made based on sequence  for 207 individual miRNAs was previously described ence see . For anyThe LNA array based expression data (log2 ratios) of 955 individual miRNAs were recently described http://dtp.nci.nih.gov/mtweb/search.jsp). Of these, 82,564 gene probes had GeneCard identifiers  and they represented 18,462 uniquely known genes in the human genome.The analyses performed in this work covered a total of 130,368 individual gene array probes in the MICROARRAY_ALL target set available at the DTP site (http://dtp.nci.nih.gov/compare). It was developed in the early 1990's to explore the results from the NCI60-cell line screen COMPARE analysis was used to generate correlations between the data in the miRNA Q data set and the gene array data. COMPARE is a computational tool available through the NCI DTP web site.  were then determined by averaging the individual correlations for each gene. To generate miRConnect-L, the expression profiles of 571 miRNAs/53 miRNA families and 18,747 genes in 59 cell lines were used to calculate a Pearson Correlation Coefficient (PCC) between each miRNA and each gene. Then, the average values of PCCs between each miRNA and each gene were calculated across all 4 gene array data sets for gene expression to normalize for variation among different microarray platforms. These averaged values were designated direct PCC (dPCC). During this process missing values were excluded. The top 2000 positive and negative dPCCs  were collected to perform the EMT, c-MYC, and RPs correlations. This resulted in the elimination of 97% of the correlations with dPCCs corresponding to values between −0.3 and +0.3.To generate miRConnect-Q, each of the 136 miRNAs with at least 30 cell lines with detectable expression and each miRNA of the 25 families was used as a seed for a single COMPARE analysis against 18,747 genes. The microarray features for all COMPARE correlations were collected. Correlations for microarray features associated with named genes . For each miRNA or miRNA family, pattern 30 consisted of the first 30 cell lines (those with the highest levels of expression), pattern 31 included all of the cell lines from pattern30 and the cell line with the next highest level of expression, pattern 32 included all of the cell lines from pattern 31 and the cell line with the next highest level of expression, and so on. The last pattern, pattern 59, consisted of all of the cell lines and completed the set of linked patterns each with 30 members. Each individual pattern was used as a seed for a single COMPARE analysis against the MICROARRAY_ALL target set. After COMPARE had been run on all 30 linked patterns, the sum of the COMPARE correlations (sum_correlation) for each microarray feature for the 30 patterns was determined. Correlations for microarray features associated with named genes (GeneCard/HUGO) were then determined by averaging the individual correlations for each gene. The top 2000 positive and negative sPCCs  were collected to perform the EMT, The only difference between the rsPCC method and the sPCC method was that rsPCC generated patterns according to randomized sorting of cell lines. Each rsPCC was repeated 10 times, and average of the calculations was taken.http://www.targetscan.org. The TargetScan data sets were used to generate predictions for microRNA families, to limit those predictions to conserved miRNA, and to generate custom predictions for families that we defined. These manipulations were based on the descriptions of the TargetScan algorithms which were included on the TargetScan 4.2 web site, with slight modifications to accommodate changes in the downloadable data as follows: Predictions were limited to those predictions that included at least one human example, context_scores >0 were not included in the determination of total_context_score, and the aggregate prediction for a miRNA family was the minimum total_context_score for any miRNA that was a member of that family.Predictions and underlying data for TargetScan version 5 were downloaded at miRConnect.org.A table of miRNA/mRNA pairs was loaded into a (MySQL) database along with their correlation values, mRNA chromosome locations, and function annotations (downloaded from the HUGO). A web interface was built to filter and extract data from this table. Genes can be shown that correlate positively or negatively with a given miRNA or miRNA family, according to the dPCC or the sPCC method. If no miRNA is selected but instead a list of mRNA gene IDs are entered, then miRNAs that correlate will be listed. For negative correlations, TargetScan total_context_score was also provided. Functional annotation search words can be used to constrain the output, and all output tables are downloadable in spreadsheet-ready form. The site contains all data on miRConnect-Q and -L and is accessible at HOX family genes and the miRNAs. In order to spotlight the miRNAs associated with HOX gene clusters, for each miRNA we summed up the dPCC/sPCC for all the members in each HOX gene clusters.The miRNA host genes were retrieved by comparing the genomic positions of miRNAs and genes as recently described Specifically expressed miRNAs in each type of cancer were screened out by Significance Analysis of Microarrays (SAM) with a manually adjusted False Discovery Rate (FDR). FDR was set as 0.01 .For the sPCC positively and negatively associated gene lists, we queried all possible pairs of miRNAs with either the top 2000 sPCC values (for positive correlations) or bottom 2000 sPCC values (for negative ones) and calculated the percent identity of overlapping correlated genes between any two miRNAs. Thus 136×136 matrices for the overlapping gene numbers were generated. Then both matrices were transformed to distance matrices by using Euclidean distance measure (R function: as.dist). Complete linkage method was used to find similar clusters (R function: hclust) for dendrogram plotting. Functional clusters of miRNAs were defined by miRNAs that shared more than 12.5% correlating genes (R function: cutree). Principal component analysis (PCA) was adopted to do a orthogonal transformation, converting correlated miRNAs into an equal size of uncorrelated variables called Principle Components (PCs). The 136×136 matrix for the positively overlapping gene numbers was used to do orthogonal transformation using the R function prcomp. The two principal components with the highest score were used to generate a PCA plot.c-MYC regulated genes. A non-redundant list of 460 genes upregulated by c-MYC and 211 genes suppressed by c-MYC was obtained from the c-MYC target gene database (http://www.myccancergene.org/) Two sets of gene signatures were used. 1) E-cadherin were serially transfected with pre-miRNAs (Ambion) up to 3 times as described E-cadherin were performed as described Mesenchymal ACHN cells which express little http://www.r-project.org/) was used to perform microarray data manipulation, dPCC calculations, sPCC/dPCC/rsPCC comparisons, miRNA target enrichment, hierarchical clustering, host gene and HOX gene analyses, PCA analysis, and gene expression signature calculations.R statistical program v2.10 Click here for additional data file.Figure S2For each of the HOX gene clusters the expression of the hosted miRNA best correlates with the expression of the HOX genes in that cluster. The sPCC values for the genes in each HOX gene cluster were cumulated for the 136 miRNAs and plotted by ranking 136 cumulative sPCC values from highest to lowest. For each HOX cluster the top ten miRNAs are listed in a table.(PDF)Click here for additional data file.Figure S3Cluster analysis of 136 miRNAs based on a pairwise comparison of negatively correlating genes using the sPCC method. The miRNAs were divided into 17 functional clusters (I–XVII). Stippled red line: threshold of 12.5% of groups that defined the 17 clusters.(PDF)Click here for additional data file.Figure S4miRNAs that are most epithelial and most mesenchymal in nature according to their positively correlated genes. (A) Ranking of 136 miRNAs according to their correlation with the expression of either E or M genes calculated by taking sPCCs related to the average of EMT signatures 1–3 (see miR-200 family members (shown in red) were found in the top 9 miRNAs most positively correlated with the expression of E genes (left stippled line). Additional E related miRNAs, miR-203, miR-7, miR-375 and miR-425* are shown in orange. The miRNAs best correlating with the expression of M genes are labeled in blue (defined by stippled line on the right). (B) Overlap in positively correlated genes between the entire miR-200 family and the miRNAs that are most epithelial in nature (red/orange) or most mesenchymal in nature (blue). 1–3 see . All 5 m(PDF)Click here for additional data file.Figure S5Principal Component Analysis of 136 miRNAs according to positively correlating genes, with the two PCs (PC1 and PC2) with the highest score plotted. The epithelial subcluster of miRNAs that regulated E-cadherin (see erin see  is label(PDF)Click here for additional data file.Figure S6Correlation of miRNA expression with that of ribosomal protein genes. The 136 miRNAs that correlated in their expression with those of ribosomal genes (RPs) were ranked according to the number of RPs that had positive or negative sPCC values with individual miRNAs. Rank order was determined by the factor: [number of positively correlating RPs] - [number of negatively correlating RPs].(PDF)Click here for additional data file.Table S1Expression of miRNAs in 59 of NCI60 cell lines sorted according to numbers of cell lines with detectable expression. miR-429 was found to be significantly expressed in more than 30 cell lines (data not shown).(XLS)Click here for additional data file.Table S2miRNA seed families represented in the Q data set. miRNAs in red = family members for which no data sets were available.(XLS)Click here for additional data file.Table S3Analysis of the top 500 conserved genes predicted by TargetScan in the human genome to be targeted by one of the 136 miRNAs.(XLS)Click here for additional data file.Table S4Identification of miRNAs with tissue specific expression. FDR: False Discovery Rate.(XLS)Click here for additional data file.Table S5miRNAs that are part of either seed families, gene clusters or are tissue specifically expressed.(XLS)Click here for additional data file.


Recent studies have linked a high fat diet to the development of breast cancer, but any genetic basis for this association is poorly understood. We investigated this association with an epistatic analysis of seven cancer traits in a segregating population of mice with metastatic mammary cancer that were fed either a control or a high-fat diet. We used an interval mapping approach with single nucleotide polymorphisms to scan all 19 autosomes, and discovered a number of diet-independent epistatic interactions of quantitative trait loci (QTLs) affecting these traits. More importantly, we also discovered significant epistatic by diet interactions affecting some of the traits that suggested these epistatic effects varied depending on the dietary environment. An analysis of these interactions showed some were due to epistasis that occurred in mice fed only the control diet or only the high-fat diet whereas other interactions were generated by differential effects of epistasis in the two dietary environments. Some of the epistatic QTLs appeared to colocalize with cancer QTLs mapped in other mouse populations and with candidate genes identified from eQTLs previously mapped in this population, but others represented novel modifying loci affecting these cancer traits. It was concluded that these diet-dependent epistatic QTLs contribute to a genetic susceptibility of dietary effects on breast cancer, and their identification may eventually lead to a better understanding that will be needed for the design of more effective treatments for this disease. Breast cancer is a particularly prevalent form of cancer that affects about 12% of all women at some point during their lifetime , with thBRCA1 and BRCA2 are well known to predispose individuals to an increased risk of breast cancer, it has become clear that the incidence of this disease primarily depends on the action of a number of polygenes . This equation shows that the LOD scores should increase linearly with increasing numbers of locus pairs. For each trait, we found this to be the case, and therefore used the intercepts and slopes calculated from these regressions to predict the threshold value for n = 3172 tests. In this manner, we estimated significant and suggestive threshold LOD values specific for each of the traits in tests both of diet-independent and diet-dependent epistasis. All estimated significant thresholds in fact exceeded 4.80, and 13 of the 14 suggestive thresholds exceeded 4.20 (see Results below).To arrive at more appropriate thresholds, therefore, we generated simulations as before, but this time for each of 3, 6, 12, 20, and 50 independent pairs of loci. We ran the epistasis models for each trait on 1000 simulated samples and identified the highest LOD score in each run. We then used the 95th (and 80th) percentile value from the LOD distributions from each of the five cases of different numbers of loci for use as the dependent variable in a linear regression. In this model, the log of the number of pairs of loci (n) served as the independent variable, allowing us to estimate the y-intercept aa and dd epistatic values appeared to predominate among the tumor and  the metastatic traits, but the overall distribution of aa, ad/da, and dd values  followed the expected 1:2:1 ratio (P = 0.49 in a chi-square test). The percentage impact of the significant epistatic components on the phenotypic variability in the traits was substantial, ranging from 15% to 41% and averaging 27.1%.We discovered a total of 28 diet-independent epistatic interactions affecting the cancer traits . Only twdd) epistatic effects on TOID in females generated by an interaction of QTLs on chromosomes 1 and 7. We calculated the expected values for each of the nine genotypes in this interaction from the coefficients produced in the epistasis solutions and the expectations given by Wolf et al. [ illustraf et al. . Note thaa, ad/da, and dd components with diet over all traits  followed the expected 1:2:1 ratio (P = 0.84 in a chi-square test). For nine of the epistasis by diet interactions, significant epistasis occurred about equally in mice fed the control versus the high fat diet (4:5). For the remaining 13 interactions, differential effects occurred in which there was significant epistasis in both dietary environments or in neither dietary environment.We discovered a total of 22 diet by epistasis interactions affecting the cancer traits . The genaa, ad, and da epistatic effects differed in sign  in male mice fed the control versus the high fat diet. In the line diagram, this effect may be seen by the varying degrees of dominance especially noticeable in the high fat diet.  previously mapped on this chromosome (at 4 cM) that also affects metastasis of mammary cancer [Some of the diet-independent epistatic QTLs may be the same as cancer loci previously mapped in other mouse populations. This seems particularly likely for epistatic QTLs on chromosomes 9 and 19. Thus we found a QTL on chromosome 9 (at 54–56 cM) epistatically interacting with QTLs on chromosomes 8 and 13 to affect TOID in males, and it colocalizes with a QTL on this chromosome at 55 cM previously found to affect tumor latency  and tumy cancer . With thaa, ad/da, and dd) present in expected (1:2:1) proportions and contributing substantially (average of 27%) to the phenotypic variation in the cancer traits. In general those traits affected by the greatest number of significant components  showed the highest percentage contributions, although just two significant components from a single epistatic interaction accounted for 15.2% of the variation in TI in males . In fact we found 22 epistasis by diet interactions affecting the cancer traits, including two that were significant at the 5% genomewise level. This level is somewhat less than the 28 instances of epistasis alone affecting the traits, although the significant and suggestive threshold LOD values  used for testing these interactions were somewhat higher than those used in the epistasis tests . Use of the liberal threshold generated 90 diet-dependent epistatic interactions affecting the cancer traits (results not shown), nearly the same as the 91 instances of diet-independent epistatic interactions. In general, therefore, we have evidence that, like single-locus effects of QTLs [Although chromosomal contributions in the diet-independent and diet-dependent epistatic interactions  and 2 apP = 0.034 in a chi-square test) whereas the reverse was true for females . In females this trend was particularly apparent for TOID where 11 significant diet by epistasis interactions occurred in the control group and none occurred in the high fat group. This trend for females is opposite to that found (in both sexes) by Gordon et al. [At the sites on the 22 pairs of chromosomes where diet-dependent epistatic interactions affected the cancer traits , signifin et al.  in theirAs an example of this, Gordon et al. , found acis-acting eQTLs scattered among all 19 autosomes, with a number being possible candidate genes for the diet-independent QTLs we discovered. Two examples are Man2a1 that is a candidate for an epistatic QTL on chromosome 17 affecting MET, and Wwc2 that is a candidate for an epistatic QTL on chromosome 8 affecting TI and AMD. Gordon et al. [cis eQTLs with diet, and detected just 15 significant instances of these interactions, the majority (9) of which were generated by significant eQTL effects in the control diet only. Among these 15 eQTLs, Gdi3, D14Ertd449e, and Notch4, respectively, appear to be reasonable candidates for diet-dependent epistatic QTLs on chromosomes 13, 14, and 17. La Merrill et al. [Vegfa, vascular endothelial growth factor A. This gene was upregulated in mice fed the high-fat diet [Vegfa is located on chromosome 17 at 23 cM, and thus is a candidate for the epistatic QTL we discovered on this chromosome (at 27 cM) interacting with a QTL on chromosome 18 to affect MET (What might be the identity of these epistatic QTLs affecting the cancer traits? Fortunately, some possibilities may be inferred from an eQTL analyses conducted by Gordon et al.  and La Mn et al.  also tesl et al.  developefat diet , and hasfat diet . Vegfa ifect MET .2 population of mice. While this was not unexpected because of occasional reports of significant interactions of cancer QTLs, our genome scan revealed a number of novel QTLs throughout the genome that participate in interactions affecting these kinds of traits. More importantly, however, we also have shown that these epistatic effects sometimes vary depending on the dietary environment. Although Gordon et al. [We have demonstrated that epistasis affects the cancer traits measured in our Fn et al.  showed t


Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a progressive disease, typically appearing in the 5th decade of life and is one of the most common monogenetic inherited human diseases, affecting approximately 600,000 people in the United States. Because ADPKD is a slowly progressing disease, I asked how fluid flow may act, via the primary cilium, to alter epithelial physiology during the course of cell turnover. I performed an experiment to determine under what conditions fluid flow can result in a change of function of renal epithelial tissue. A wildtype epithelial cell line derived the cortical collecting duct of a heterozygous offspring of the Immortomouse (Charles River Laboratory) was selected as our model system. Gentle orbital shaking was used to induce physiologically relevant fluid flow, and periodic measurements of the transepithelial Sodium current were performed. At the conclusion of the experiment, mechanosensitive proteins of interest were visualized by immunostaining. I found that fluid flow, in itself, modifies the transepithelial sodium current, cell proliferation, and the actin cytoskeleton. These results significantly impact the understanding of both the mechanosensation function of primary cilia as well as the understanding of ADPKD disease progression.Although solitary or sensory cilia are present in most cells of the body and their existence has been known since the sixties, very little is been known about their functions. One suspected function is fluid flow sensing- physical bending of cilia produces an influx of Ca Cilia grow from the mature mother centriole of the mother-daughter pair of centrioles in the cell's centrosome Cilia are microtubule-based projections from the cell body  Smell, taste, and vision occurs via modified cilia: olfactory or gustatory sensory cilia and outer segments of rod cell, respectively  is a progressive disease, typically appearing in the 5++-permeable cation channel in renal epithelia ++ influx after deletion of the ciliary motor Kif3a and this reprogramming cannot be ascribed to faulty development th decade) appearance of cysts as well as the simultaneous appearance of numerous focal cysts through accumulated mutations in mechanosensitive proteins. Abnormalities in ciliary associated proteins result in altered cell functions and disease progression.Genetic evidence is quite strong that ciliary dysfunction is an important determinant of renal epithelial cell biology and cystogenesis: 1) The gene products of PKD1 and PKD2 (polycystin 1 and polycystin 2) Evidence for flow as an environmental factor for ADPKD is currently indirect, and is based mostly on the purported function of the primary cilium Because ADPKD is a slowly progressing disease, I asked how fluid flow may act to alter cellular physiology during the course of cell turnover. Cell division in renal epithelia occurs not just during growth or repair of the nephron, but also constitutively. Although not completely understood, it is suspected that renal epithelial cells can de-differentiate, proliferate, and re-differentiate to restore functional integrity of the nephron Previously, my laboratory has shown that a cell line derived from the Immortomouse- principal cells of the cortical collecting duct- respond to fluid flow by changing the transepithelial sodium flux, and that this response requires the presence of an intact primary cilium We previously demonstrated that ciliary-mediated mechanosensation is very sensitive to flow and that an intact cilium is required to generate a cellular response to flow. Additionally, some of the data suggested that the cellular response involved cellular memory- cells that lost the primary cilium did not attain original levels of transepithelial sodium current after regenerating the cilium.The first set of results  comparesSeveral results obtained previously Interestingly, monolayers in cohort 5 which differentiated under quiescent conditions for an extended period of time (10 days) before being exposed to flow also exhibited a graded response to changes in flow conditions, similar to cohort 4. Unfortunately, both cohorts 5 and 6 experienced significant contamination during the experiment and are largely excluded from the results.Examination of the transepithelial sodium flux of Cohorts 1 and 2 are less definitive (data not shown); Cohort 2 appeared to present a graded response to flow while Cohort 1 did not, consistent with the data for cohorts 3 and 4.Comparing the response of matched monolayers from cohorts 3 and 4 during the final stage of the flow protocol is shown in Some cells were returned to the differentiation conditions  while others were not. Ratioing the measured sodium current from paired monolayers returned to differentiation conditions to those that were not measured the degree to which monolayers maintained the ability to sense flow. This appears to be a long-lived feature of the cells, as shown by data presented in The results provided here provide insight to the state of the cell only at the endpoint of the flow protocol; they provide information regarding the terminal state of the epithelial tissue. Note, although the various proteins were tagged with a wide spectrum of fluorescent secondaries as detailed in the methods section, for ease of comparison in images both cytoskeletal proteins (acetylated α-tubulin and actin) are rendered in green, both membrane proteins (Polycystin-2 and Somatostatin Receptor-3) are rendered in red, and the nucleus is rendered in blue.2) was not normally distributed, as verified by a Q-Q plot 2 and 1640 +/− 160 cells/mm2). Because cell division halts when the cells become terminally differentiated, this data indicates that some cohorts take longer to differentiate than others- and further, the delay was sufficient for at least an additional cell cycle (8 hours).This data provides suggestive evidence about the interplay between differentiation and fluid flow conditions. Cohorts 1 and 2 were exposed to flow either prior to differentiation or in the very early stages of differentiation. Cells hyperproliferated when flow conditions changed , while cells did not proliferate when flow conditions did not change . Cohort 1C did not show evidence of hyperproliferation, but the actin cystoskeleton of all the sub-groups of Cohort 1showed profound phenotypic differences with respect to all other cohorts (discussed below). Cohorts 3 and 4 were resistant to changes in flow conditions: the only group to hyperproliferate was subjected to maximal changes in flow conditions . Of special interest are the results for cohorts 5B and 6- both showed evidence of hyperproliferation in spite of flow changes occurring well after (10 days) the onset of differentiation. The data presented here leads us to conclude that cells in a dynamic environment (i.e. the fluid flow rate is variable) can remain in a dedifferentiated pro-growth state, while cells exposed to a static environment- an environment where the fluid flow does not vary- stop proliferating and differentiate. These results should be considered together with other recent results Comparison of all monolayers either exposed to flow or not at the conclusion of the experiment show that in the presence of apical flow, increased numbers of focal adhesions are formed. This should be compared with the electrophysiological results during the same time period. Both cohorts 3 and 4 (which the exception of 4B) maintained the ability to sense flow, while group 4B lost the ability to sense flow. The visual appearance of the actin cystoskeleton provides evidence that focal adhesions (or integrin binding) may be correlated with the ciliary-mediated ENaC flow sensing response.Based on considerable evidence presented in the literature in vivo chronic stimulation within the mouse nephron  changes in tubule flow drive increased cellular proliferation and 2) the cessation of flow causes increased salt transport. In this model, a tubule experiencing a dynamic flow environment will experience underregulated growth, leading to an overall decrease in flow rate, leading to increased salt resorption and hypertension.Placing the results in the context of renal function, it is clear that there is significant interplay between applied flow conditions and salt resorption. The results show a clear response when the flow conditions change, and salt transport is decreased when flow is present. Furthermore, flow conditions present during differentiation affect the mechanosensation response later in time. It must be noted that under normal physiological conditions, tubule flow is highly dynamic due to changes in blood pressure over the course of a heartbeat. Importantly, the timescale of flow changes in this experiment are much longer and correlate to slow-acting chronic changes rather than the rapid changes associated with cardiac function.Of note is the long-time behavior of cohort 4B: from the data presented in The actin images, taken with the protein localization images, show that actin remodeling is potentially independent from the ADPKD/BBS signaling pathway. Actin cytoskeleton remodeling is an acute response that occurs on a time scale of less than 10 days. Except for one case (Cohort 1C) which was exposed to high levels of flow at the onset of differentiation, the final state of the actin cytoskeleton is independent of the past history of flow conditions. It is not clear what the physiological relevance of this result is at this time, but the data provides evidence that actin remodeling may be an essential component of mechanosensation.While the actin cystoskeleton shows extensive remodeling in response to physiological flow conditions, SSTR3 does not show any change in cellular distribution while PC2 does, in agreement with previous results The method used to induce apical flow is that same as presented in an earlier study Three types of mechanical forces due to the shaking are readily identified: 1) Shear stress on the apical membrane due to fluid movement relative to cells; 2) Drag force on the cilium due to fluid movement; and 3) Buoyancy induced by centripetal forces and density differences between the cilium and apical fluid, often referred to as “body force” because this force is distributed throughout the volume of the cilium. Calculations of the shear and drag forces require information on the fluid velocity profile between wall and cilia tips, in addition to fluid density, viscosity and ciliary geometry, while those for the buoyancy force require additional information about the density difference between cilium and fluid.The fluid flow induced by gentle orbital shaking, particularly when the fluid layer above the cells is thin, constitutes a complex problem. To the best of my knowledge, neither an analytical nor a numerical solution of the flow field is available in the literature.1’ and ‘c2’ were determined by measurement and are unique to the orbital frequency. Once the velocity profile is known, the shear stress (τ) at the apical surface of the monolayer is defined by Equation 2:2. If this area is assumed as effective apical surface area, then the shear force on the entire cytoskeleton per cell is about 29 fN. However, because the cilium is required for flow sensing In our earlier study The drag force on a cilium can be calculated from fluid velocity transverse to the cilium assuming the cilium to be a rigid cylinder capped by a hemisphere and is given by Equation 3 The buoyancy force comes about because of the relative motion between the monolayer frame and laboratory frame. Even if monolayer and fluid co-move (or if the cilium is free to pivot and is freely advected by the fluid), there will be a resultant force on the cilium due to centripetal acceleration. This force, averaged over time, is given by Eq. 4.Knowledge of the limits of applied forces is important for exploring the physiological roles of cilia in mechanosensation and ciliary pathophysiology in human disease, such polycystic kidney disease. The calculations were restricted to limit estimates because, as indicated above, no mathematical solution of the flow field is currently available for the type of geometry present in orbital shaking. We also need to emphasize that the elastic interaction between a cilium and the apical fluid is the subject of much research and a definitive picture has yet to emerge. I assume here, given the smallness of the applied forces, that the cilia do not move. If the cilia either flex or deflect in response to an applied force, then the applied force is even smaller than is calculated here, and so I consider the calculations to provide a maximal value.Calculations were carried out for cilia with a length of 2.7 µm and a diameter of 0.2 µm. Following the calculations in The primary conclusion is that the predominant force acting on the cilium is due to drag- the flow of fluid past the cilium.2; Millipore Corp, Billerica, MA) to promote a polarized epithelial phenotype. Cells were grown to confluence at 33°C, 5% CO2 and then maintained at 39°C, 5% CO2 to enhance differentiation. The growth medium consisted of the following : Dulbecco's Modified Eagle Medium w/o glucose and Ham's F12 at a 1∶1 ratio, 5 mM glucose, 5 mg/ml transferrin, 5 mg/ml insulin, 10 µg/ml epithelial growth factor (EGF), 4 mg/ml dexamethasone, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.06% NaHCO3, 2 mM L-glutamine, 10 ng/ml mouse interferon-γ, 50 µM ascorbic acid 2-phosphate, 20 nM selenium, 1 nM 3,3′,5-triiodo-L-thyronine (T3), 5%fetal bovine serum (FBS). For differentiation, FBS, insulin, and interferon-γ were omitted from the apical medium and insulin, EGF, and interferon-γ from the basal medium. The apical amount of medium was restricted to 100 µl per filter insert so that the apical fluid was thin enough to allow sufficient O2 to diffuse to the monolayer. All monolayers were routinely monitored for electrical resistance and transepithelial potential.Experiments were carried out with a mouse cell line derived following sc) assuming an ohmic relationship.Transepithelial voltage and resistance measurements were performed using an Endohm chamber , connected to a MilliOhm  voltohmmeter containing DMEM plus penicillin-streptomycin (10 mg/ml) and gentamicin (50 mg/ml). Cells were placed in the Endohm chamber for no more than 1 min, during which time the transepithelial voltage remained constant. The transepithelial voltage and resistance were converted to short-circuit current equivalent (Isc) with identical electrolyte solutions on both sides of the epithelium. The current flow through ENaC is conveniently quantified by sensitivity of Isc to 10 µM of apical amiloride, a well understood diuretic drug. Similarly, calculation of Isc from transepithelial potential and resistance provides a quantitative measure of ENaC activity. More than 95% of Isc was inhibited by 10 µM apical amiloride and thus Isc is considered to be proportional to the activity of epithelial sodium channels (ENaC) in the apical plasma membrane.Sodium transport via ENaC is a well understood process that is described by the classical and, by now, text book Ussing model of sodium absorption: Sodium entry at the apical membrane is by electrodiffusion through the ENaC channel and exit at the basolateral membrane is by active transport via Na,K-ATPase in the basolateral plasma membrane. Due to the presence of potassium channels in the basolateral plasma membrane, Sodium transport by this process is completely electrogenic and can be quantified by measuring the short-circuit current , and 5% fetal bovine serum (FBS). The monolayers were then stained with primary antibodies against proteins of interest , actin stained using phalloidin- Oregon green, polyclonal antibodies against Polycystin-2 anti-goat (H-280) and Somatostatin Receptor Type-3 (SSTR3) anti-rabbit (W-15), both supplied by Santa Cruz Biotechnology. The secondaries used to visualize the primary antibodies were Cy5 for acetylated α-tubulin , DyLight 405 nm (Polycystin-2) and Texas Red (SSTR3), both supplied by Thermo Scientific. The stained filter was cut out of the culture insert and transferred to a microscope slide, monolayer side up. The filter was mounted in VectaShield  with DAPI. A # 1 ½ coverslip was placed on top of the monolayer- if needed, small spacers were used to prevent the coverslip from contacting the monolayer. The slides were then sealed with nail polish and stored for imaging at 4°C.Image stacks were acquired with a Leica DM6000 B upright microscope using a 63X numerical aperture 1.47 Plan Apo lens with a Lumen 200 Pro source (Prior Scientific) and a Rolera-MGi Plus intensified camera (QImaging) under control of MicroManager, an open-source microscopy software package Cells were subjected to orbital motion by placing monolayers on top of a shaker table  placed within a standard laboratory incubator. Monolayers without shaking were maintained in the same incubator. Shaken monolayers were subjected to orbital motion at a fixed frequency (either 0.9 Hz (slow) or 5 Hz (fast)) determined by a stopwatch. Electrophysiological measurements or fixation for immunocytochemistry were carried out on cells immediately after removal from the orbital shaker.Broadly speaking, monolayers were exposed to a particular initial condition (flow or no flow) for 10 days, subjected to a second flow condition  for 10 days, and finally were returned to either slow or no flow conditions until fixation . Each grOne consequence of this protocol is that each initial cohort had some subgroups that followed the same experimental protocol as monolayers in other cohorts: for example, monolayers 1A1 were never exposed to orbital motion, similar to monolayers in groups 3A1 and 5A1. Similarly, monolayers 2B2, 4B2, 5B2 were continuously shaken at the same frequency throughout . Thus, the protocol established internal positive and negative controls.The dynamic viscosity of apical media was measured with a Cannon-Fenske Routine Viscometer  with the apparatus and media equilibrated to 37°C. The density of the media was measured by weighing a controlled volume . In both cases, the apparatus was calibrated by measurements on double distilled water to ensure accuracy.t-test, two-tailed heteroscedastic, as appropriate. Values of p<0.01 are considered significant. Groups with fewer than 3 monolayers were omitted from presented results.Data is presented with +/− 1 standard deviation error bars. Statistical analysis was done by Student's The primary finding of interest here is that cells differentiated in the presence of flow behave very differently from cells differentiated in the absence of flow. Additionally,our cell line displays highly physiologically relevant behavior in the presence of fluid flow: the transepithelial sodium current is modulated, the actin cystoskeleton remodels, and certain membrane-bound proteins localize to the primary cilium. Taken together, our results begin to show a pathophysiological mechanism by which tubular cells hyperproliferate and form fluid-filled cysts: salt transport is decreased, leading to an accumulation of ultrafiltrate on the apical side of the epithelium. The presented data also supports the use of this cell line as a disease model. Future work will involve subjecting cells to a more defined flow by culturing the cells in a flow chamber rather than subjecting the cells to orbital motion. Additionally, further work determining the protein localization of Polycystin-1 (which cleaves in the presence of flow), STAT6  will be performed.


In images of textured three-dimensional surfaces, pattern changes can be characterized as changes in orientation and spatial frequency, features for which neurons in primary visual cortex are classically selective. Previously, we have demonstrated that correct 3-D shape perception is contingent on the visibility of orientation flows that run parallel to the surface curvature. We sought to determine the relative contributions of orientation modulations (OMs) and frequency modulations (FMs) for the detection of slant and shape from 3-D surfaces. Results show that 1) when OM and FM indicate inconsistent degrees of surface slant or curvature, observer responses were consistent with the slant or curvature specified by OM even if the FM indicated a slant or curvature in the opposite direction to the same degree. 2) For slanted surfaces, OM information dictates slant perception at both shallow and steep slants while FM information is effective only for steep slants. Together these results point to a dominant role of OM information in the perception of 3-D slant and shape. The visual system is well able to extract information about 3-dimensional (3-D) shape from monocular cues. One potentially powerful monocular cue is the pattern or texture on a surface, which when projected in perspective, results in systematic changes in the texture in the image. These changes have commonly been referred to as texture gradients  Recent adaptation studies have shown support for neural mechanisms that extract orientation flow information that exhibit invariance to the nature of the texture pattern The goal of the current study is to examine the contributions of changes in orientation and frequency to the detection of slant and shape from 3-D surfaces projected in perspective. Our hypothesis is that the visibility of orientation and frequency information in perspective images is directly linked to the perception of 3-D slant and shape. We used planar and corrugated surfaces texture-mapped with grating patterns. Planar surfaces were slanted out of the fronto-parallel plane, and corrugated surfaces varied in concavity/convexity. Examples of the planar stimuli are shown in All research followed the tenets of the World Medical Association Declaration of Helsinki and informed written consent was obtained from the observers after explanation of the nature of the study. The research was approved by the Queens College Institutional Review Board.All stimuli were generated in MATLAB, converted to bitmap format in Adobe Photoshop CS5 and presented on a calibrated 22″ Mitsubishi Diamond Pro 2070 flat screen CRT monitor with a 1024×768 pixel resolution at a refresh rate of 100 Hz. The monitor was driven by a Cambridge Research Systems ViSaGe (CRS) Visual Stimulus Generator running on a 3.2 GHz Pentium 4 PC. Experimental code was written in MATLAB 7.4 using the CRS Toolbox. Observer responses were recorded using a CRS CB6 infrared response box.Observers’ head positions were fixed with the use of a chinrest situated 1 m from the stimulus monitor. In all conditions, the center of the screen was level with the observer’s eye. When one stimulus was on screen, it was centered, or if two stimuli were presented simultaneously they were both equally spaced 3.25 deg to the left and right of center. Viewing was monocular in a dimly lit room, with no audio feedback except to indicate that a response had been registered.Li and Zaidi 2. This field of view was chosen to allow for a consistent presentation of stimuli across both experiments. A black central fixation cross subtending 17×17 arc min was present at the center of the screen for the entire duration of all sessions.All stimuli were presented in a circular aperture subtending 6.5 deg (11.36×11.36 cm) on a grey background with a mean luminance of 54 cd/minconsistent slanted condition, stimuli were generated by mapping developable planar surfaces with different textures, slanting them around a vertical axis of rotation and projecting them in perspective. Surfaces were patterned with 2 cpd horizontal and vertical gratings at 50% contrast obtainable on the display monitor, and 2 cpd horizontal-vertical plaids at 100% contrast. Slanting the surfaces about a vertical axis and viewing them in perspective produces systematic changes in orientation (orientation modulations or OM) in the horizontal gratings and systematic frequency modulations (FM) in the vertical gratings  (see In the nts) see . For FM-nts) see .All sessions began with a 60 sec fixation period to a mean grey screen followed by a tone to signal the start of the trials. An audio beep coincided with the presentation of each test stimulus. Stimuli were presented for 250 msec each. Following each stimulus presentation, a 500 msec Gaussian noise mask was presented to minimize potential afterimages of the test stimuli. The screen then remained at the mean grey until the observer made a response. Each session lasted approximately 15–20 minutes.inconsistent slanted condition, observers performed a single interval task in which they were asked to indicate the direction of slant for a planar surface textured with a plaid containing both OMs and FMs. Both OM-fixed and FM-fixed stimuli were randomly interleaved into one session. There were a total of 280 trials .In the inconsistent corrugated condition was analogous in design to the above condition, in which observers were given a single interval task but asked to indicate the sign of surface curvature (convex or concave) for a plaid textured corrugated surface. As in the above condition, OM-fixed and FM-fixed stimuli were randomly interleaved into one session. There were a total of 440 trials .The The goal of this second experiment was to quantify the contributions of orientation and frequency to the perception of slant when the two specify consistent slants. We first measured the minimum amount of slant required to perceive pattern changes in OM and FM-only stimuli. We then compared these thresholds to the minimum slant required to perceive the 3-D slant of a surface containing both OM and FM information.We sought to measure the perception of OM and FM patterns and determine how perceptions of these patterns, which are 2-D in nature, relate to the perception of 3-D surface slant. Thus there were two different types of tasks – tasks that required judgments about 2-D patterns and tasks that required judgments about 3-D surface slant. We examined pattern and slant percepts for two surface conditions: in the 0-deg condition, percepts were measured for surfaces around the fronto-parallel plane; in the 30-deg condition, percepts were measured for surfaces deviating in slant around an existing ±30 deg slant.In the 0-deg condition, observers were presented with stimuli varying in slant around the fronto-parallel plane patterned with OMs, FMs, or both in a plaid . StimuliIn the 30-deg condition, observers were presented with stimuli varying in slant around an existing ±30 deg slant also patterned with OMs, FMs, or a plaid. In each trial, two stimuli were presented simultaneously to the left and right of fixation; one stimulus was a base slant set at a ±30 deg slant, while the other was a test slant that deviated from the base slant and was always more steeply slanted than the base slant. Observers were required to indicate the stimulus that contained a greater change in OM or FM pattern, or that appeared more slanted in depth. There were 210 trials in each session for the . The test slant presented as well as the side on which the base and test slant were presented onscreen were randomized from trial to trial.The motivation for using a single interval task for the 0-deg condition was to ensure that observers judged the FM gradient rather than the overall frequency or effective contrast of the stimulus. As surface slant is varied, so does the average frequency and effective contrast of the FM stimuli, and these changes are more visible for surfaces varying around the fronto-parallel plane over the range of slants required to determine thresholds. Thus, in a two-interval paradigm around fronto-parallel, it would be possible for observers to use average frequency or contrast as a cue. Although these potential confounds still exist for the 30-deg condition, the range of slants required to detect pattern changes in the FM stimuli (and therefore the range of average frequencies across these stimuli) was greatly reduced compared to the 0-deg condition, and an additional control experiment described in the Results section indicated observers were not using effective contrast as a cue in this condition.Although our instructions to observers were specifically worded in the context of judging ‘pattern’ (2-D) vs. ‘slant’ (3-D), the response choices were identical across the two surface conditions . Thus, it is difficult to ensure that observers were specifically making 2-D vs. 3-D judgments, e.g. observers might have been indicating the direction of line convergence when asked to make judgments about surface slant, and/or when asked to make judgments about the direction of line convergence, they could have been indicating the direction of perceived slant. Two things should be noted here: 1) As may be illustrated in Three main sets of test stimuli were generated by mapping developable planar surfaces with different textures, slanting them around a vertical axis of rotation and projecting them in perspective. For the OM and FM stimuli, surfaces were mapped with 2 cpd horizontal or vertical gratings at 50% contrast obtainable on the monitor . For theA preliminary study was run to determine the appropriate range of slants for each condition and surface texture to be used in a method of constant stimuli. For each of the OM, FM, and plaid patterns in the 0-deg condition, 18 test stimuli were generated by slanting the patterns around a vertical axis of rotation, with half the stimuli slanted leftward and the other half slanted rightward. Slant angles of the OM stimuli were ±1, 2, 4, 6, 8, 10, 12, 14 and 16 deg; slant angles of the FM stimuli were ±1, 8, 16, 24, 32, 40, 48, 56, and 64 deg; and slant angles of the plaid textured surfaces were ±1, 3, 6, 9, 12, 15, 18, 21 and 24 deg.For each of the OM, FM, and plaid patterns in the 30-deg condition, 14 test stimuli were generated deviating from a fixed base slant of ±30 deg. For both the OM stimuli and the plaid surfaces, the stimuli were +2, 4, 6, 8, 10, 12, and 14 deg deviating from the fixed base slant of ±30. For the FM stimuli, the slant angles were ±1, 2, 3, 4, 5, 6, 7 deg deviating from the fixed base slant of ±30.For generality, the 0- and 30-deg conditions were repeated using stimuli that were rotated about a horizontal axis (i.e. floor/ceiling slant).All sessions began with a 60 sec fixation period to a mean grey screen followed by a tone to signal the start of the trials. An audio beep coincided with the presentation of each test stimulus. Following each stimulus presentation, there was a 500 msec Gaussian noise mask. The screen then remained at the mean grey until the observer made a response. Each session lasted approximately 15–20 minutes.In the 0-deg condition, stimuli were each presented for 250 msec. For the OM and FM patterns, observers were asked to indicate the direction of convergence in OMs for the horizontal gratings, or the direction of bar width gradient decrease for the FMs in vertical gratings. For plaid stimuli, observers were asked to indicate the direction of surface slant for a horizontal-vertical plaid.In the 30-deg condition, stimuli were presented for 800 msec, with the extra time being necessary as observers were being asked to discriminate between two stimuli presented side by side. Observers were asked to indicate which of the two stimuli contained either the greater degree of OM or FM change, or the greater degree of surface slant in the plaid surface.Three of the authors and 2 naïve observers participated in both experiments. All had normal or corrected-to-normal visual acuity.For the inconsistent slanted condition, the percentage of trials reported consistent with the pattern (OM or FM) at a fixed base slant of ±30 deg was plotted vs. the range of slants specified by the other pattern . Data foThe same pattern of results was observed for the inconsistent corrugated condition. For each of the three patterns in the 0-deg condition, the percentage of correct trials (i.e. reported consistent with the simulated direction of pattern (OM/FM) change or surface slant) was plotted vs. the degree of surface slant. For the 30-deg condition, the percentage of correct trials indicating which of two stimuli presented had the greater pattern change or surface slant was plotted against the degree of surface slant from the fixed base slant. The percent correct for positive and negative slants were averaged together at each slant value, with the threshold extrapolated from a Weibull fit of the averaged data. As an example, Data averaged across the five observers are shown in In the 0-deg condition, the highest slant thresholds were consistently observed for the FM pattern tasks regardless of axis of rotation. No significant difference was observed between the amount of surface slant needed to identify the direction of pattern change for OMs and that needed to detect the surface slant of the plaid. In the 30-deg condition, thresholds for determining which of two stimuli had greater OM change was consistent with that needed to judge surface slant of the plaid. Interestingly, the thresholds for FM stimuli in the 30-deg conditions were significantly lower than that of the OMs irrespective of axis of rotation. For surfaces mapped with a plaid texture that were rotated around a vertical axis, there was no significant difference in the amount of surface slant required for observers to detect the direction of surface slant in the 0-deg condition and that needed to discriminate which of two stimuli contained a greater degree of surface slant in the 30-deg condition. However, for surfaces rotated around a horizontal axis , the amount of surface slant needed to detect slant around fronto-parallel was significantly greater than that around 30-deg. These results indicate that slant perception appears to be dictated by the detectability of OMs for slants around the fronto-parallel plane. At steeper slants, OMs and FMs appear to be similarly effective at conveying surface slant.As the effective contrast of the FM stimuli decreased with increasing slant, one concern was that observers may have been utilizing differences in effective contrast as a cue rather than perceived FM gradients. An additional experiment was conducted using the method of limits in which observers were presented with an FM pattern at 50% contrast at the maximum slant for these stimuli used in the 30-deg condition (±37 deg). These were presented together with FM stimuli of the different slants used in the 30-deg condition , in which the contrast was varied in ascending and descending series for each slant. Observers were asked to indicate which of the two stimuli was higher in contrast, and thresholds for equated contrast were estimated from 6 series of trials (3 ascending and 3 descending) for each slant. The contrast level of all the FM stimuli in the 30-deg condition which observers judged as perceptually similar to the contrast of the FM pattern at the maximum slant was not significantly different. Thus we do not think that effective contrast was a confounding factor in our FM stimuli.Our results show that when the OMs and FMs on a slanted or corrugated surface specify inconsistent directions of slant or curvature, the percept is dictated by the OMs. In addition, slant perception is dictated by the detectability of OMs for slants around the fronto-parallel plane, and both OMs and FMs are similarly effective at conveying surface slant around steeper slants. For surfaces around fronto-parallel, an increase in surface slant organically results in changes in orientation that are more readily visible than changes in frequency. The amount of surface slant needed to detect changes in slant was therefore more consistent with that needed to detect a pattern change for OMs than FMs. For steeper slants, the amount of slant needed to detect FMs was actually less than that needed to detect orientation changes. The marked drop in FM thresholds with increasing slant suggests that for slanted surfaces, a small amount of slant results in more visible changes in FMs relative to OMs. There is likely a combination of mechanisms responsible for extracting both OMs and FMs for the perception of slanted surfaces, but our results do not distinguish their specific contributions at steeper slants.Use of the plaid stimulus introduces an additional, potentially useful cue of orthogonality whereby deviations from orthogonality of the horizontal and vertical plaid in the image increase with surface slant Interestingly, for surfaces around fronto-parallel in the 0-deg condition, slant thresholds for the plaid surfaces were higher than pattern thresholds for the OMs across all observers, although this trend was not significant when averaged across observers. Previous physiological work has shown that striate neurons in cat and primates have a decreased response if a stimulus at the preferred orientation is superimposed by an orthogonal stimulus, a process known as cross-orientation suppression Previously we have shown that in isolation, FMs resulting from developable surface texture mappings, such as the planar and corrugated surfaces used in this study, can lead to misperceptions of surface shape, specifically concave surfaces tend to appear convex Our results suggest that the perception of 3-D slant and shape from texture must involve the neural extraction of OMs  and FMs (at steep slants). Results from adaptation studies suggest that mechanisms that extract OMs are invariant to how the OMs are defined over a range of spatial frequencies and thus inevitably lie in extra-striate visual areas


Previous studies have found that common genetic variants on chromosome 8q24 are associated with the risk of developing colorectal neoplasia. We conducted a hospital-based case-control study, including 435 cases and 788 unrelated controls to investigate the associations between common variants on 8q24 and the risk of colorectal cancer in a Chinese population. We also evaluated the association of rs6983267 with colorectal neoplasia in the published literature via a meta-analysis study. We found that rs6983267 was significantly associated with the risk of colorectal cancer in the Chinese population, with an adjusted odds-ratio (OR) for the GT heterozygotes and GG homozygotes of 1.30  and 1.66 , respectively, compared to the TT homozygotes, with a P-trend value of 0.003. No association was found for the other three loci . In the meta-analysis of the published genetic association studies, the rs6983267 variant was found to be associated with an increased risk of colorectal neoplasia. The heterozygous GT carriers showed a 20% increased risk of colorectal neoplasia  with a summary OR for homozygous GG carriers of 1.39  compared to the TT genotype carriers. We found no significant differences between the association of rs6983267 and colorectal cancer and colorectal adenomas. In summary, our study confirms that the variant rs6983267 is a risk factor for colorectal neoplasia in various populations, including the Chinese population. APC, MLH1, MSH2, TGFBR2 and SMAD4) have been identified Colorectal cancer (CRC) is the third most common cancer and the fourth most frequent cause of cancer death worldwide et al genotyped six variants in 1,124 individuals with invasive CRC and 4,573 controls that had been previously identified as having an underlying risk for prostate cancer due to alterations on 8q24 et al conducted a genome-wide association study of 550,000 tag SNPs in 930 familial colorectal cancer patients and 960 controls and found that rs6983267 had the strongest association with CRC risk e.g. rs6983267, rs10808556 and rs10505477) were associated with CRC risk. The variations in the other regions were found to be associated with prostate, breast and ovarian cancers Among these loci, variants on 8q24 have shown strong evidence of an association with the risk of CRC in different populations. Haiman The constantly improving standard of living in China has brought about extensive changes to the lifestyle and diet of the average Chinese citizen over the past three decades, and has led to a corresponding increase in the incidence and mortality of CRC in China Most of the participants in the study have been described previously A 5-mL peripheral venous blood sample was obtained from each subject after written informed consent was obtained. Each participant was personally interviewed by trained interviewers to complete a Semi-quantitative Food Frequency Questionnaire (SQFFQ), which collected demographic information and information about dietary, and smoking habits , alcohol use  and other lifestyle factors The study was approved by all of the ethics committees of the participating hospitals . All of the samples were collected with a written informed consent provided by the participants, and all protocols were approved by the human research ethics committees of the participating hospitals.2 = 0.95) according to the Hapmap database, and they were also found to be associated with an increased risk of prostate, breast and ovarian cancers in many population studies Genome-wide association studies and gene-based candidate studies have confirmed that chromosome 8q24 is a susceptibility region for CRC. It has also been shown that specific loci on 8q24 are associated with specific cancers Genomic DNA was extracted from 2.5-mL of whole blood with a Promega DNA Purification Wizard kit according to the manufacturer's instructions. The genotyping methods have been reported previously In order to explore the association between rs6983267 and the risk of colorectal neoplasia, a meta-analysis was conducted. We performed a comprehensive and systematic bibliographic search updated to February of 2011 based on the MEDLINE and PubMed databases. We used the terms “8q24” and “rs6983267” in combination with “colorectal neoplasia”, “colorectal cancer” or “colorectal adenoma” to search the database to identify the studies regarding the association between the rs6983267 polymorphism and the risk of colorectal cancer. References were also checked to identify any missing studies. The details of the studies were thoroughly examined in order to exclude potentially overlapping data. If the same participants were used in different papers, only the largest and most complete study was included here. The studies included were those that provided data about rs6983267 and the risk of colorectal cancer or colorectal adenomas and could be case-control, cohort or cross-sectional studies that were reported in English. The studies included should also provide sufficient data about the frequency of the genotypes. Individual authors were also contacted for further data when the criteria were not met. The flow chart that tracks the selection process for the studies and the reasons for exclusion is presented in etc), they were considered individually. Pooled allelic effects were estimated both under a fixed effects model and a random effects model, and the pooled OR with its 95% CIs was used as the summary measurement.For each study, the following information was recorded: first author, publication year, study design, study location, study population/ethnicity, total number and sources of cases and controls, sub-group and disease categories, total number of cases and controls, and the allele frequency in the study. When there were sub-group studies described in the paper  and Student's t-test (for continuous variables) were used to evaluate the differences in demographic characteristics and selected variables. Hardy-Weinberg Equilibrium (HWE) was assessed by the χ2 test (1 degree of freedom (d.f)). The common homozygote was used as the reference to calculate the genotype-specific odds ratio (OR) and its 95% confidence intervals (CI) with or without adjustment for age, sex, smoking status and alcohol use under the unconditional Logistic regression statistic model. The statistical powers of the study were calculated using the Power software under the assuming of two sided test with alpha level is 0.05 with the log-additive genetic model The prevalence of each of the alleles was measured in cases and controls. The χ2 statistic. Publication bias was graphically represented by funnel plotting and was assessed both by Egger's linear regression http://www.r-project.org/).For meta-analysis, the pooled OR and its 95% CI were calculated using the standard inverse variance weighting method for the fixed effects model and the DerSimonian-Laird method for the random-effects model. Heterogeneity between studies was assessed using the Cochrane Q-test in combination with the IThe characteristics of cases and controls are given in Statistically significant associations were obtained only for the previously reported SNP rs6983267 . Compare2 = 0%), and no evidence of a significant publication bias was found . For the GG homozygotes, we noted that there was a significant increase in colorectal neoplasia, with the SOR of 1.40 (95% CI = 1.33–1.46) under the fixed effects model and 1.39 (95% CI  = 1.32–1.48) under the random effects model compared to the TT carriers (2 = 33.2%). However, there still no significant publication bias . Sensitivity analyses were also performed by removing the individual studies sequentially, and we found that none individual studies dramatically affected the overall polled ORs.For the meta-analysis regarding the association between colorectal cancer risk and the variant rs6983267, we identified twenty-three reports regarding the association between rs6983267 and colorectal neoplasia. After detailed evaluation, sixteen studies including a total of 36,761 cases and 38,901 controls were used for the meta-analysis  [5], [11carriers . HoweverTo determine whether the association between variant rs6983267 and colorectal neoplasia could differ for colorectal adenomas and colorectal cancer, we examined the association between the variant and colorectal cancer and the variant and colorectal adenomas. Among the fourteen identified reports, two examined the association between the variant and colorectal adenomas, with a total of 4,197 cases and 5,109 controls et al that there are five different haplotype blocks within this region. The rs6983267 locus and its highly correlated locus, rs10505477 (r2 = 0.95 in CHB population for Hapmap database), which are located between a 128.47 and 128.54 Mb region, have been found to be associated with prostate, colorectal and ovarian cancers Variants on 8q24 (position 128.14 Mb to 128.62 Mb) were found to be associated with various types of cancer. It was reported by Ghoussaini In the meta-analysis of rs6983267 and colorectal neoplasia, sixteen eligible papers that examined the risk of colorectal carcinoma, including two that also reported the association between the locus and colorectal adenomas risk were examined. We found that the GT heterozygotes had a 20%  and the GG homozygotes showed a 39%  increased risk of colorectal neoplasia. The SORs determined for colorectal neoplasia were similar in magnitude to those observed for colorectal cancer and colorectal adenomas . These fAlthough the variant rs6983267 had been found to be associated with an increased susceptibility to colorectal neoplasia, whether the rs6983267 SNP is the causal locus is still uncertain. Several recently published studies have shown that rs6983267 is located within a transcriptional enhancer region and affects a binding site for TCF4 , a transcription factor which is activated in most CRCs. TCF4 interacts with β-catenin to activate the transcription of Wnt target genes, which are part of a key pathway involved in CRC initiation There are several potential limitations to the current study. First, for a hospital-based case-control study, subjects were recruited based on their outcome (with CRC or without CRC) rather than their exposure. The potential CRC risks for the controls are immeasurable, and may have caused a cause bias in studying the etiology of the disease. It could also have led to spurious results in the gene/environmental interaction studies. Second, the sample size was relatively small. We cannot exclude the possibility there is an association between rs16901979, rs1447295 and rs7837688 and colorectal cancer in the Chinese population due to the inadequate statistical power of our study , although many other studies have also reported no significant association for these loci with CRC in other populations In summary, we observed a statistically significant association between SNP rs6983267 on 8q24 and the risk of colorectal cancer in a Chinese population. Moreover, a meta-analysis of previous studies conducted in different populations confirmed the association between this locus and colorectal neoplasia risk in different populations and ethnic groups. These results provide a more complete picture of the role of this polymorphism in the risk of colorectal neoplasia, and may give genetic insight into possible strategies for prevention of colorectal neoplasia.


This study attempted to repeat experiments performed 20 years ago that detected exceptionally high strength electromagnetic fields (100 nT) from the hands of several energy healers. The equipment utilized in this replication attempt was far more sensitive, and the magnetic shielding used to block confounding fields from electrical lines far more effective, than in the original studies.Using a Magnes 2500 WH SQUID at Scripps Research Institute, San Diego, the electromagnetic field from the hands and heart of each of 3 Reiki masters was measured for 5 minutes when they were: (i) not practicing Reiki; (ii) sending Reiki to a distant person; and (iii) sending Reiki to a person in the room. Similar measurements were made on 4 Reiki-naïve volunteers before and after they received Reiki attunements enabling them to run Reiki energy through themselves.For all subjects, under all conditions, sensors closest to the heart and the hands produced spikes of 2pT corresponding to the heartbeat. Recordings from two Masters and one volunteer showed a low intensity sine wave oscillation of 0.25-0.3Hz (intensity 0.1-0.5pT) whether or not they were practicing Reiki. This oscillation probably reflected respiratory sinus arrhythmia, as judged by comparison with recent previous studies. These signals were not detected in the original studies. No segments of electromagnetic field intensity greater than 3pT were seen in any recording in the current study.These results suggest that practicing Reiki does not routinely produce high intensity electromagnetic fields from the palms. The previous findings could have been from a few exceptional healers. Alternatively, it is possible that energy healing is stimulated by attunement to an external environmental radiation, such as the Schumann resonance, which was blocked in the present study by the strong magnetic shielding in the room containing the SQUID.