While much cross-sectional data is available, there have been few longitudinal investigations of patterns of electronic media use in children. Further, the possibility of a bi-directional relationship between electronic media use and body mass index in children has not been considered. This study aimed to describe longitudinal patterns of television viewing and electronic game/computer use, and investigate relationships with body mass index (BMI).This prospective cohort study was conducted in elementary schools in Victoria, Australia. 1278 children aged 5–10 years at baseline and 8–13 years at follow-up had their BMI calculated, from measured height and weight, and transformed to z-scores based on US 2000 growth data. Weight status  was based on international BMI cut-off points. Weekly television viewing and electronic game/computer use were reported by parents, these were summed to generate total weekly screen time. Children were classified as meeting electronic media use guidelines if their total screen time was ≤14 hrs/wk.Electronic media use increased over the course of the study; 40% met guidelines at baseline but only 18% three years later. Television viewing and electronic game/computer use tracked moderately and total screen time was positively associated with adiposity cross-sectionally. While weaker relationships with adiposity were observed longitudinally, baseline z-BMI and weight status were positively associated with follow-up screen time and baseline screen time was positively associated with z-BMI and weight status at follow-up. Children who did not meet guidelines at baseline had significantly higher z-BMI and were more likely to be classified as overweight/obese at follow-up.Electronic media use in Australian elementary school children is high, increases with age and tracks over time. There appears to be a bi-directional association suggesting that interventions targeting reductions in either screen time or adiposity may have a positive effect on both screen time and adiposity. High levels of electronic media use during childhood have detrimental effects on child and adulChildren's television viewing patterns have been well described in cross-sectional studies. Girls and boys appear to watch similar amounts of television , and oldPatterns of other electronic media use in children have received less attention, perhaps because children spend less time on these activities ,14,15. BTo date, most investigations of electronic media use patterns in elementary school children have come from cross-sectional studies. Longitudinal descriptions of electronic media use and changes in usage over time in individual children have been scarce. The available data suggests that television viewing tracks quite strongly within the middle childhood period (correlations range from r = 0.46 to r = 0.73) -20, and Longitudinal associations between electronic media use and adiposity in childhood have been investigated in different ways in different studies, making comparisons difficult. Unlike the consistent weak positive associations reported in cross-sectional studies for all forms of electronic media use , findingThis paper aims to (1) describe longitudinal patterns of television viewing and electronic game/computer use in a large population-ascertained sample of Australian children as they move from the early to late elementary school years and (2) investigate relationships between television and electronic game/computer use and BMI trajectories.This cohort was derived from the Health of Young Victorians Study (HOYVS), conducted between September and December 1997. At baseline, HOYVS employed a two-stage stratified random sampling design to draw a sample of children representative of the state of Victoria, Australia. Twenty-four elementary schools were selected, with a probability proportional to school size , to be representative of the school sectors  and the geographic distribution of the state. At each school, one class at each year level (preparatory to grade six) was randomly selected for the study except where total school enrolment did not exceed 240 students, in which case the entire school was included. Children in grades preparatory to three (aged 5–10 years) in the baseline study were included in the follow-up study, conducted between October 2000 and June 2001 when children were aged 8–13 years. The average time between assessments was 3.2 years (SD = 0.2 years).Both studies received approval from the Royal Children's Hospital Ethics in Human Research Committee and the relevant education authorities, and parents provided written informed consent in both waves.2) and transformed into standardised (z) scores based on gender and exact age using the US Centers for Disease Control and Prevention 2000 growth chart data [Children had their height and weight measured by trained researchers at baseline and follow-up. Investigation of anthropometric reliability found no evidence of systematic bias for intra- or inter-rater comparisons. Body mass index (BMI) was calculated (weight (kg)/height (m)art data . Childreart data .At baseline and follow-up, parents completed a self-administered questionnaire reporting on their child's television viewing and electronic game/computer use for an average school day and average non-school day. At baseline, six-point ordinal response scales were used, with duration taken to be the midpoint of each category  except the '7 or more hours' category (coded as 7.5 h). At follow-up, open ended continuous response scales ('___ hours') were used. Weekly scores were calculated by summing hours reported for an average school day (multiplied by five) and average non-school day (multiplied by two). Weekly television viewing time and weekly electronic game/computer use were summed to create a total screen time variable. Change in television time, electronic game/computer use time and total screen time scores were generated by subtracting weekly scores at baseline from weekly scores at follow-up. In addition, children were classified as meeting electronic media use guidelines if their total screen time did not exceed 14 hours per week. Parents provided standard demographic information.Baseline characteristics of responders and non-responders were compared using independent samples t-tests for continuous variables and chi-square statistics for categorical variables. Differences in outcome variables by gender were assessed using independent samples t-tests and differences by age were assessed using linear regression analyses. Pearson's correlations assessed the relationship between outcome variables at baseline and follow-up. Differences in the proportions of children meeting guidelines for electronic media use by gender were assessed using the chi-square statistic, and by age using logistic regression analyses.Cross-sectional relationships between screen time and BMI category or z-BMI were assessed using linear regression, adjusted for age. Cross-sectional relationships between proportions of children meeting electronic media use guidelines and BMI category or z-BMI were assessed using logistic regression, adjusted for age.Only children with complete BMI and screen time data at both time points were included in analyses of temporal relationships. For analyses of temporal relationships BMI categories were collapsed to non-overweight and overweight/obese. Pearson's correlation coefficients were used to assess the relationship between z-BMI at baseline and follow-up and between total screen time at baseline and follow-up. Independent samples t-test statistics compared differences in mean z-BMI between children who did and did not meet electronic media use guidelines. Pearson's chi-square statistics assessed differences in the proportions of children classified as overweight/obese who did and did not meet electronic media use guidelines. Longitudinal relationships between z-BMI or BMI category and screen time were assessed using linear regression, logistic regression and analysis of variance analyses, as appropriate, each adjusting for child age. Longitudinal relationships between z-BMI or BMI category and electronic media use guidelines were assessed using Pearson's chi-square statistic or independent samples t-test analyses as appropriate.2; p = 0.02), and more non-responders were classified as overweight or obese  at baseline. In addition, non-responders were more likely to live in urban areas  and to have parents born in a non-English speaking country .Of the 1943 children who participated in the baseline study, 204 were unable to be located and 170 declined to participate at follow-up. Thus 1569 children provided data at follow-up, a retention rate of 81% of the cohort. Baseline characteristics of the 1569 responders and 374 children lost to follow-up (non-responders) were compared. Responders and non-responders were similar in terms of gender, age, maternal and paternal education levels, weekly television viewing, weekly electronic game/computer use and total screen time. However, the mean BMI of non-responders was higher than that of responders , and usage rose with age (p < 0.001 at both time points). Baseline and follow-up electronic game/computer use were moderately correlated (r = 0.34). Usage rose substantially over the three years (by 4.6 hrs/wk (SD = 6.7)), with greater increases for boys than girls (p < 0.001) but no differences by age.Table Similar changes were observed for total screen time, which increased by an average of 5.7 hrs/wk (SD = 10.4) over three years, more so for boys than girls (p = 0.006). Total screen time at baseline and follow-up were moderately correlated (r = 0.46). At baseline, 40% of children met the Australian guideline of two hours or less of electronic media use per day (≤14 hrs/wk) but only 18% met this guideline at follow-up. More girls than boys met the guideline (p < 0.001 at both time points) and the odds of meeting the guideline decreased with age .total screen time increased across BMI categories at both baseline and follow-up, and boys at follow-up only.Table Examining relationships between weekly screen time and z-BMI yielded stronger associations. At both baseline and follow-up, television viewing time increased with increasing z-BMI for boys  and girls . Similarly, a positive association was observed between electronic game/computer use and z-BMI for boys  and girls , although not all associations reached significance. Total screen time was strongly associated with z-BMI at both time points for boys  and girls . The proportion of children who met electronic media use guidelines decreased with increasing z-BMI at baseline and follow-up for both boys  and girls , although the association for boys at follow-up did not reach significance.BMI and screen time data were available at both time points for 1151 children who form the sample for this section of the paper. Baseline and follow-up z-BMI were highly correlated (r = 0.84). Total screen time at baseline and follow-up were moderately correlated (r = 0.45). The vast majority (85%) of children remained in the same BMI category  between baseline and follow-up and most (67%) remained in the same electronic media use guideline category (meet or do not meet guidelines). However, more than one quarter (28%) of all children were classified as meeting electronic media use guidelines at baseline but not follow-up. These findings were similar for boys and girls. Mean z-BMI and the proportion of children classified as overweight/obese was higher for children who did not meet electronic media use guidelines than for those who did Table .Baseline screen time predicted follow-up z-BMI  and BMI category  after adjustment for child age. This suggests that for every extra hour per week a child is engaged in screen-based activities, their odds of being overweight three years later increases by 3%. Children spending more time in screen-based activities and those failing to meet electronic media use guidelines at baseline tended to have higher z-BMIs and were more likely to be classified as overweight/obese at follow-up . Children classified as overweight/obese at baseline had two hrs/wk more screen time at follow-up than non-overweight children ; the difference was less than two hrs/wk for girls  and three hrs/wk for boys . Baseline z-BMI did not predict change in screen time and there was no difference in screen time change between children classified as non-overweight and overweight/obese at baseline. There was a tendency for girls whose screen time increased to also show increases in z-BMI , but this trend was not seen for boys .Patterns of screen time (both television and electronic games/computer use) appear well-established by the early elementary school years and track quite strongly through middle childhood. Of concern, children's screen time increased with age and children became less likely to meet recommended guidelines as they got older. By late elementary school, less than 20% of this sample met the recommendation of spending less than two hours per day using electronic media. Previous research has established that adolescents spend more time in screen based activities than their younger counterparts . Thus itCross-sectional relationships between screen time and BMI were stronger than the relationships observed longitudinally. This trend was also observed in a meta-analysis  reportinDespite equivocal findings in the literature , screen The relationship between screen time and BMI was bi-directional: baseline screen time predicted higher follow-up BMI and baseline BMI predicted higher follow-up screen time. Previous studies have tended to focus on whether screen time predicts adiposity. The findings of this study are important as they suggest that interventions targeting either reduction in screen time or adiposity may have a positive effect on both screen time and overweight. Further research is required to confirm this bi-directional relationship.This study is limited by its observational nature to be able to suggest directions of associations but not infer causality. The subjective measures of screen time employed may have resulted in underreporting of these behaviours by parents and such reporting bias may have differed by the weight status of the child. Yet such reporting bias is likely to have resulted in under-rather than over-estimation of the screen time behaviours and the association between screen time and adiposity. While the response scales for the screen time measures differed between baseline  and follow-up (continuous), both were converted to continuous scales to allow for comparative analysis. A higher proportion of overweight than non-overweight children were lost to follow-up. While this resulted in a smaller proportion of overweight children in the sample, it is unlikely to have influenced the relationships observed between BMI and screen time as the baseline screen time behaviours of responders and non-responders did not differ. The strengths of this study include its large sample size and population-based sampling design, and its high response and retention rates. The vast majority of previous studies of electronic media use have focused exclusively on television viewing. This study also included a measure of electronic game/computer use which was also found to be a prevalent behaviour.Television viewing and electronic game/computer use are prevalent pastimes for elementary school children which become more prevalent as the children get older. These pastimes appear to be associated with overweight and may represent a useful means of assessing the risk of overweight in clinical practice. The apparent bi-directional nature of the association between electronic media use and overweight suggests that interventions designed to reduce the prevalence of one may have a positive effect on both.The author(s) declare that they have no competing interests.KH participated in the design and coordination of the study, performed the statistical analyses and drafted the manuscript. MW participated in the design and coordination of the study and contributed to the development of the manuscript. MG contributed to the development of the manuscript. EW participated in the design and coordination of the study and contributed to the development of the manuscript. All authors read and approved the final manuscript.


Activated rodent macrophages are capable of expressing an inducible form of this enzyme (iNOS) in response to appropriate stimuli, i.e., lipopolysaccharide (LPS) and interferon-γ (IFNγ). Other cytokines can modulate the induction of NO biosynthesis in macrophages. NO is a major effector molecule of the anti-microbial and cytotoxic activity of rodent macrophages against certain micro-organisms and tumour cells, respectively. The NO synthesizing pathway has been demonstrated in human monocytes and other cells, but its role in host defence seems to be accessory. A delicate functional balance between microbial stimuli, host-derived cytokines and hormones in the microenvironment regulates iNOS expression. This review will focus mainly on the known and proposed mechanisms of the regulation of iNOS induction, and on agents that can modulate NO release once the active enzyme has been expressed in the macrophage.L-Arginine is converted to the highly reactive and unstable nitric oxide (NO) and L-citrulline by an enzyme named nitric oxide synthase (NOS). NO decomposes into other nitrogen oxides such as nitrite         (NO


Tumorigenesis is a multi-step process in which normal cells transform into malignant tumors following the accumulation of genetic mutations that enable them to evade the growth control checkpoints that would normally suppress their growth or result in apoptosis. It is therefore important to identify those combinations of mutations that collaborate in cancer development and progression. DNA copy number alterations (CNAs) are one of the ways in which cancer genes are deregulated in tumor cells. We hypothesized that synergistic interactions between cancer genes might be identified by looking for regions of co-occurring gain and/or loss. To this end we developed a scoring framework to separate truly co-occurring aberrations from passenger mutations and dominant single signals present in the data. The resulting regions of high co-occurrence can be investigated for between-region functional interactions. Analysis of high-resolution DNA copy number data from a panel of 95 hematological tumor cell lines correctly identified co-occurring recombinations at the T-cell receptor and immunoglobulin loci in T- and B-cell malignancies, respectively, showing that we can recover truly co-occurring genomic alterations. In addition, our analysis revealed networks of co-occurring genomic losses and gains that are enriched for cancer genes. These networks are also highly enriched for functional relationships between genes. We further examine sub-networks of these networks, core networks, which contain many known cancer genes. The core network for co-occurring DNA losses we find seems to be independent of the canonical cancer genes within the network. Our findings suggest that large-scale, low-intensity copy number alterations may be an important feature of cancer development or maintenance by affecting gene dosage of a large interconnected network of functionally related genes. It is generally accepted that a normal cell has to acquire multiple mutations in order to become a malignant tumor cell. Considerable effort has been invested in finding single genes involved in tumor initiation and progression, but relatively little is known about the constellations of cancer genes that effectively collaborate in oncogenesis. In this study we focus on the identification of co-occurring DNA copy number alterations  in a series of tumor samples. We describe an analysis method to identify DNA copy number mutations that specifically occur together by examining every possible pair of positions on the genome. We analyze a dataset of hematopoietic tumor cell lines, in which we define a network of specific DNA copy number mutations. The regions in this network contain several well-studied cancer related genes. Upon further investigation we find that the regions of DNA copy number alteration also contain large networks of functionally related genes that have not previously been linked to cancer formation. This might illuminate a novel role for these recurrent DNA copy number mutations in hematopoietic malignancies. PIK3CA and KRAS genes was observed Tumor development is generally thought to be a process in which healthy cells transform into malignant tumor cells through the step-wise acquisition of oncogenic alterations Besides single basepair mutations or retroviral integrations, the activity of genes can also be perturbed by DNA copy number alterations that arise as a result of genomic instability, which is frequently observed in tumor cells n) DNA is performed on oligonucleotide- or Bacterial Artificial Chromosome (BAC) based microarray platforms. For each probe on the microarray, the ratio of signal intensities of tumor versus normal DNA is a measure of the relative DNA copy number of the corresponding genomic region in the tumor sample. Platforms designed to identify single nucleotide polymorphisms (SNPs) can also infer CNAs by comparing the raw probe intensity values measured in a tumor sample with a reference sample.DNA copy number alterations (CNAs) may be measured on microarray platforms In order to extract those DNA copy number aberrations that preferentially occur together, we developed an analysis framework. The basic premise of our analysis is to define a pair-wise score for any given pair of genomic locations present in the dataset. This scoring index will only be high if both genomic locations are recurrently aberrated in multiple independent samples within the tumor panel, and if they co-vary similarly over the different samples . Using aThe raw data consist of non-discrete measurements of the average DNA copy number of the population of cells present in the measured sample. The signal consists of a measurement of a heterogeneous population of tumor cells, which may contain many populations potentially carrying different mutations and copy number alterations, as well as normal (diploid) cells. To reduce heterogeneity as much as possible we choose to analyze a collection of hematopoietic tumor cell lines, which on a per-sample basis can be considered clonal. There were several other reasons for analyzing this particular dataset. First, it is a high resolution dataset of well-characterized, clinically relevant samples. Although these samples are cell lines, they are widely used as a model system for the diseases from which they have been derived. Second, this collection of samples includes cell lines derived from T- and B-cell leukemias carrying rearranged T-cell receptor and immunoglobulin loci, respectively. We therefore should be able to separate these two distinct lymphoid malignancies based on co-occurring DNA copy number losses at the T-cell receptor and immunoglobulin loci. During T- and B-cell development, these loci undergo DNA recombination and gene deletion in a process known as V(D)J-recombination. The human genome contains three specific T-cell receptor loci  on two different chromosomes that determine their variability. B-cells have three different loci  on three different chromosomes that undergo recombination to generate a diverse repertoire of immunoglobulins. Since T- and B-cells only undergo recombination of their respective loci after lineage commitment, it is unlikely that T-cell receptor loci are recombined in B-cells and vice-versa. If our approach is successful at finding co-occurring losses, it should identify the co-occurring rearrangements at the T-cell receptor alpha/delta and beta/gamma loci in T-cell leukemias. Similarly, we should be able to pick up co-occurring losses at the IgG kappa, lambda and heavy chain loci in B-cell malignancies.A classic example of finding associations in a large (binary) dataset is association rule mining. Identification of cooperating events in continuous data requires a different approach than binary association rule mining. First we developed a method to score for co-occurrence between two continuous measurements . We thenDNA copy number measurements at two different genomic loci can be visualized in a 2D space, with each axis representing measurements at a certain genomic locus. A point in this space represents a sample in which both loci were measured. We use the covariance of the two measurements to score for co-occurring loci. This score only rewards a high value to a truly co-occurring and co-varying pairs of measurements .For thisσ parameter of the Gaussian function used to convolve the score matrix  we expected to retrieve these co-occurring losses in the small (2 Mb) scale analyses. Since we disregarded co-occurrences on the same chromosome we expected to find five co-occurring losses. Indeed, four of the five expected co-occurring losses are present in the top 50 peaks of the Scale 2 analyses . Figure While the recovery of the V(D)J-related recombination loci as co-occurring losses serves as a positive control for our analysis approach, we are mainly interested in identifying cooperating genes or regions that might play a role in cancer. To see whether the locations we recover are linked to this disease, we analyzed whether the co-occurring genomic loci are enriched for genes known to play a role in cancer. As a reference gene set we used the Cancer Gene Census list While finding enrichment for cancer genes is an encouraging result, this does not explain the possible cooperation between two loci. We expect that the co-occurring loss of two regions points to a functional relationship between the constituents of the genomic loci. A co-occurrence between two genomic regions can point to many different kinds of interactions between the genes residing in both regions, e.g. biochemical interactions of the protein products or functional collaboration of two cancer genes in tumorigenesis. We therefore decided to employ interaction data to shed further light on the genes present in the co-occurring regions. We translated the co-occurring pairs of genomic loci to pairs of gene sets, and we investigated the functional relationships of their protein products using the STRING database In order to keep control of the complexity, we considered in our co-occurrence analysis only radially symmetric kernels, i.e. Gaussian kernels with diagonal, equal variance covariance matrices. This implies that asymmetric co-occurring regions – where a small locus co-occurs with a large locus – will not be optimally detected. Since an asymmetric co-occurring region typically consists of a series of symmetric co-occurring regions detected on a smaller scale (just like a rectangle can be constructed from a collection of squares), we set out to construct larger co-occurring regions from the results of the smaller scales using a hierarchical clustering approach. For details see Supplemental 8). This resulted in a 1000×1000 distance matrix. On this distance matrix we performed single linkage hierarchical clustering. The resulting dendrogram was cut at 1 * 107 bp (5 kernel widths). The resulting clusters are unique genomic loci and were represented as nodes in a graph. Clusters were then linked if a co-occurrence was found between individual loci of different clusters. These links are represented as edges in a graph. The result of the clustering analysis is shown in Briefly, we collected the loci involved in the top 500 co-occurrences of the Scale 2 analysis. This resulted in 1000 genomic loci. For each pair of loci, we calculated the genomic distance in base pairs. The distance between two loci on different chromosome arms was set to a default high value  , the MAPK pathway suppressor GPS2, the nuclear co-activator (and known tumor-suppressor) p300 and a gene of unknown function, CBFA2T3. All interactions found are based on physical binding and co-occurrence in Pubmed abstracts.We investigated the remaining 113 interactors for any interesting interactions that might be a target of this collection of co-occurring losses. Within the total network of interactors we found a sub-network centered on the nuclear co-repressor  (TRAC1) . This inTrp53 or p19-ARF deficient mice Cbfa2t3 is shown in Cbfa2t3 was not flagged as a common integration site, several viral integrations near this gene were found. Remarkably, two individual tumors harbored a bi-allelic integration near the transcription start site of Cbfa2t3, suggesting functional inactivation of this candidate tumor suppressor gene. Indeed, bi-allelic integration is thought to be a hallmark of tumor suppressor genes in IM screens To see whether we could find more information regarding the putative tumor suppressor function of the different interactors, we tested if we could corroborate our findings with data from a large retroviral insertional mutagenesis (IM) screen where hematopoietic tumors were induced through Murine Leukemia virus (MuLV) infection of wild-type mice or Given that we find this sub-network of interactors in a co-occurring network of DNA copy number losses and the recovery of inactivating insertions in a retroviral IM screen, we conclude that this network might be a putative tumor suppressor network.In this study we present a genome-wide analysis for finding collaborating DNA copy number changes on different chromosomes. Using our 2D kernel convolution framework we can score and find co-occurring DNA copy number changes in a high quality, high resolution aCGH dataset. Using a dataset of hematological cell lines we are able to recover DNA copy number alterations specific to the cell lineage of the samples. Furthermore we uncover cancer-related networks of co-occurring DNA copy number changes.Several studies have investigated concerted copy number changes in aCGH data. In studies on lung cancer n results, residing in the extremes of the results distribution, thus minimizing the chance of including false positives. The top n lists allowed us to generate workable results which we have validated extensively with other sources of evidence.The output of our analysis does not include a measure of significance. Constructing a background distribution based on permutations of the DNA copy number data would mean re-running our analysis thousands of times, a task which remains computationally infeasible at this stage. Furthermore, the multiple-testing problem would have to be properly addressed, given that the number of tests is the square of the number of grid points in the 2D space. Due to the complexity of the analysis procedure (minimum operation and kernel smoothing) the definition of an analytical null distribution has remained elusive. Therefore, we have chosen to work with top While we were able to use a distributed computing solution for our analysis, we were fortunate to have the required computational architecture at our disposal. Since the problem basically consists of repeating the same action many times it could be well-suited to software optimization or a hardware based solution where the most time-consuming actions are handled by a dedicated processing unit.When looking for areas in the 2D pair-wise space highly enriched for co-occurrence scores we convolve this space with a 2D-Gaussian kernel. The sigma parameter of this function is a representation of the size of the aberrations we expect to recover. Currently we make the implicit assumption that the co-occurring aberrations have the same size by using a symmetric kernel for the convolution. This could be relieved by allowing for an asymmetrical (ellipsoid) Gaussian kernel for all combinations of scales used. Clearly, this comes at the cost of increased computational complexity. Here we resolve this issue by concatenation of the results obtained in a small scale. In this way we can recover co-occurring losses of different sizes that give a better enrichment for functional interactions when combined with the single peaks obtained in a higher scale analysis.In our analysis of a set of cell lines derived from hematological malignancies we found enrichment of cancer related genes and functional interactions in co-occurring DNA copy number changes. Our results suggest that tumorigenesis requires elimination of multiple gatekeeper genes and gain of multiple oncogenes as demonstrated by the presence of many functional interactions between the loci in the gain-gain and loss-loss core networks.Haploinsufficiency is a well known characteristic of several tumor suppressor genes, where simple reduction of gene dosage by loss of gene copies at the DNA level can already promote oncogenic transformation TP53 gene, often thought to be the single target of this deletion TP53 is no doubt an important target of the DNA copy number loss, our analysis indicates that the concomitant loss of other genes near TP53, as well as co-occurring losses on the other genomic loci may together account for the full tumorigenic effect.We show that the 17p loss and its co-lost regions are highly enriched for functional relationships, which are not fully explained by the presence of the Loss of the loci on 17p, 9p, 9q, 13q, 16q and 22q has been reported previously for several types of hematological malignancies represented in our dataset TP53, RB1 and CDKN2A (INK4a/ARF). We found one sub-network of genes around NCOR1 which might be an example of other tumor suppressor genes that are affected by the concerted loss of these genomic loci.Not all of the gene-gene interactions defined by the 17p network involve the well-known canonical cancer genes NCOR1, is a well-known transcriptional co-repressor that associates in a ligand-independent manner with nuclear receptors SMRT, for recruitment of HDAC proteins to the DNA to induce transcriptional silencing. Its role in cancer is not well-established. NCOR1 null mice die in early embryogenesis NCOR1 is known to increase proliferation in hepatocytes NCOR1 decreases AKT phosphorylation, thus countering its pro-survival signal NCOR1 - might increase proliferation and promote survival.The hub of this interaction network, NCOR1 and its partner genes  have been based on co-occurrence in PubMed abstract and true physical binding CBFA2T3 is a close relative of ETO, which is a target of the recurrent AML1-ETO translocation that occurs in acute myeloid leukemia. It has been shown that the fusion gene AML1-ETO actually interferes with the CBFA2T3-NCOR1 interaction, and that its oncogenic effect derives from that inhibition Cbfa2t3 is recurrently targeted by bi-allelic retroviral integrations, which are predicted to cause functional inactivation of Cbfa2t3PPARA is a member of the Peroxisome proliferator-activated receptors, and has been implicated in hepatocellular carcinoma development in rodents PPARG, exhibit a tumor suppressor-like phenotype, it is possible that PPARA can act as a tumor suppressor in hematological malignancies. GPS2 is a known suppressor of JNK signaling MAP kinase signaling pathway. Deregulation of this pathway is a well-known phenomenon in cancer NCOR1 and the known tumor suppressor p300, our data suggest a selective advantage for loss of multiple constituents that interact with NCOR1 since they all may have tumor suppressor-like activities.All interactions between BRCA1/2-related breast cancer.Many studies focus on a single hematological malignancy in which a single combination of aberrations might be important NCOR1 that may be a novel network of tumor suppressor genes that are affected by the observed co-occurring losses. The observation that DNA copy number changes may affect gene dosage of larger numbers of cancer-relevant genes deviates from the classical view where mutations in a few (5–7) cancer genes lead to tumor development. Our data support the notion of cancer-related networks or pathways, where multiple collaborating genes are deregulated simultaneously to induce oncogenesis. Such a network view of oncogenesis is an important step towards developing effective drug targets because it increases the number of potential targets. However, this view also implies that multiple molecules need to be targeted simultaneously in order to achieve optimal therapy response and to reduce the risk of therapy resistance.We have developed a method for genome-wide analysis of collaborating DNA copy number changes and their corresponding networks. Using this approach we have identified a loss-loss network centered around a region on human chromosome 17p. This network is highly enriched for functional relationships and hints at a more complex system of tumor suppression in which many different genes are affected simultaneously to induce cancer. We show one example of a sub-network around the nuclear co-repressor Datasets consisting of array-based copy number measurements are continuously increasing in size. If probe level interactions are evaluated, the analysis space is of dimensionality Let the aCGH profile of the Negative and positive log2 values respectively denote loss or gain of DNA in the test sample versus the reference sample. We regard both situations separately, which prevents the negative and positive values cancelling each other through summation later in the algorithm. We separate gains and losses by only retaining grid positions with positive values for the gains or negative values for the losses. The absolute values of the separated matrices are then used in the downstream steps. The remaining grid positions are set to zero. More specifically, the gains matrix, The first component of the co-occurrence score is the continuous variant of the AND Boolean logic function: the minimum operation. For two grid points, Since we believe the co-occurrence score to be a smooth variable, and since neighboring co-occurrence values can therefore be employed to reduce the noise locally, we convolve the co-occurrence score matrix with an isotropic 2D Gaussian kernel function. In practice this implies sampling the 2D Gaussian kernel function on a square grid consisting of With 39 unique chromosome arms in the human genome (disregarding the p-arms of the acrocentric chromosomes and the sex chromosomes), three different scales and 4 triangular pair-wise matrices to evaluate  we compute 8892 different CCMs. To solve this problem computationally we used a large distributed computing cluster. Our choice of resolution of the genomic grid was bounded by the memory present on the nodes. We set th peak represents two co-occurring loci, For each CCM we determine the top N peaks for each combination of gains and losses. The nhttp://www.sanger.ac.uk/genetics/CGP/Census/). The reference list of all genes was retrieved from the Ensembl website, with a filter to keep only genes with bio-type = ‘protein_coding’. This left 18840 genes. All CGC genes that could not be mapped back to the reference gene set were excluded. The CGC genes that were annotated as ‘recessive’ were used as the tumor-suppressor genes and ‘dominant’ as oncogenes. Enrichment for all CGC genes, the tumor-suppressor subgroup and the oncogene subgroup in the gene sets determined by the co-occurrence analysis was calculated using a Fisher's exact test.The list of CGC genes was obtained from the CGC website . Clusters are represented as nodes in a graph. Edges between nodes are drawn if any co-occurrence relationship is found between loci present in the nodes.For all pairs of co-occurring loci, http://www.sanger.ac.uk/genetics/CGP/CellLines/). A list of the cell lines included in this dataset can be found in The case we subjected to analysis was a dataset containing 105 cell-lines derived from hematological origin. The aCGH measurements were done on 1.8 million probe Affymetrix SNP 6.0 arrays. After data pre-processing we were left with 95 samples. These cell lines are a subset of the Cancer Genome Project cancer cell line project Click here for additional data file.Dataset S1This file contains information about the cell lines used in this study.(0.03 MB XLS)Click here for additional data file.


P< 0.001; Pearson's r = 0.95). There was absence of cytoplasmic staining and only nuclear staining  was demonstrated in the tumour cells. In normal epithelium of the nasopharynx, the basal layer was stained. An inverse relationship was observed between the level of MT expression and the apoptotic index in the NPC tissues . The results suggest that overexpression of MT in NPC may protect the tumour cells from entering into the apoptotic process and thereby contribute to tumour expansion. Preferential localization of MT in the nuclei of NPC cells may possibly enhance radioresistance since radiotherapy is known to eradicate tumour cells by free radical-induced apoptosis. © 2000 Cancer Research CampaignThe expression of metallothionein (MT), an intracellular ubiquitous low molecular weight protein thiol with antioxidant properties, was studied in nasopharyngeal cancer (NPC) and correlated with the apoptotic index. Immunohistochemical staining of randomly selected, formalin-fixed and paraffin-embedded normal and malignant nasopharyngeal tissues were analysed for the expression of MT using the commercially available E9 antibody directed against MT I and MT II isoforms. The corresponding apoptosis labelling indices were evaluated by the TUNEL method. Localization of MT at the ultrastructural level was studied by immunogold labelling. All the tumour sections (17 specimens) showed MT-immunopositivity. A direct correlation between the percentage of MT-positive cells and the staining intensity was noted (


Previous studies of natural killer (NK) activity in the peripheral blood of breast cancer patients have failed to show a reduction in cytotoxicity, an observation at variance with results obtained in other malignancies. Interpretation of the data however is complicated by the presence of treated and post-mastectomy patients in the groups studied.In this study, lymphocytes from preoperative blood samples of untreated women with benign and malignant breast disease were tested at various effector-to-target ratios for cytotoxicity activity against the NK sensitive erythromyeloid cell line, K562.P=0·02). When the carcinoma group was further divided into pre- and postmenopausal patients, the reduction was found to be a feature only of premenopausal women (P=0·002). The levels of NK activity in patients with benign breast disease were not significantly different from those in controls, irrespective of menstrual status. There was no correlation between NK activity and tumour size, oestrogen-receptor or lymph-node status in the carcinoma patients.A significant reduction in NK activity was observed between carcinoma patients and the control group (P=0·001). This finding, coupled with the variation in NK activity shown between pre- and postmenopausal breast carcinoma patients, suggests that hormonal effects in conjunction with malignancy determine the level of NK activity in breast cancer.A preliminary analysis of NK activities in the control group suggests that women donating blood in the first half of their menstrual cycle show significantly reduced NK activity in comparison with those in the second half (


Our goal is to determine if there is an impaired ability to take up bodies in hepatocytes from ethanol-fed animals compared with control hepatocytes. An ethanol-induced impairment in the ability to remove apoptotic cells in the liver parenchyma could lead to an accumulation of bodies thereby disturbing the hepatic architecture and contributing to the initiation of fibrosis, specifically by activation of the non-parenchymal cell population. The work described in this present study was performed to characterize the in vitro system using flow cytometry to examine uptake of apoptotic bodies by healthy hepatocytes. Our long-term goal is to determine whether inadequate removal of apoptotic cells, presumably via altered receptor-mediated endocytosis (RME), plays a role in the course of pathogenesis of alcoholic liver injury.The spectrum of liver disease after alcohol abuse is similar to many other types of liver diseases in that damage to the hepatocyte, or parenchymal cell, is the cardinal event in the pathogenesis of injury. Damage to the liver, however, involves not only the hepatocyte but also the nonparenchymal cells: the Kupffer, endothelial, and stellate cells which release inflammatory and fibrogenic factors (such as cytokines and chemokines) that lead to altered liver pathology. We have studied extensively the effects of ethanol administration on protein trafficking in the liver, focusing most of our work on the hepatocyte and the process of endocytosis by these cells, using the endocytic pathway of the asialoglycoprotein receptor (ASGP-R) as a model -8. CurreHepatocytes were obtained from male Wistar rats using a collagenase perfusion method that is routinely performed in our laboratory -8. EthanRat hepatoma H4IIE cells were cultured in William's E media. Apoptosis was induced in sub-confluent cells in the presence of 10 mg cisplatin/ml for 20–24 hours at 37 degrees C.The generated apoptotic cells were labeled according to the manufacturer's (Sigma) protocol with the fluorescent probe, 5–6-carboxytetramethyl-rhodamine-succinimidyl-ester (TAMRA). The extent and characteristics of apoptotic cells obtained by the cisplatin treatment was determined by Hoechst 33342 microscopic evaluation, Annexin V-FITC staining, viability staining (trypan blue) and ethidium bromide and acridine orange  and lectin binding: all were performed by standard procedures.5) were cytospun onto glass slides, stained with Wright-Giemsa stain, and examined using a confocal laser-scanning microscope (Carl Zeiss LSM 410 invert with an argon-krypton laser with DIC capabilities) at the appropriate wavelengths.TAMRA-labeled apoptotic or non-apoptotic H4 cells (5–10 million) were incubated with freshly isolated rat hepatocytes (3–5 million) in the presence or absence of 200 mM N-acetylgalactosamine and galactose , 200 mM glucose (Glu) or anti-ASGP-R antibody or non-immune serum . To quantify the phagocytosis of apoptotic cells, we employed a flow cytometric method. Briefly, after incubation of the fluorescently labeled apoptotic or non-apoptotic cells with isolated rat hepatocytes cells at 37 degrees C, cell aliquots were analyzed by flow cytometry using the FACSCalibur (Becton Dickinson) with emission for the TAMRA-labeled cells. Fluorescence-activated cell sorting of TAMRA-positive rat hepatocytes was performed using a FACStarPLUS system (Becton Dickinson) with emission collected using a 575/26-band pass filter. Identification of the phagocytosed apoptotic cells was verified through confocal microscopy of sorted cells. The cells (approximately 1.0 – 10in vitro assay indicates that the ASGP-R is involved in the recognition and uptake of apoptotic cells and suggests an association with alterations in clearance of cells by the ASGP-R. Confirmation of apoptotic cell uptake by the rat hepatocytes was performed by fluorescent microscopy analysis of sorted hepatocytes after flow cytometric analysis as well as by confocal microscopy. We feel that this experimental design provides an excellent means to examine apoptotic cell uptake in isolated cell populations. We will utilize this technique with hepatocytes from ethanol-fed animals to determine if there is an impaired uptake after ethanol administration, due to impaired RME by the ASGP-R. In future studies, we will also examine the time course for these impairments as well as a zonal distribution of the effects, since we have previously shown that altered RME is seen first and foremost in the perivenular region of the liver [We initially performed ligand internalization studies with freshly isolated hepatocytes to establish that the endocytosis results were representative of the previous experiments we performed for protein trafficking studies in our laboratory -8. We thhe liver . In addi


The use of chemotherapy is considered standard therapy in patients with locally advanced non-small-cell lung cancer that cannot be treated with radiotherapy and in those with metastatic non-small-cell lung cancer and good performance status. This approach is also accepted in patients with earlier stage disease, when combined with radiotherapy in those with non-resectable locally advanced disease, or in the preoperative setting. Randomised clinical studies and meta-analyses of the literature have confirmed the beneficial survival effect of platinum-based chemotherapy. Cisplatin and carboplatin have been successfully used with other drugs in a wide variety of well-established two-drug combinations while three-drug combinations are still under investigation. Cisplatin and carboplatin use is limited by toxicity and inherent resistance. These considerations have prompted research into new platinum agents, such as the trinuclear platinum agent BBR3464, the platinum complex ZD0473 and oxaliplatin. These compounds could be developed in combination with agents such as paclitaxel, gemcitabine or vinorelbine in patients with advanced and/or refractory solid tumours.British Journal of Cancer (2002) 87, 825–833. doi:10.1038/sj.bjc.6600540www.bjcancer.comCancer Research UK© 2002  Lung cancer has the highest mortality rate of any major malignancy in the developed world, causing an estimated 1 million deaths worldwide annually . In the Non-small-cell lung cancer (NSCLC) represents approximately 75–80% of all lung cancer . Fewer tThis review outlines current treatment options for patients with NSCLC with emphasis on the use of platinum-containing regimens. This disease is inherently resistant to chemotherapy and is associated with lower response rates than many other malignancies  and the Surgery or radiotherapy is the standard option for patients with early stages of NSCLC. Chemotherapy has shown benefit when used alone in patients with stage IV disease, in combination with radiotherapy in patients with locally advanced disease and in the preoperative setting in those with early stages of NSCLC.Surgery provides the best chance for cure of localised disease. It is therefore the treatment of choice in stages 0, I and II NSCLC . With vePrimary radiotherapy (with curative intent) can be considered in patients with inoperable stages I or II of the disease and sufficient pulmonary reserve. Analysis of one randomised and 26 nonrandomised studies in more than 2000 patients receiving radical radiotherapy for stage I or II disease found that 5-year survival rates ranged from 0 to 42% . PrimaryThe poor efficacy and considerable toxicity of chemotherapy caused great pessimism for many years regarding this approach, as only a small impact on survival was observed.During the 1980s, cisplatin and carboplatin were studied extensively in NCSLC ,b. RandoThe first generation agents in NSCLC  produced response rates ranging from 15 to 25% when used as monotherapy  but, witN7 positions of guanine and adenine in intact DNA. The main DNA lesions produced by both cisplatin and carboplatin, accounting for a total of 95% of platinum-DNA adducts, are at the G-G, A-G and G-X-G intrastrand crosslinks  and renal function of the patient  with which they are being combined and the status of the patient. However, cisplatin is usually given at a dosage of 50–120 mg mAnalysis of the Southwest Oncology Group (SWOG) database of 2531 patients with extensive NSCLC (1974–1988) showed the use of cisplatin to be an independent predictor of improved outcome , with stTwo drug combinations Two types of trials have been conducted to compare monochemotherapy with cisplatin-containing two agent chemotherapy: comparisons with cisplatin monotherapy and comparisons using monotherapy with the non-platinum agent. The relative benefits of combination therapy over monotherapy, shown in many publications, resulted in combination therapy becoming recognised standard practice  and survival distribution  in favour of combination therapy but no significant difference between treatments in 1-year survival rate (31 vs 36%) (vs 30%) and a significantly longer time to progression  with combination therapy; the median survival was 9 months for the whole study population , doxorubicin and 5-fluorouracil (AFP), cyclophosphamide and bleomycin (CBP), vindesine (VP), etoposide (EP), or vindesine and mitomycin-C (MVP). Median survival ranged from 21.6 to 26.6 weeks. The MVP regimen showed a trend towards a higher response rate than the other regimens in certain trials with no benefit on survival  increased repair of platinum-induced DNA damage (increased nucleotide excision repair or loss of DNA mismatch repair); (b) glutathione or metallothionein drug deactivation; (c) reduced cellular uptake of the platinum; (d) altered apoptosis , have prompted research into new platinum agents that have improved toxicity profiles, may circumvent resistance mechanisms, and have administration schedules that are acceptable to physicians and patients.in vitro studies, and have potential in the treatment of solid tumours , in patients with solid tumours unresponsive to previous antitumour treatment. Phase II trials are currently underway.BBR3464 is a trinuclear platinum complex that binds to DNA more rapidly than cisplatin and forms long-range interstrand and intrastrand crosslinks. Phase I studies show diarrhoea and neutropenia to be dose-limiting toxicities, without significant nephro-, neuro- or pulmonary toxicity  therapy. Platinum drugs are still considered of crucial interest based on clinical studies and the results of meta-analyses, with the inconvenience of the observed toxicity and the inherent resistance. These observations have prompted the development of second generation drugs and newer platinums  and any relative benefits for these approaches will be investigated in the ongoing trials.


Hypermethylation of promoter CpG islands with associated loss of gene expression, and hypomethylation of CpG-rich repetitive elements that may destabilize the genome are common events in most, if not all, epithelial cancers.The methylation of 6,502 CpG-rich sequences spanning the genome was analyzed in 137 ovarian samples  ranging from normal tissue through to stage IV cancer using a sequence-validated human CpG island microarray. The microarray contained 5' promoter-associated CpG islands as well as CpG-rich satellite and Alu repetitive elements.Results showed a progressive de-evolution of normal CpG methylation patterns with disease progression; 659 CpG islands showed significant loss or gain of methylation. Satellite and Alu sequences were primarily associated with loss of methylation, while promoter CpG islands composed the majority of sequences with gains in methylation. Since the majority of ovarian tumors are late stage when diagnosed, we tested whether DNA methylation profiles could differentiate between normal and low malignant potential (LMP) compared to stage III ovarian samples. We developed a class predictor consisting of three CpG-rich sequences that was 100% sensitive and 89% specific when used to predict an independent set of normal and LMP samples versus stage III samples. Bisulfite sequencing confirmed the NKX-2-3 promoter CpG island was hypermethylated with disease progression. In addition, 5-aza-2'-deoxycytidine treatment of the ES2 and OVCAR ovarian cancer cell lines re-expressed NKX-2-3. Finally, we merged our CpG methylation results with previously published ovarian expression microarray data and identified correlated expression changes.Our results show that changes in CpG methylation are cumulative with ovarian cancer progression in a sequence-type dependent manner, and that CpG island microarrays can rapidly discover novel genes affected by CpG methylation in clinical samples of ovarian cancer. Ovarian cancer remains the most deadly gynecologic malignancy. There were an estimated 22,430 new cases and 15,280 deaths in the United States in 2007[Aberrant DNA methylation at CpG islands, often in close proximity to transcription start sites, is associated with the epigenetic regulation of genes through altered transcription factor binding and chromatin structure. WidesprWe have used a 6,560 element CpG island microarray to analyze the epigenomic profile of clinical samples of ovarian tissues ranging from benign to late stage tumors. We identified CpG methylation changes that progressed with disease severity and distinguished stage III ovarian cancer from normal or LMP ovarian tissue. We confirmed the microarray results with bisulfite sequencing, 5-aza-2'-deoxyazacytidine re-expression, and correlation with previously published expression profile data.Ovarian tissue samples were obtained from The University of Iowa Gynecologic Oncology Tumor Bank. Samples were collected with informed consent in accordance with the standards of the Institutional Human Subjects Protection Review Board and were surgically staged according to FIGO staging guidelines. Tumor samples were taken from primary tumors only, with no prior exposure to chemotherapy. Normal tissue and tissue samples of low malignant potential were also collected for comparison to tumor. Tissues were snap frozen at the time of surgery in liquid nitrogen and high molecular weight tumor DNA extracted with Trizol reagent by following the manufacturer's instructions . The DOT quantitative test was used to estimate the DNA concentration.The CpG island clones, their preparation for printing, and microarray production were performed as previously described.Genomic DNA was cut be MseI , and then a catch-linker was ligated to the MseI fragments. The fragments were then cut with a methylation-specific restriction enzyme, McrBc . Mock-cut reference samples were exposed to the same conditions and reagents as the digested samples; however, no GTP was added to drive the restriction digest. Twenty nanograms of the mock-digested or twenty nanograms McrBc-cut genomic DNA was then amplified by PCR using primers specific to the linkers and purified with the QIAquick PCR purification kit . Fluorescent Cy3 or Cy5 dye was incorporated into the PCR product using the BioPrime DNA labeling system .Two-color fluorescence hybridizations analogous to expression microarrays were used to compare DNA digested with the methylation-specific enzyme McrBc to mock-digested reference DNA as described previously. After lThe data from scanned microarray images were extracted using GenePix software. Median pixel intensity of each spot was used for analysis. To normalize Cy3 and Cy5 signal intensities we used the "interactive linear regression" approach with minor modifications. Raw dat. All other settings were default.Class prediction using five other methods was performed in BRB ArrayTools using sequences univariately significant at p < 0.001. Only data from the training set was used to develop the classifier. Cross-validation was done using the leave-one-out method and repeating the entire analysis for each iteration of the cross-validation including determination of which sequences were univariately significant. The permutation p-value for the cross-validated mis-classification rate was estimated by analysis of 2000 permutations in which the sample labels were scrambled and the class prediction analysis performed. The permutated p-value is the proportion of the random permutations that gave as small a cross-validated misclassification rate as was obtained with the real class labels. Additional information on how BRB ArrayTools implements the algorithms and performs cross-validation can be found in the manual: Five micrograms of genomic DNA was modified with sodium bisulfite as previously described. The NKXThe resulting PCR product was cloned into a TA cloning vector according to the manufacturer's instructions . Forty-seven positive recombinants from each of ten samples  were isolated using a Qiaprep Spin Plasmid Miniprep kit  according to the manufacturer's instructions and sequenced on an ABI automated DNA sequencer by the Genomic Analysis, Technology, & Sequencing Core at the University of Arizona. The methylation status of individual CpG sites was determined by comparison of the sequence obtained with the known target sequence. The number of methylated CpGs in each sample was counted and averaged over each tissue class. The means for normal and tumor tissue were compared using an unpaired Welch's t-test.2 atmosphere with 10% fetal bovine serum and 100 mg/mL penicillin and streptomycin. OvCar 3 cells were maintained in the same conditions as ES3 except the media was RPMI 1640. 5-aza-2'-deoxycytidine  was added to the culture media at a concentration of 0, 2, or ten micromolar for six days with drug added every other day starting on day one. On day six cells were washed with 4 C phosphate buffered saline and collected for RNA isolation using Qiagen's RNeasy Midi kit according to manufacturer's instructions.ES2 cells were maintained in McCoy's media at 37 C under 95%/5% air/COt method, as described previously[PCR amplification was performed using Taqman primer/probes specific for NKX2-3 obtained from Applied Biosystems ; the primer probe sequences are available upon request. PCR was performed with the ABI Prism 7000 sequence detection system following Applied Biosystem's PCR Master Mix protocol. Real-time PCR was carried out in triplicate on five nanograms of cDNA using parameters recommended by Applied Biosystems. Relative expression was determined by applying the comparative Creviously.137 samples of benign and malignant ovarian tissues  from various stages and histopathologies were analyzed to develop a broad view of DNA methylation changes with progression of ovarian cancer  was performed. 2,042 CpG-rich clones with significant differences in methylation by stage at p = 0.01  adjusted) were identified, and the list reduced to 659 clones by selection for at least a 1.5-fold difference in methylation between any two stages  algorithm as implemented in BRB-ArrayTools was used to develop a classifier using all CpG-rich sequences on the microarray. The PAM algorithm optimizes the size of the classifier such that the fewest number of elements are included without decreasing performance.The PAM algorithm produced a classifier at a threshold value of 6.26 that consisted of three CpG-rich clones, all of which were hypomethylated in stage III tumors relative to normal or LMP tissue Figure . On the A separate class prediction analysis was performed in which the training and test sets were analyzed using several class prediction methods implemented in BRB ArrayTools. The training set was used to select sequences that were differentially methylated between the classes at the p < 0.001 significance level, and resulted in a classifier with 911 sequences. The 911-sequence classifier was then tested using five class prediction methods: compound covariate predictor, diagonal linear discriminant analysis, 1-nearest neighbor, 3-nearest neighbors, and support vector machines. Cross-validation using the leave-one-out method was performed to determine the mis-classification error rate of each class prediction method. To prevent bias in estimation of the mis-classification error rate, the entire analysis was repeated during each iteration of the cross-validation, including determination of which sequences were significant on the reduced training sample. The sensitivity and specificity of the class prediction methods in cross-validation were nearly identical: 94% and 89% respectively for the compound covariate predictor, diagonal linear discriminant analysis, and 1-nearest neighbor methods; and 94% and 93% for the 3-nearest neighbors and support vector machines methods  methylation differences between the normal and LMP samples and the Stage III cancers, 373 had a greater than 1.5-fold change  cancers using more sensitive methods for measuring altered CpG methylation. Early detection of ovarian cancer should improve patient treatment, and is the focus of current research using protein detection in blood samples. FurtherTo validate the CGI microarray results, we confirmed hypermethylation at a NKX2-3 CpG island in ovarian tumor samples relative to normal tissue using bisulfite sequencing. In addition, NKX2-3, was reactivated in two ovarian tumor cell lines following treatment with the demethlyating agent 5'-aza-2'deoxycytidine. Although we did not measure the methylation status of the NKX2-3 CpG island in the cell lines, re-expression of the gene following treatment with 5'-aza-2'deoxycytidine and confirmation of methylation in patient samples suggests that NKX2-3 is epigenetically silenced during ovarian tumorigenesis. As a further confirmation of the CpG island microarrays, we identified seven clones whose methylation profile predicted their loss or gain in expression with disease progression by analyzing our methylation data in conjunction with Wu et al.'s expression data. These results demonstrate that CpG island microarrays can be used to identify novel targets of epigenetic control in tumor samples.We found patterns of DNA methylation that distinguish tumor samples from benign tissue, and used this data to discover genes affected by epigenetic regulation. We showed that the changes in methylation are cumulative with increasing stage, thus the methylation changes may provide a marker for early detection of disease. Our results extend previous studies that suggested simultaneous hypo- and hyper-methylation occurred with tumor progression by measuring both in the same tumor cells.LMP: low malignant potential; FDR: False Discovery Rate; ANOVA: analysis of variance; PAM: prediction analysis of microarrays; SVM: support vector machines.There are no financial competing interests to disclose. The authors state that no financial support has been provided by any organization that stands to gain or lose financially from publication of the work presented here. The authors do not hold stock in any organization that may benefit from this publication, nor are any patents relating to this work held or applied for.GSW: experiment conception and design, data analysis, manuscript preparation; BWF: experiment conception and design, data analysis; NH: data generation; KDG: experiment design and critical discussion, samples; FED: experiment conception and design, data analysis; SR: critical discussion, experiment conception and design, data analysis, samples.The pre-publication history for this paper can be accessed here:M = (R/G) versus average intensities A = (R*G)/2 for three representative hybridizations.Three M versus A plots  methylation differences between any stage. All data normalized to the median of the 10 normal ovarian tissue samples. Average methylation for each stage is shown. Clones associated with a gene (rank 1–4) have the gene's accession number listed.Click here for fileThe cumulative loss of DNA methylation with ovarian cancer progression reverts temporarily in endometroid and serous papillary adenocarcinomas, but not mucinous adenocarcinomas. The 659 CpG-rich clones with significant  methylation differences between any two stages are graphed by histopathology and tumor stage. A reversion to a more normal methylation state can be seen for the sequences with overall loss of methylation in the progression from stage I to stage II in the endometroid and serous papillary adenocarcinomas. Each CpG-rich clone is represented by one line. Lines are colored by their average methylation in Stage IV papillary serous adenocarcinoma relative to the median of the ten normal samples; blue indicates loss of methylation, and red indicates gain of methylation.Click here for fileCumulative loss and gain of DNA methylation in the progression from low to high grade ovarian cancer. The 659 CpG-rich clones with significant  changes in methylation between any two stages of cancer are graphed by grade. Each CpG-rich clone is represented by one line. Lines are colored by their average methylation in grade 3 tumors relative to the median of the ten normal samples; blue indicates loss of methylation, and red indicates gain of methylation. The average methylation of the normal and low malignant potential samples is shown combined as "benign".Click here for fileData for all CpG-rich clones on the CGI microarray with replicates spots averaged. All data normalized to the median of the 10 normal ovarian tissue samples. Average methylation for each stage is shown. Clones associated with a gene (rank 1–4) have the gene's accession number listed.Click here for fileThe results of the BRB ArrayTools analysis are shown, including cross-validation results, and results on the test for each of the five class prediction methods, as well as the list of the 911 sequences that compose the classifier. An additional sheet shows the methylation data for the 911 sequences across all disease stages with all data normalized to the median of the ten normal ovarian tissue samples. Average methylation for each stage is shown. Clones associated with a gene (rank 1–4) have the gene's accession number listed.Click here for fileThe 373 CpG-rich clones used by the class prediction methods with 1.5-fold changes in DNA methylation between normal and LMP, and stage III samples graphed by tumor stage. Each CpG-rich clone is represented by one line. Lines are colored by their average methylation in Stage IV relative to the median of the ten normal samples; blue indicates loss of methylation, and red indicates gain of methylation.Click here for file


Spinal cord injury leads to neurological dysfunctions affecting the motor, sensory as well as the autonomic systems. Increased excitability of motor neurons has been implicated in injury-induced spasticity, where the reappearance of self-sustained plateau potentials in the absence of modulatory inputs from the brain correlates with the development of spasticity.Here we examine the dynamic transcriptional response of motor neurons to spinal cord injury as it evolves over time to unravel common gene expression patterns and their underlying regulatory mechanisms. For this we use a rat-tail-model with complete spinal cord transection causing injury-induced spasticity, where gene expression profiles are obtained from labeled motor neurons extracted with laser microdissection 0, 2, 7, 21 and 60 days post injury. Consensus clustering identifies 12 gene clusters with distinct time expression profiles. Analysis of these gene clusters identifies early immunological/inflammatory and late developmental responses as well as a regulation of genes relating to neuron excitability that support the development of motor neuron hyper-excitability and the reappearance of plateau potentials in the late phase of the injury response. Transcription factor motif analysis identifies differentially expressed transcription factors involved in the regulation of each gene cluster, shaping the expression of the identified biological processes and their associated genes underlying the changes in motor neuron excitability.This analysis provides important clues to the underlying mechanisms of transcriptional regulation responsible for the increased excitability observed in motor neurons in the late chronic phase of spinal cord injury suggesting alternative targets for treatment of spinal cord injury. Several transcription factors were identified as potential regulators of gene clusters containing elements related to motor neuron hyper-excitability, the manipulation of which potentially could be used to alter the transcriptional response to prevent the motor neurons from entering a state of hyper-excitability. The mammalian central nervous system has limited capability for regeneration. Spinal cord injury therefore leads to neurological dysfunctions affecting the motor, sensory as well as the autonomic systems . In the Under normal physiological conditions the motor output (gain) can be modulated by activation of channels in the motor neurons that conduct persistent inward currents, resulting in plateau potentials and sustained firing, leading to enhanced and prolonged muscle contraction -14. The Cluster analysis of the gene expression time series identifies 12 time profiles reflecting combinations of early and late transcriptional regulations. Ontology analysis shows that these clusters contain groups of genes that define over-represented ontologies, indicating that each cluster profile reflects the timing of distinct biological processes as the motor neurons respond to the injury. Genes previously implicated in the development of the plateau potentials in injury-induced spasticity  are alsoSpinal cord injury was inflicted by a complete spinal cord transection at the second sacral segment (S2), in effect disconnecting the spinal networks caudal to the lesion from the remaining part of the central nervous system. The injury causes complete paralysis of the tail, with no effect on bladder, bowel or hind limb functions ,32. The ® Rat Genome 230_2.0 Arrays  were hybridized with RNA samples originating from motor neurons of uninjured control animals (n = 4) as well as animals 2 (n = 6), 7 (n = 5), 21 (n = 8) and 60 (n = 8) days post injury. A conglomerate classifier based on three well-established adjusted ANOVA test-statistics for microarray analysis  was used to identify significantly differentially expressed genes used for subsequent clustering, identifying 3,708 genes with a set false discovery rate (FDR) threshold of 0.02 . The consensus clustering algorithm aims at producing robust clustering results by averaging over multiple clustering runs such that the sensitivity to settings and initialization are diminished. The algorithm works as follows (for more details see [for gene ails see ). Initiaails see .In the present study, we found 12 consensus clusters to be optimal in terms of representing distinct expression time profiles (each containing approximately 150-600 genes). A full list of the differentially expressed genes grouped according to their cluster ID is provided in Additional file The DAVID online ontology-cluster tool ,68 was uTo identify transcription factors that may be involved in regulation of the identified gene clusters we performed motif analysis on each cluster looking for transcription factor binding sites in the core promoter sequences of their constituent genes. For this analysis we obtained the sequences from the Ensembl database of 1000 bp upstream and 200 bp downstream of the coding region of all differentially expressed genes using biomaRt. Each set of promoter sequences pertaining to a distinct time profile was analyzed for over- and under-represented transcription factor binding sites using ASAP  with posWe note that the presented findings resting on this methodology are based on predictions and that more than one transcription factors may bind to the same target. This means that over-representation of a certain binding site for one transcription factor in a gene promoter region may not always predict the binding of the exact same factor.2+, Na+, Cl- and K+ channels as well as genes relating to glutamatergic, GABAergic, glycinergic, cholinergic, serotonergic, adrenergic and dopaminergic receptors were identified and are shown in Table 2+ binding genes relating to calmodulin and IP3 were also included in this table.Significantly differentially expressed genes relating to motor neuron excitability were extracted along with their corresponding cluster ID, belonging to the same categories of ion channels and neurotransmitter receptors as described in . In parthttp://www.r-project.org/ and Bioconductor http://bioconductor.org. Cyber-T source code was obtained from the website http://cybert.microarray.ics.uci.edu/. Open source software was used for clustering  and promoter analysis . Separate scripts for integration of R and bioconductor data formats with these programs were developed and are included as supplementary material  and RMA expression summaries. Quantile-quantile plots B and D illustrate the variation of the distribution tails of the distributions plotted in A and C. The nice overlap of both PM and RMA distributions validates the normalization procedure and the quality of the data obtained from different animals at different time points following spinal cord injury. A. Plot of linear-quantile-normalized PM distributions of each microarray. B. Quantile-quantile plot: linear-quantile-normalized PM distributions (shown in A) plotted against the average PM distribution. C. Distributions of normalized RMA expression summaries, based on PM values from A and B, which has undergone further quantile normalization. D. Quantile-quantile plot: normalized RMA expression summary distributions (shown in C) plotted against the average RMA distribution.Click here for fileR scripts source file. An R source file containing scripts for probe annotation, export and import of expression values to and from ClustreLustre and cluster plots, extraction of sequences using biomaRt for ASAP analysis.Click here for fileR scripts example. Text file illustrating an example of how to run scripts Click here for file


An in vitro autophosphorylation assay has been used to demonstrate that there is considerable variation in met associated protein kinase among human tumour cell lines. Of particular note was the very high level of autophosphorylation of the 140 kD met protein (p140met) in experiments with A431 human cervical carcinoma cells. In contrast in experiments with Daoy human medulloblastoma cells we failed to detect phosphorylation of p140met; instead a high level of phosphorylation of a 132 kD protein was observed. To help understand the basis for the variation in kinase activity and to learn more about the structure of the mature met protein we have analysed p140met in SDS-polyacrylamide gels under non-reducing conditions. Under these conditions the met protein had an apparent molecular weight of 165,000 indicating that the mature met protein may exist as an alpha beta complex in which p140met (designated the beta subunit) is joined by disulphide bonds to a smaller, 25 kD, alpha-chain. We have identified a potential proteolytic cleavage site with the sequence Lys-Arg-Lys-Lys-Arg-Ser at amino acids 303-308 in the human met protein that may account for cleavage of the met protein into alpha and beta subunits.


The pathogenic role of brain derived neurotrophic factor (BDNF) in the incisional pain is poorly understood. The present study explores the role of the BDNF in the incision-induced pain hypersensitivity.A longitudinal incision was made in one plantar hind paw of isoflurane-anesthetized rats. Dorsal root ganglias (DRG) and spinal cords were removed at various postoperative times (1–72 h). Expression pattern of BDNF was determined by immunohistochemistry and double-labeling immunofluorescence. Lidocaine-induced blockade of sciatic nerve function was used to determine the importance of afferent nerve activity on BDNF expression in the DRG and spinal cord after incision. BDNF antibody was administered intrathecally (IT) or intraperitoneal (IP) to modulate the spinal BDNF or peripheral BDNF after incision.After hind-paw incision, the BDNF was upregulated in the ipsilateral lumbar DRG and spinal cord whereas thoracic BDNF remained unchanged in response to incision. The upregulated BDNF was mainly expressed in the large-sized neurons in DRG and the neurons and the primary nerve terminals in the spinal cord. Sciatic nerve blockade prevented the increase of BDNF in the DRG and spinal cord. IT injection of BDNF antibody greatly inhibited the mechanical allodynia induced by incision whereas IP administration had only marginal effect.The present study showed that incision induced the segmental upregulation of BDNF in the DRG and spinal cord through somatic afferent nerve transmission, and the upregulated BDNF contributed to the pain hypersensitivity induced by surgical incision. Brain derived neurotrophic factor (BDNF) is a 12.4-kDa basic protein initially isolated from pig brain and widely expressed in the peripheral and central nervous system. In the dorsal root ganglia (DRG), BDNF is expressed in the small- and medium-sized neurons and anterogradely transported to the central terminals of the spinal cord where BDNF is located in large dense-cored vesicles in terminals of primary afferent fibers in groups I and II glomeruli ,4. SeconPost-operative pain, a unique and common form of acute pain, could induce the rest pain and incident pain  . GrowingAt 1 hour after hind-paw surgical incision, the expression of BDNF was increased dramatically in the ipsilateral dorsal horn of the lumbar spinal cord as compared with that in the contralateral side  in the DRG were increased 117% at 1 hour Figure  and 163%To further determine whether the upregulation of BDNF was also from glial cells, double labeling of BDNF either with S-100 or GFAP, both of which are the markers for satellite cells and Schwann cells in DRG, were performed. As shown in Figure To further investigate the role of nerve transmission in the increased BDNF after hind-paw incision, sciatic nerve block was performed by lidocaine injection just before incision. Successful block was confirmed by the nociceptive testing. Blocking the sensory impulses by lidocaine dramatically reduced BDNF protein level in spinal cord Figure  and 5B aThe upregulation of BDNF in the DRG and spinal cord following incision suggests that BDNF may be involved in the incisional pain. Mechanical allodynia is the typical feature of human surgical wounds as well as the rat model of incisional pain in which allodynia sustained more than 1 day. To explore the role of BDNF in the development of mechanical allodynia induced by surgical incision, IP administration or IT injection of anti-BNDF antibody were performed before surgical incision. As shown in Figure There are several interesting findings in the present study that sought to identify the role of BDNF in the incisional pain. First, hind-paw incision induced transient activation of BDNF expression, mainly localized in the nerve terminals and neurons in the lumbar DRG and spinal cord; Second, the increased BDNF expression was changed to large-sized neurons from the small-sized neurons in the DRG in response to surgical incision; Third, sciatic nerve block by local anesthetic can prevent the upregulated BDNF in the DRG and spinal cord; Finally, IT administration, but not IP administration of anti-BDNF antibody dramatically reduced the mechanical allodynia developed by the incision.Like inflammatory pain and neuropathic pain, incisional pain also induced the upregulation of BDNF in the DRG and spinal cord in the present study. However, the expression pattern of BDNF in the incisional pain was distinct from those in the inflammatory pain and neuropathic pain. In neuropathic pain, the increased BDNF in the spinal cord was mainly expressed in the activated microglia . On the In the present study, IT injection of BDNF antibody attenuated the mechanical allodynia after incision strongly suggesting that BDNF was implicated in the incision-evoked mechanical allodynia. However, it remains elusive the underlying mechanism that BDNF contributes to the incision-evoked pain hypersensitivity. Ample evidence demonstrated that incision-evoked pain hypersensitivity was mainly mediated by AMPA/kainite-dependent mechanisms, but not NMDA receptor-dependent mechanism. This is because the AMPA antagonist but not NMDA receptor antagonist can reverse the incision-evoked pain hypersensitivity -13. HoweIt has been reported that NGF contributed to the neuropathic pain and inflammatory pain, and was also associated with incision-evoked pain hypersensitivity . The incConsiderable evidence shows that inflammatory mediators in the spinal cord are activated segmentally in response to surgical incision. It has been reported that bilateral hind paw incision induce mild increase of cyclooxygenas 2 (COX-2) in the lumbar spinal cord but not cervical segments ,24. Our In the present study, the IT injection but not IP injection of anti-BDNF antibody substantially inhibited incision-evoked mechanical allodynia suggesting that the spinal BDNF may play more important role for the incision-evoked pain hypersensitivity. Thus, blocking the upregulated spinal BDNF may be a novel potential therapeutic approach to treat post-operative pain. It has been well known that alleviation of the post-operative pain, on the other hand, can reduce the anxiety and provide subjective comfort, and should also blunt the autonomic and somatic reflex response and thus restore organ functions and enable mobilization and food intake, thereby helping to improve the outcome. Multimodal analgesia has been suggested to treat post-operative pain in which combination of different modalities, working at different pain mechanisms and reduce the side effect. In this regard, the opioids, paracetamol, NSAIDs, and COX-2 inhibitors are widely used as the pharmaceutical interventions to treat pain . In the The present study demonstrated that incision induced the segmental upregulation of BDNF in the DRGs and spinal cords through afferent nerve transmission. The expression pattern of BDNF was distinct from those in the inflammatory pain and neuropathic pain suggesting that BDNF have a differential role in the incision-evoked pain hypersensitivity with neuropathic pain and inflammatory pain. IT injection of anti-BDNF antibody, but not systemic administration of anti-BDNF antibody dramatically reduced the developed mechanical allodynia following incision indicating that spinal BDNF play an important role in the incision-evoked mechanical allodynia. Thus, inhibition of the spinal BDNF may be a new approach to treat post-operative pain.The study was carried out on male Wistar rats (150–250 g) obtained from Central South University Animal Services . The experimental protocol was approved by the Animal Care and Use Committee of Central South University and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of rats used and their suffering.Animals were briefly anesthetized with 1.5% isoflurane in oxygen, and an incision was made in one hind paw as described previously . Using set al [Mechanical allodynia was assayed using nylon von Frey filaments according to the "up-down" algorithm described by Chaplan et al . The nocIsoflurane-anesthetized rats were implanted with an IT catheter modified from a method previously described . After aThoracic (T1–T2) and lumbar segments (L4–L5) of the spinal cord and DRGs from the control and experimental rats were fixed for 4 hours with 2% paraformaldehyde after perfusion and cryoprotected by immersion in 20% sucrose in phosphate buffer (pH 7.4) overnight. Transverse sections of the spinal cord were cut at 30 μm using a cryostat and mounted on 3-aminopropyl triethoxy-silane-coated slides, rabbit anti-BDNF antibody  were incubated at room temperature overnight. The secondary reagents used for localization were biotinylated goat anti-rabbit IgG and ABC kit . The peroxidase reaction was visualized using 3,3'-diaminobenzidine tetrahydrochloride  as a peroxidase substrate.1 conjugate  for 1 h in the dark at room temperature. After further washes, the preparations were mounted in Vectashield mounting medium . Negative controls were performed routinely by incubating the sections without either the primary or secondary antibodies.Types of cells expressing BDNF was identified using double labeling. The first primary antibodies were polyclonal rabbit anti-BDNF (dilutions 1:100), and the second primary antibodies were either mouse anti-OX-42 , anti-GFAP , anti-NeuN  or anti-S-100 . The respective first and second primary antibodies were applied to the same sections simultaneously. After the overnight incubation, sections were then placed in 5 μg/ml Alexa Fluor 488 goat anti-mouse and 594 goat anti-rabbit IgGvs. a hypersensitive 20 g threshold in control incision animals).Ten minutes before foot incision, animals were injected ipsilaterally with local anesthetic using a 25-gauge needle placed into the sciatic notch. Animals were injected with lidocaine hydrochloride 1% or normal saline, applying 0.5 ml into each sciatic notch. Sensory nerve block was confirmed by von Frey filament testing of mechanical hyperalgesia, which demonstrated an elevation of withdrawal force threshold to 144 g or greater in rats receiving local anesthetic block of the sciatic nerve  was used for the image analysis. Samples were viewed under a light microscopy (Motic). Images were captured using a digital camera (Nikon) attached to the microscope. Capture parameters were initially established and kept unmodified for all images. Five sections from each specimen and five visual fields for each section were randomly quantified. In order to define the density of BDNF immunoreactivity (-IR), we evaluated the relative optic density (OD) of positive staining. For analysis of BDNF-IR cells, cells were confirmed positive-labeled if their density level was over five times background levels . Under 2post hoc Dunnett testing for the BDNF expression or a two-way analysis of variance followed by Bonferroni testing for nociceptive testing. A value of p < 0.05 was considered as significant difference.Statistics analysis was performed using software SPSS 13.0 (SPSS Inc). The OD of the BDNF-IR products and percentage of BDNF-IR neurons were expressed as mean ± SD. Analysis was accomplished using a one-way analysis of variance (ANOVA) followed by The authors declare that they have no competing interests.LCQ participated in the experiments and performed data analysis and wrote the draft of the manuscript. XJM and LD performed the animal models, IT catherarization and nociceptive testing. LCQ and ZJY performed the immunohistochemistry and the analysis of the OD. DRP conceptualized the hypothesis, designed and supervised the experiments, directed the data analysis, and revised the manuscript. All the authors have read the manuscript and agreed to submit to the journal.


However, the molecular basis of the observed effects has not been fully revealed. Although previous studies have documented the inhibition of nitric oxide and cyclooxygenase (COX) activity in vitro by plant and fruit extracts added directly into the culture medium but whether concentrations of bioactive compounds sufficient enough to exert such inhibitory effects in vivo can be achieved through oral consumption has not been reported. In the present study we determined the effect of rabbit plasma obtained after ingestion of a polyphenol rich extract of pomegranate fruit (PFE) on COX enzyme activity ex vivo and the IL-1β-induced production of NO and PGE2 in chondrocytes in vitro. Plasma samples collected before and 2 hr after supplementation with PFE were tested. Plasma samples collected after oral ingestion of PFE were found to inhibit the IL-1β-induced PGE2 and NO production in chondrocytes. These same plasma samples also inhibited both COX-1 and COX-2 enzyme activity ex vivo but the effect was more pronounced on the enzyme activity of COX-2 enzyme. Taken together these results provide additional evidence of the bioavailability and bioactivity of compounds present in pomegranate fruit after oral ingestion. Furthermore, these studies suggest that PFE-derived bioavailable compounds may exert an anti-inflammatory effect by inhibiting the inflammatory cytokine-induced production of PGE2 and NO in vivo.Several recent studies have documented that supplementation with pomegranate fruit extract inhibits inflammatory symptoms  Hydrolysable tannins are predominant polyphenols found in pomegranate juice and account for 92% of its antioxidant activity . PomegraRibes nigrum inhibit cyclooxygenase-2 (COX-2) and lipoxygenase activity and production of prostaglandins E2 (PGE2) in vitro, suggesting that the primary effect of delphinidins  may be against inflammatory responses . Ouriewed in  but in hiewed in , at leasiewed in . These fin vitro [in vivo efficacy of pomegranate constituents and/or their metabolites that become bioavailable after oral ingestion PFE. It is also important to point out that the polyphenolic content of the PFE powder (34 mg/Kg) employed in this study was equivalent to the polyphenolic content of 175 ml of pomegranate juice indicating that this is feasible in terms of human nutrition. Inhibition of COX activity by constituents and/or metabolites that became bioavailable via systemic circulation provide the first direct evidence of pomegranate extract-derived active principles in the plasma that significantly inhibited the COX-2 activity (P < 0.05). After the oral ingestion of a single dose of PFE the inhibition of COX-1 and COX-2 induced by rabbit plasma samples indicated a COX-2/COX-1 ratio of 0.8 which is suggestive of selective inhibition of COX-2 [in vitro assays to inhibit COX activity fails to address the question whether sufficiently high concentrations of these flavonoids could be achieved in vivo to exert the same effect [There are large numbers of phytochemicals consumed in our diet and among them polyphenols constitute the largest group. Although direct inhibitory effect of plant extracts or components on COX enzyme activity have been reported by several investigators -55 but iin vitro . In thisof COX-2 . Selectiof COX-2  but seleE powder  mg/Kg emof COX-2 . AlthougE powder  mg/Kg eme effect .Results of the present study also highlight the effectiveness of bioavailable pomegranate fruit constituents and/or metabolites present in the blood plasma to inhibit the IL-1β-induced NO production in articular cartilage chondrocytes. Biological activities of polyphenols present in popular medicinal plants and herbs have been studied extensively including inhibition of inflammatory stimuli-induced responses in different cell and tissue types [reviewed in ]. NO plaThese studies provide evidence to show that bioavailable constituents and/or metabolites of PFE exert an anti-inflammatory effect by inhibiting the activity of eicosanoid generating enzymes and the production of NO. This further suggests that consumption of PFE may be of value in inhibiting inflammatory stimuli-induced cartilage breakdown and production of inflammatory mediators in arthritis.The authors declare that they have no competing interests.MS carried out the experimental work, collected and interpreted the data, KG carried out the experimental work, collected and interpreted the data, ZR carried out the experimental work, collected and interpreted the data, KAK participated in literature search and drafting of the manuscript, TMH conceived of the study, its design, coordination and drafting the manuscript.All authors have read and approved the final manuscript.