The mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.XLacZ+/- X-inactivation mosaics was replaced after birth by radial stripes, indicating activation of LSCs. Stripe patterns  were independent between paired eyes. Wound healing in organ culture was analysed by mosaic analysis of XLacZ+/- eyes or time-lapse imaging of GFP mosaics. Both central and peripheral wounds healed clonally, with cells moving in from all around the wound circumference without significant cell mixing, to reconstitute striping patterns. Mosaic analysis revealed that wounds can heal asymmetrically. Healing of peripheral wounds produced stripe patterns that mimicked some aberrant striping patterns observed in unwounded corneas. Quantitative analysis provided no evidence for an uneven distribution of LSC clones but showed that corrected corneal epithelial stripe numbers declined with age (implying declining LSC function) but stabilised after 39 weeks.The initial pattern of corneal epithelial patches in Striping patterns, produced by centripetal movement, are defined independently and stochastically in individual eyes. Little cell mixing occurs during the initial phase of wound healing and the direction of cell movement is determined by the position of the wound and not by population pressure from the limbus. LSC function declines with age and this may reflect reduced LSCs numbers, more quiescent LSCs or a reduced ability of older stem cells to maintain tissue homeostasis. The later plateau of LSC function might indicate the minimum LSC function that is sufficient for corneal epithelial maintenance. Quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function, tissue maintenance and repair. These m tissues ,5.XLacZ+/- mosaics from our group have shown that LSCs become active after birth [The corneal epithelium is an excellent model system for the study of tissue maintenance and repair because it is a discrete 5–6 cell thick epithelium replenished by a regionalised stem cell population, which is confined to the basal layer of the limbus at the periphery of the cornea . These ler birth  and idenOur previous mosaic analysis, suggesting that LSCs become active after birth, was based on a transition from a pattern of patches to one of radial stripes. The sequence of events shown in Fig. LSCs are involved in wound healing, as well as normal tissue homeostasis, and they are up-regulated to replace the lost cells . Cornealex vivo healing response [The source of the cells and the extent of cell mixing during the early phase of wound healing can be investigated experimentally by a combination of mosaic analysis and organ culture. Wound healing during 24-hour organ cultures reproduces the initial rapid movement of surrounding corneal epithelial cells to cover the exposed stroma but after 24-hours the experimental wound is only covered by a single layer of cells and the epithelium does not stratify during this initial response -21.in vivo or in organ culture where wounding of a multi-layered stratified epithelium allows more scope for cell mixing during wound healing. This is because cells from the upper epithelial layers could contribute to the cell monolayer that forms during the initial movement phase. It has also been suggested that cells at the wound margin become less adhesive and may detach from the epithelial sheet [ex-vivo wound healing [The alternative experimental approach of wounding cultured corneal epithelial cell monolayers is thought to cause a loss of spatial constraints and induce motility of sheets of epithelial cells rather than individual cells . Howeveral sheet , so prom healing  have bee healing  but it hQuantitative analysis of distributions of the two cell populations in mosaic tissues  can provide more information than qualitative mosaic analysis -27 but tLacZ transgene, the stripes of β-gal-positive cells are elongated patches formed from one or more β-gal-positive coherent clones whose descendents have migrated centripetally. (The terms 'patch' and 'coherent clone' are defined in the Methods section.) A stripe spans the corneal radius, so its length is not affected by the number of LSCs and is not relevant to the analysis. The stripe width, however, is variable and depends in part on the number of adjacent corneal epithelial coherent clones belonging to the same cell population . Clearly, an individual stripe is more likely to be made up of multiple adjacent β-gal-positive corneal epithelial coherent clones when the proportion of β-gal-positive cells in the corneal epithelium is higher. This source of variation in stripe width can be factored out by dividing the observed mean width of β-gal-positive stripes by the function 1/(1-p), where p is the proportion of β-gal-positive cells around the circumference [In an adult corneal epithelium, showing mosaic expression of a mference ,28. The Although, the corrected stripe number is related to the number of active LSC coherent clones it does not provide a direct estimate of LSC numbers. This is partly because the proportion of LSCs that are active may vary and also because the number of LSCs per LSC coherent clone may vary. For example, variation in the number of LSCs per LSC coherent clone may occur because of differences in the extent of cell mixing during development of the surface ectoderm, from which the corneal and limbal epithelia develop  LSC function declines with age, (ii) little cell mixing occurs either during normal maintenance of the corneal epithelium or during wound healing, (iii) the main driving force during wound closure is not population pressure from centripetally streaming cells produced by LSCs and (iv) quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function in tissue maintenance and repair.XLacZ+/- mosaic mice showed that the initial pattern of randomly orientated patches seen at 3 weeks was replaced by radial stripes by postnatal week 10  was used for the wound healing study because corneal stripes were clearer in eyes with weaker GFP expression in the lens. Histological sections showed that the GFP-positive stripes were localised to the corneal epithelium not the stroma  transgenic reporter mice [Two approaches were used to study the dynamics of epithelial migration during e tissue ). X-gal ter mice . Using tTime-lapse recording of the radial GFP stripes allowed visualisation of the movement of GFP-positive cells towards the centre of wounds during the initial 24 h of the wound healing response. GFP-positive stripes extended towards the centre of the previously wounded region and stripes remained contiguous throughout the wound healing response Fig.  and 2. W-1. However, tracking of individual stripes revealed that the wound actually closed asymmetrically  to 0.80 μm min-1 (stripe 3) for the 6 different stripes measured. Method B gave very similar results (0.31 μm min-1 for stripe 6 and 0.82 μm min-1 for stripe 3). To our knowledge this is the first report of differential rates of cell movement at the leading edge of a circular wound in the corneal epithelium and highlights the advantages of using a mosaic analysis.The mean rate of migration calculated from the time taken for the wound shown in Fig lly Fig. . BecauseXLacZ+/- mosaic corneal epithelia (see Methods) and used to compare LSC function at different ages up to 52 weeks and in different regions around the circumference. This showed that the corrected stripe number declined with age, up to but not beyond 39 weeks  or the corrected stripe numbers .XLacZ+/- and PAX6-GFP stripes remained contiguous during healing shows that wounds healed in a clonal manner, suggesting a coordinated tissue response to wound healing stimuli, without any significant cell mixing. This differs from interpretations of experiments on adult skin epithelial wound healing, suggesting quite extensive cell mixing [XLacZ+/- and PAX6-GFP corneal wound healing experiments.The fact that both l mixing  and may l mixing ,44. HoweXLacZ+/- mosaic eyes  shows that the main driving force is not population pressure from cells produced by LSCs so is likely to be population pressure from the wound-margin or other forces, such as chemotaxis or electric fields. Peripheral wound healing in mosaic corneas produced a new pattern of stripes, which converged within the wound. As the original central region was not wounded the healing of peripheral wounds produced a second centre of stripe convergence. The resultant bi-convergent striping pattern resembles the abnormal striping patterns seen previously in some Use of time-lapse confocal microscopy for studies of wound healing allows rates of cell movement to be monitored throughout wound healing and provides a simple means of identifying the time of wound closure.  The coAlthough previous reports suggest there is an uneven distribution of LSCs and TACs around the circumference, no clear consensus has emerged. Analysis of holoclones produced by human limbal cells suggested there were more colony forming cells (putative LSCs) in the superior and temporal than the inferior and nasal regions  but thisOur quantitative analysis implies that LSC function declines with age to 39 weeks and then stabilises. An age-related decline could occur either if some LSCs were lost  or if some LSCs become quiescent, resulting in a reduction in the number of active LSC clones , were obtained from the MRC Mammalian Genetics Unit, Harwell, UK. Hemizygous XLacZ+/- females F1 females) show mosaic transgene expression, after X-gal staining for β-gal reporter activity, due to random X-inactivation during development. Mice of the transgenic reporter lines Y001deltaDRR and Y223 (showing mosaic expression of PAX6-GFP) were used to visualise stripe dynamics by time-lapse confocal microscopy. The Y223 line carries 6 copies of a yeast artificial chromosome (YAC) containing the human PAX6 locus [CAGGS-Cre line [H253 strain mice , ubiquitX6 locus  in whichCre line  resultinCre line .Whole eyes dissected at 3 to 52 weeks after birth were fixed and stained for β-gal reporter activity as described previously . To prepXLacZ+/- corneas. Eyes were then cultured for 24 hours and stained with fluorescein at 0, 6 and 24 h to visualise and photograph the wound margin [XLacZ+/- eyes. They were then cultured in a controlled environmental chamber  surrounding the stage of a Leica inverted confocal microscope. During culture the eye was supported on a specially designed stand so the cornea faced downwards. Z-stack images were collected at intervals over a 24 h period using a ×5 objective. Confocal images were analysed using ImageJ, a shareware image analysis software package [As described previously 1 mm diameter wounds through the full epithelial thickness were made in d margin . Eyes we package ,48. The  package  developepatch is a group of cells of like genotype (or phenotype) that are contiguous at the time of consideration. A descendent clone is any group of clonally related cells irrespective of whether they have remained contiguous throughout development. A coherent clone is a group of clonally-related cells that have remained contiguous throughout development. The number of coherent clones per patch depends partly on the proportions of the two cell populations in the mosaic. For one-dimensional mosaics (strings of two cell populations) the number of coherent clones per patch of cell population 'A' is estimated as 1/(1-p) (where p is the proportion of cell population 'A' in the mosaic) [For analysis of variegated, mosaic patterns, it is important to distinguish between different types of patches and clones. We use three terms defined previously ,50-53. A mosaic) ,28,53.p), where p is the proportion of β-gal-positive cells around the circumference [The observed mean width of β-gal-positive stripes in the corneal epithelium was corrected for the probability that stripes would contain multiple adjacent β-gal-positive corneal epithelial clones. This involved dividing the observed mean width by the function 1/. Each region was equivalent to half of the circumference . The nasal conjunctival epithelium was sutured to mark its orientation and eyes were removed and stained. Stripes were counted and measured separately in different hemispheres in two ways: nasal versus temporal and superior versus inferior. Stripes on the nasal-temporal or superior-inferior boundaries were included in both regions. To correct for overlapping stripes, the corrected stripe number per region was calculated by dividing half the circumference by the corrected mean stripe width separately for each of the four regions.The clonal analysis of striping patterns between 15 and 52 weeks was carried out as described previously ,5. To dePAX6-GFP: Y001deltaDRR (PAX6-GFP) transgenic mice; TAC: transient amplifying cell ; XLacZ+/-: female X-inactivation mosaic mice, hemizygous for the H253 X-linked nLacZ transgene.ANOVA: analysis of variance; β-gal: β-galactosidase; CI: confidence interval; GFP: green fluorescent protein; LSC: limbal stem cell; The authors declare that they have no competing interests.RM devised the temporal mosaic analysis of wound healing, carried out the quantitative clonal analysis and histology, contributed to the design of the study and drafted the manuscript. TR carried out the wound healing (in conjunction with RM). DK produced and provided the Y001deltaDRR mice. JW and SM conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.PAX6-GFP mosaic corneal epitheliumTime-lapse confocal microscopy of healing of a 1 mm diameter central wound in a . Time-lapse video showing the complete healing of a central wound in the corneal epithelium between 5.25 and 18.75 hours post wounding. At 18.75 hours the wound appears completely healed and stripes extend to the centre of the previously wounded region.Click here for filePAX6-GFP mosaic corneal epitheliumTime-lapse confocal microscopy of healing of a 1 mm diameter peripheral wound in a . Time-lapse video showing incomplete healing of a peripheral wound in the corneal epithelium between 5.25 and 18.75 hours post wounding. At 18.75 hours the wound is only partially healed, stripes extend towards the centre of the previously wounded region.Click here for fileThe effects of age on mean corrected stripe number in the corneal epithelium of X-inactivation mosaic mice.Click here for fileComparisons of corneal epithelial stripe numbers in different regions (at 23–24 weeks).Click here for file


Integration of biological knowledge encoded in various lists of functionally related genes has become one of the most important aspects of analyzing genome-wide functional genomics data. In the context of cluster analysis, functional coherence of clusters established through such analyses have been used to identify biologically meaningful clusters, compare clustering algorithms and identify biological pathways associated with the biological process under investigation.. The package integrates routines for calculating gene specific functional coherence scores and the open source interactive Java-based viewer Functional TreeView (FTreeView).We developed a computational framework for analytically and visually integrating knowledge-based functional categories with the cluster analysis of genomics data. The framework is based on the simple, conceptually appealing, and biologically interpretable gene-specific functional coherence score (CLEAN score). The score is derived by correlating the clustering structure as a whole with functional categories of interest. We directly demonstrate that integrating biological knowledge in this way improves the reproducibility of conclusions derived from cluster analysis. The CLEAN score differentiates between the levels of functional coherence for genes within the same cluster based on their membership in enriched functional categories. We show that this aspect results in higher reproducibility across independent datasets and produces more informative genes for distinguishing different sample types than the scores based on the traditional cluster-wide analysis. We also demonstrate the utility of the CLEAN framework in comparing clusterings produced by different algorithms. CLEAN was implemented as an add-on R package and can be downloaded at Our results indicate that using the gene-specific functional coherence score improves the reproducibility of the conclusions made about clusters of co-expressed genes over using the traditional cluster-wide scores. Using gene-specific coherence scores also simplifies the comparisons of clusterings produced by different clustering algorithms and provides a simple tool for selecting genes with a "functionally coherent" expression profile. The iustering  using thustering . Similarustering , gene liustering ,8 and epustering , as wellustering . IntroduThe first two concept defining papers ,5 also hA frequently encountered problem in analyzing genome-wide experimental data is to choose among results produced by different clustering algorithms. Criteria such as homogeneity and separation are relatively straightforward to compute but are mostly of theoretical interest. A more relevant criterion from a biological perspective is the overall functional coherence of resulting gene clusters. Most of the methods developed to date for this purpose require specification of the number of clusters ,20. CompWe developed an analytical framework and flexible computational infrastructure for integrating knowledge-based functional categories into the cluster analysis of gene expression data. The framework is based on the simple, conceptually appealing and biologically interpretable gene-specific functional coherence CLustering Enrichment ANalysis (CLEAN) score derived by correlating the clustering structure as a whole with functional categories of interest. The CLEAN score is gene-specific and it differentiates between the levels of functional coherence for genes within the same cluster. The statistical significance of coherence scores is established by comparing them to the empirical null-distribution obtained by randomly permuting gene identifiers. The corresponding computational infrastructure is based on an open-source R package for the data analysis and open-source Java viewer for visually integrating and analyzing expression data and associated knowledge-based functional categories.We investigate the reproducibility of the findings based on the CLEAN scores, and demonstrate its utility in comparing the functional coherence of clusterings produced by different algorithms and in selecting genes with informative expression patterns. Being gene-specific, the CLEAN score facilitates easy comparisons of functional coherence of different hierarchical structures  and selection of genes based on functional coherence of their expression pattern without the need to fix the number of clusters. On the other hand, we demonstrate that differentiating between the levels of functional coherence for genes within the same cluster leads to significant improvements in reproducibility of findings across independent microarray datasets when compared to traditional cluster-wide analyses. Furthermore, genes selected based on the CLEAN score produced more precise sample groupings than genes selected using the cluster-wide score.Given a hierarchical clustering of genes based on their expression profiles and a set of functional categories (e.g. Gene Ontologies), the CLustering Enrichment ANalysis (CLEAN) score for a gene is calculated as follows Figure :1. Fisher's exact test for enrichment is calculated for all functional categories containing the gene and for all possible clusters containing this gene.  of enrichment tests across all pairs of clusters containing the gene and functional categories containing the gene (see methods for details).2. The CLEAN score is then computed as the maximum -logThe clustering-specific null-distribution of the CLEAN score is established by randomly permuting gene identifiers. Statistically significant scores are then used to facilitate selection of genes or gene clusters, as well as the assessment of functional coherence and the comparison of clustering results produced by different algorithms. The integrated clustering viewer/browser, Functional TreeView (FTreeView), is used for integrative browsing and visual display of gene clusters and associated functional categories.When multiple category types are used, the joint CLEAN score is calculated as the maximum of CLEAN scores for each category type. Here we focus on three sets of functional categories: Gene Ontology (GO) categories , Kyoto E10 for all clusters containing the gene regardless of whether the enriched functional categories contain the gene or not  score to serve as a surrogate for this traditional type of analysis. The cwCLEAN score is defined as the maximum of -logThe CLEAN score provides a tool to compare the functional coherence of clustering results produced by different clustering algorithms on a gene-by-gene basis without requiring a pre-defined number of gene clusters. We used four independent large-scale breast cancer gene expression datasets -31 to deFor each clustering algorithm the total number of genes (y-axis) with the CLEAN score higher than the given threshold was plotted against all possible threshold levels (x-axis). There are two conclusions that can be immediately made based on results in Figure We used the same four independent breast cancer gene expression datasets . The heatmap in Figure 10) of the Fisher's Exact tests for the same contingency tables were also all positive implicating the higher reproducibility of CLEAN scores  than for the cwCLEAN score .We repeated a similar type of analysis for a mouse and human datasets profiling gene expression in different tissues (79 human and 61 mouse tissue types). . After mReproducibly identifying genes whose expression patterns can delineate biologically meaningful groups of samples has been an important problem in computational biomedicine. We focus on the situation when the identity of samples belonging to different groups or even the number of the groups is not known in advance. In this case, the informative genes have to be selected in an unsupervised fashion. By studying the problem of classifying samples from different tissue types in the integrated mouse-human dataset, we demonstrate the utility of using the CLEAN score to select informative genes. We first identify genes with statistically significant CLEAN scores in mouse and human tissue expression profiling datasets. Then we show that expression profiles of these genes facilitate better separation of samples from different tissue types than expression profiles of genes not having statistically significant CLEAN scores. Furthermore, we demonstrate that the improvements in precision are significantly larger when using the CLEAN score than when using the cluster-wide cwCLEAN scores.We created a total of 6 different gene lists and assessed their abilities to distinguish different tissue types in the combined human-mouse expression dataset. Gene lists were as follows:All 10,287 genes present in both microarray platforms.Genes with significant CLEAN scores in both human and mouse datasets.Genes with not significant CLEAN scores in both human and mouse datasets.Genes with significant cwCLEAN scores in both human and mouse datasets.Genes with not significant cwCLEAN scores in both human and mouse datasets.Genes identified by applying the Cancer Outlier Profiler Analysis (COPA)  analysisTissue samples were then clustered based on each of these gene lists using Euclidean distance, average linkage hierarchical clustering. Co-clustered pairs of samples derived from the same tissue type (regardless of whether they are human or mouse derived) were considered true positives, and co-clustered pairs of samples derived from different tissues were considered false positives. By cutting the hierarchical tree structure at all possible levels and each time recording the number of true and false positives we determined the receiver operating characteristic (ROC) for each of the gene lists. Since the number of positive pairs (232) is small compared to the number of negative pairs , we used the ratio of number of false positive pairs divided by the total number of positive pairs, as recently described , insteadk-means [We developed an open-source R package  that perk-means . The pack-means , and use-60) as well as two KEGG pathways, and putative targets of the Interferon Consensus Sequence-binding protein (ICSBP) transcription factor. FTreeView is available as a stand-alone or as a Web Start application from our server .In addition, we extended the Java-based expression data viewing software TreeView  to interIntegrating biological knowledge encoded in lists of functionally related genes into the analysis of genome-wide functional genomics data is an increasingly important aspect of analyzing genomics data. In the context of cluster analysis, such integration is necessary for selecting meaningful clusters of genes, and for the adequate biological interpretation of patterns defined by such clusters. We developed a computational framework for analytically and visually integrating knowledge-based functional categories with the cluster analysis of genomics data. The framework is based on the gene-specific functional coherence score derived by correlating the clustering structure as a whole with functional categories of interest. The statistical significance of coherence scores is established by comparing them to the empirical null-distribution obtained by randomly permuting gene identifiers.We established the reproducibility of the CLEAN score across related gene expression datasets, and its utility in comparing the functional coherence of different clusterings and in unsupervised selection of genes that discriminate between biologically meaningful groups of samples. When compared to the commonly used cluster-wide assessment of functional coherence, the CLEAN score exhibits higher reproducibility across different microarray datasets. Genes selected based on the CLEAN score produced more precise sample groupings than genes selected using either cluster-wide score or by using COPA algorithm.It is important notice that by using the CLEAN score instead of the traditional cluster-wide approach one cannot use the guilt-by-association principle  to hypotOne way to think about the differences between CLEAN and cwCLEAN score in terms of differences between assuming functional coherence based on co-clustering  vs. having some additional pre-existing evidence of functional relationship (CLEAN). Our results in this context implicate that in the case of breast cancer and tissue datasets the previous evidence of functional relationship is overall more reliable than guilt-by-associations relationships arising only from the cluster analysis on their own. It is possible that in some other situations, new functional relationships would dominate the existing one and the opposite would be the case. Calculating the difference between the two scores can quickly implicate novel functional relationships arising from the data analysis alone.A systematically different approach to integrating the experimental data and prior knowledge is to incorporate the functional information into the clustering algorithm itself -46. WhilWe directly demonstrate that integrating prior biological knowledge encoding in the lists of functionally coherent genes improves the reproducibility of clustering results. We also demonstrate that our gene-specific functional coherence score, which differentiates between the levels of functional coherence for genes within the same cluster, shows higher reproducibility than the cluster-wide score. The CLEAN score also produced more informative genes for distinguishing different sample types than the cluster-wide score. This implicates that the gene-specificity of the CLEAN score is a fundamentally different and, at least in some circumstances, better approach for integrating biological knowledge with results of the cluster analysis than previously used cluster-based scores.Raw data files (Affymetrix HG-U133A CEL files) of four independent human breast cancer datasets  [We applied a mild variation filter using Cancer Outlier Profiler Analysis   to selec• Context-Specific Infinite Mixture Models (CSIMM) . For any• Pearson Correlation of gene expression values as the similarity measure.• Euclidian Distance based on per-gene normalized expression values as the distance measure.All three hierarchical clustering algorithms used Average Linkage.Each clustering analysis was then repeated after further variance based re-scaling each dataset by dividing expression levels by their standard deviation for each gene and each datasets separately. When computing the Pearson correlation, expression values are implicitly divided by the standard deviation. Thus, this additional normalization step did not significantly affect Pearson's correlations.All statistical analyses were performed using the statistical programming environment R version 2.7.1  and BiocClustering Enrichment Analysis (CLEAN) is based on testing every possible cluster within a gene clustering for statistically significant enrichment of biological categories. A background gene list  is given as well as a hierarchical clustering of some or all genes in the background list. The method was implemented as described in Algorithm 1.Algorithm 1. Clustering Enrichment Analysis (CLEAN).1. Define one or more sets of biological categories with sufficient representation in the background gene list2. Determine all possible gene clusters within a given size range3. For each gene cluster3.1 For each functional category, determine the 2 × 2 contingency table and perform Fisher's Exact testq-values, that is the adjusted Fisher p-values, to account for multiple testing3.2 Compute q-values3.3 Record categories the cluster is significantly enriched with and corresponding 4. Compute the cluster-wide cwCLEAN scoreq-value for each gene cluster4.1 Determine the minimum q-value starting with the lowest (i.e. most significant) q-value.4.2 Sort gene clusters by minimum q-value to avoid 'spill-over' effect, i.e. remove gene clusters whose significant functional enrichment score is likely due to a single subtree.4.3 Prune cluster supersets with less significant q-value over all remaining clusters the gene is member of4.4 For each gene, find the minimum q-value over all category sets (e.g. GO and KEGG)4.5 For each gene, find the minimum 10-transformed minimum per-gene q-value.4.6 The cwCLEAN is the resulting -log5. Compute the gene-specific CLEAN scoreq-value over all clusters and all categories the gene is member of.5.1 For each gene, find the minimum q-value over all category sets (e.g. GO and KEGG)5.2 For each gene, find the minimum 10-transformed minimum per-gene q-value.5.3 The CLEAN is the resulting -logA functional category is defined as a non-empty set of genes representing a biological concept such as "cell cycle", "immunological synapse" or "cytokine-cytokine receptor activation". The method is designed to accommodate any set of functional categories such that each category is comprised of a list of genes that has user-specified minimum overlap with the background gene list. Here, sets of categories were either downloaded from publically accessible databases such as Gene Ontology (GO) , Kyoto EMore specifically, functional categories based on GO and KEGG were downloaded as R packages ,37 whileFor compatibility, all gene identifiers were converted to Entrez gene IDs, and matched across species where necessary using the HomoloGene database . SubsequGiven a hierarchical gene tree, a list of all possible gene clusters is obtained by recursively traversing the tree structure and at each node recording the list of corresponding genes. The size of clusters is limited to a user-specified range. Here, clusters smaller than 10 genes and larger than 1,000 were disregarded.q-values  are computed to control the false discovery rate (FDR) [q-value not greater than a user-defined cutoff are considered significant. The default q-value cutoff is 0.1.To determine whether a functional category is over-represented in a given gene cluster, i.e. more genes of the functional category are present in the cluster than expected by chance, a two-by-two contingency table  is constructed and Fisher's Exact Test is performed. The procedure is repeated for each category within a category set  and te (FDR) . Categork-means [k of clusters, a mutually exclusive set of gene clusters is already given and step 2 in Algorithm 1 is skipped. If the user specifies a range K of cluster numbers, step 2 in Algorithm 1 is preceded by Algorithm 2 which generates a hierarchical gene clustering as a means to average over multiple runs of the non-hierarchical clustering algorithm.The procedure can be extended to non-hierarchical methods such as k-means . For a fAlgorithm 2. Averaging gene clusterings over n runs of a non-hierarchical clustering algorithmk in K, run the non-hierarchical clustering algorithm1. For each i2. For each gene j ≠ i, count the number nij of clusterings where i and j are in the same cluster2.1 For each gene j ≠ i, compute pij = nij/n, where n is the size of K2.2 For each gene pij as a similarity metric and average linkage as a summary method to generate a hierarchical clustering.3. Use the .An R package to perform CLEAN and an open-source Java tool to interactively display gene expression data, gene and sample clustering, gene annotation, and functional cluster annotation can be freely downloaded from the authors' web-site CG: co-regulation groups; ChIP: Chromatin Immunoprecipitation; CLEAN: Clustering Enrichment Analysis; COPA: Cancer Outlier Profiler Analysis; CSIMM: Context-Specific Infinite Mixture Models; cwCLEAN: cluster-wide CLEAN score; FDR: false discovery rate; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; OR: odds ratio; PPP: posterior pairwise probability; PWM: position-weight matrix; RMA: Robust Multichip Average; ROC: Receiver Operating Characteristic.JF developed the methods and the R package, performed all analyses, interpreted results and drafted the manuscript. MM conceived the methodology and provided guidance in the development, design, analysis, interpretation of results, and drafting of the manuscript. VJ developed FTreeView and ZH constructed some of the functional categories used in the analysis.


Exercise therapies generate substantial costs in computer workers with non-specific work-related upper limb disorders (WRULD).To study if postural exercise therapy is cost-effective compared to regular physiotherapy in screen-workers with early complaints, both from health care and societal perspective.Prospective randomized trial including cost-effectiveness analysis; one year follow-up. Participants: Eighty-eight screen-workers with early non-specific WRULD; six drop-outs. Interventions: A ten week postural exercise program versus regular physiotherapy. Outcome measures: Effectiveness measures: Pain: visual analogous scale (VAS), self-perceived WRULD (yes/no). Functional outcome: Disabilities of Arm, Shoulder and Hand- Dutch Language Version (DASH-DLV). Quality of life outcome: EQ-5D.Economic measures: health care costs including patient and family costs and productivity costs resulting in societal costs. Cost-effectiveness measures: health care costs and societal costs related to the effectiveness measures. Outcome measures were assessed at baseline; three, six and twelve months after baseline.At baseline both groups were comparable for baseline characteristics except scores on the Pain Catastrophizing Scale and comparable for costs. No significant differences between the groups concerning effectiveness at one year follow-up were found. Effectiveness scores slightly improved over time. After one year 55% of participants were free of complaints. After one year the postural exercise group had higher mean total health care costs, but lower productivity costs compared to the physiotherapy group. Mean societal costs after one year (therefore) were in favor of postural exercise therapy . After one year, only self- perceived WRULD seemed to result in acceptable cost-effectiveness of the postural exercise strategy over physiotherapy; however the probability of acceptable cost-effectiveness did not exceed 60%.Considering societal costs related to QALYs, postural exercise therapy had a probability of over 80% to be cost-effective over a wide range of cost-effectiveness ceiling ratios; however based on a marginal QALY-difference of 0.1 over a 12 month time frame.Although our trial failed to find significant differences in VAS, QALYs and ICERs based on VAS and QALYs at one-year follow-up, CEACs suggest that postural exercise therapy according to Mensendieck/Cesar has a higher probability of being cost-effective compared to regular physiotherapy; however further research is required.ISRCTN 15872455 The prevalence of Work-Related Upper Limb Disorders (WRULD) in the Dutch working population is estimated about 19-30% ,3.WRULD can result in decreased productivity, increased medical consumption and consequently increased costs. A recent study estimates the total yearly costs due to specific and non-specific WRULD in the Netherlands at about 2.1 billion Euros, consisting of medical costs, costs due to decreased productivity, absenteeism related to WRULD and disability pensions .It can be assumed that WRULD is associated with a decreased quality of life .Of all WRULD complaints, it is estimated that specific disorders are responsible for about 13-37% of them. The majority concerns non-specific WRULD .In the Netherlands non-specific WRULD is treated within various medical and paramedical disciplines . PosturaVery little reliable research is available regarding the effectiveness of exercise and other treatments in non-specific WRULD -11.The same goes for cost-effectiveness studies ,13 in whThe high prevalence, costs and decreased quality of life signify a large impact of non-specific WRULD.Beyond effectiveness studies, cost-effectiveness studies in relation with quality of life are needed in patients with non-specific WRULD to be able to improve health care and to lower the costs.As the department of rehabilitation of the Maastricht University Hospital acts as a tertiary referral centre for non-specific WRULD complaints a cost-effectiveness study among computer screen-workers with early stages of non-specific WRULD was set up.In the current study we tested if postural exercise therapy according to Mensendieck/Cesar is cost-effective with respect to pain, disability and quality of life as when compared to regular physiotherapy in computer screen-workers with early stages of non-specific WRULD, both from a health care and societal perspective.A prospective randomized clinical trial was set up among computer screen-workers with early non-specific work-related upper limb disorders . RecruitWithin two weeks after eligible patients were selected and invited to participate by the occupational physician, baseline assessments were performed at one of two locations, either the Maastricht University Hospital or the Institute for Rehabilitation Research in Hoensbroek, a small town in the south-eastern region of the Netherlands.Participants were randomized to the PE group or the RP group in strata depending on the duration of the complaints (cut-off point six weeks). Blocks of four were generated for each stratum by means of a computer generated random sequence table.Randomization was concealed because a research assistant, who was not involved in the selection of the participants, allocated participants to groups using a list of random numbers which was generated before commencement of the trial. Because both interventions were active, blinding of participants and therapists was not possible. In both groups, the ten week intervention started within one week after baseline measures were completed. Outcome measures were collected at baseline and at three, six and twelve months where the same questionnaires were completed using a computer under the supervision of a research assistant. The research assistant instructed participants about the questionnaires, which had to be completed by using a computer in the participant's usual manner. The computer workstation was custom-made for this purpose for each participant. Only the pain outcome measure was assessed by the participants filling in the forms by pen during four sequential working days . AlthougThis research project was approved by the Medical Ethical Committee of the University Hospital of Maastricht.Computer screen-workers with early non-specific WRULD . Early nTo be eligible for this study, participants had to fulfil the following inclusion criteria:- were computer screen-worker at the time of first complaints and being employed in present job for at least three months- had non-specific WRULD with symptoms existing longer than two weeks but shorter than three months- aged between 20 and 45 yearsExcluded were patients not fulfilling the inclusion criteria and patients with non-specific WRULD during other daily activities such as teeth brushing and car driving, patients with specific WRULD , patients with other diseases of musculoskeletal system , pregnant patients, patients who were on sick leave and patients who already had received therapy for their complaints or who had received postural exercise therapy during the last five years.One group of participants received PE therapy, in the Netherlands known as Mensendieck and Cesar. PE therapy according to Bess Mensendieck and Maria Cesar do not differ basically and both therapies and their training programs have been assimilated since the fusion of both societies in 2004 ,11. PE tThe other group of participants received RP and was treated by four physiotherapists who attended a WRULD-course. They did not make use of applications or massage techniques. Active muscle training and fitness exercises were part of the therapy. The focus was on improvement of muscle condition for long-lasting static postures.All participants in both treatment arms received ten weeks of therapy according to protocol .The PE group received in total one and a half hours more therapy compared to the RP group, although the last group received six more sessions Table .Treatments were paid for by health care insurance companies.At baseline, besides the effectiveness measures (see further) sex, age, number of working hours and level of education were assessed.Participants were labeled as "highly educated" if they had at least a bachelor's degree.As this syndrome is related to work, the number of working hours was registered. Because onset and course of non-specific WRULD are influenced by physical, psychosocial and personal risk factors, ,16-18 vaThe following variables were assessed:1. The validated Groningen Fitness Questionnaire  was used2. The Dutch version of the Job Stress Survey (JSS)  was used3. The Dutch version of the Multidimensional Perfectionism Scale of Frost (MPS-F) measures (neurotic) perfectionism ,21.4. The Dutch version of the State-Trait Anxiety Inventory (STAI) measures state-respectively trait anxiety 5. The Dutch version of the Pain Catastrophizing Scale (PCS) has been used to measure to which extend people who suffer from pain experience catastrophizing thoughts .As a primary outcome measure we used the horizontal numerical visual analogous ten cm scale (VAS) according to Jensen  to measuAs a secondary outcome measure we used the Disabilities of the Arm, Shoulder and Hand- Dutch Language Version (DASH-DLV) questionnaire  to measuTo measure the generic quality of life we made use of the EQ-5D of the EuroQol Group . This quAll scales are commonly used internationally and are reliable and validated.Costs are defined from the societal perspective. These are subdivided into health care costs (including out of pocket costs for the patient and family) and productivity costs. Only costs related to WRULD are included in the analyses. Costs are determined by multiplying the volume reported on each cost item by the estimated costs per unit Table . Out of The number of PE and RP sessions during the ten week intervention period registered by the therapists is used to calculate the health care costs of intervention sessions during this period.Costs concerning productivity loss are based on the reported sick leave from work due to non-specific WRULD. Data concerning absenteeism are collected in a questionnaire concerning employment and absence through illness . ProductIn the cost calculation one general cost price per lost hour of productivity is used for all patients. The number of days absent from work is related to the number of working days and working hours reported by the individual patient. Economic data are gathered three times during the one year follow-up period by the questionnaires, each time measuring the last two months prior to the questionnaires. These assessments took place at the same time as the effectiveness measures. The costs are based on these questionnaires and extrapolated to the costs during the full one year follow-up period.The handbook of Oostenbrink et al  is used Those cost prices of health care services not mentioned, being ergo therapy, psychology and care by occupational doctor are obtained from professional organizations. Prices of medication are obtained from the Dutch College of Health Insurance .Cost prices of devices are obtained from the reporting of patients. If the cost price of a relevant device is not reported by the patient, a suitable minimum, maximum and mean cost price is estimated. Cost price estimates of devices are obtained from an organization specialized in devices for ergonomic work places. For some of the cost items there was one mean cost price, for other items a mean cost price is calculated based on a minimum and maximum cost price. All cost prices are indexed to 2005 by using the price index numbers of the Dutch Central Bureau of Statistics  .In the cost-effectiveness analyses the VAS, self-perceived WRULD and DASH-DLV are related to the health care costs. The QALY is related to the societal costs including productivity costs, in a cost-utility analysis.The expected improvement in pain in the PE group was set at 60% and for the RP group at 40%, implying a minimal clinical relevant difference of 20%, correlating with 20 mm difference on the horizontal VAS-scale. These expected improvements in pain were based on past clinical experience in our department of rehabilitation of the Maastricht University Hospital . With anData were analyzed by a blinded statistician using SPSS 13.0 for Windows Data were checked for missing values and normality. Missing values have been replaced by mean imputation and by LOC-F (Last Observation Carried Forward) method. Each follow-up moment was analyzed separately and the analyses were carried out according to the intention to treat principle. Differences in baseline characteristics and baseline values of the outcome measures between PE and RP group were tested with an independent samples t-test (α = 0.05). In the event of significant differences between the two groups in baseline characteristics, adjustments were made in the statistical analyses .2-test has been used to analyze the answer on the question put dichotomously: "do you still experience non-specific WRULD complaints, yes or no".The primary effectiveness measure, the horizontal VAS according to Jensen, has been analyzed at each follow-up moment by a t-test. Scores on DASH and EQ-5D questionnaires have been dealt with in the same way. The χThe costs of both patient groups were compared by the bootstrapping method making use of confidence intervals in percentiles. By bootstrapping samples of the same size as the original data are drawn with replacement from the observed data . In our The economic evaluation concerns cost-effectiveness and cost-utility analyses. The incremental cost-effectiveness ratios (ICER) are calculated based on the measured costs and outcome parameters. Health care costs are related to the medical outcome parameters and societal costs, including productivity costs are related to the QALY. Bootstrapping is performed and the simulated ratios indicate the uncertainty of the ICERs of the observed data. The ICERs resulting from bootstrapping are plotted on a cost-effectiveness acceptability curve (CEAC) indicating the probability for a range of ceiling ratios (society's maximum willingness to pay for one unit of effectiveness) that the cost-effectiveness of the PE treatment is acceptable .Table 313 potential participants reacted or on the advertisements or were recruited by occupational physicians. Participants were selected and diagnosed between May 2003 and February 2005 and each participant had to complete a short questionnaire. The Saltsa-report  has beenFinally 28% of these potential participants (i.e. 88 participants) have been included in this study, meeting the inclusion criteria and willing to participate. Information about the routing of the participants through the trial is presented in Figure Many potential participants had to be excluded for more than one reason.Forty-four participants were randomized to each arm of the trial.Both groups were comparable at baseline for nearly all variables except the score on the Pain Catastrophizing Scale Table .At baseline all data were available of the 88 participants. Between baseline assessment and three months assessment there were six drop outs for various reasons, four of the PE group and two of the RP group. Besides three non-compliant participants in the PE group, there was one participant who followed therapy, but resigned immediately after the therapy. One of the two non-compliants from the RP group wanted the other intervention after three treatment sessions, being the reason for his drop out.After accounting for participants who stopped attending because they were free of complaints, compliance was 94% in the PE therapy group and 96% in the RP group .Table Table th and 97.5th percentile based on bootstrap replications.Table th and 97.5th percentile based on bootstrap replications.Table At baseline the health care costs and productivity costs are about the same. The costs for the patient and family and the societal costs have a rather small cost difference of €28 between the two patient groups .The PE group has higher treatment costs during the follow-up period compared to the RP group. Although other health care costs were lower in the PE group, the total health care costs indicate that the PE group has higher costs opposed to the RP group during the period after baseline. Concerning all other follow-up cost items the PE group is less costly than the RP group.During the intervention period the total health care costs of the PE group are higher . There were no differences between both groups in costs over 1-year follow-up . The health care costs mainly consist of the costs of the treatments for PE therapy or RP during the ten weeks intervention period. Concerning the other health care costs, the PE group is less costly than the RP group with €29 versus €125 at one year after baseline  and €6 versus €26 just after the intervention periodThere were no differences between both groups in costs at three months respectively 1-year follow-up .During the follow-up period only few patients (four patients in the RP group and two patients in the PE group) of both treatment groups had productivity loss due to their non-specific WRULD complaints. At one year after baseline the mean costs due to productivity loss were €317 in the PE group and €920 in the RP group . At three months incremental cost was -€106; 95% CI; -318, 0.The mean societal costs one year after baseline are €1797 for the RP group, which is about €622 more  than the mean societal costs of €1176 of the PE therapy group. The difference between the mean health care and the mean societal costs per patient of the RP group in relation to the PE group is mainly attributable to the higher productivity costs in the RP group.th and 97.5th percentile based on bootstrap replications.Because of a possible bias due to group differences at baseline  in combination with small differences at follow-up regarding VAS-scores, DASH and EQ5D, the  ICERs of the PE versus the RP treatment are calculated at three months and one year after baseline based on change scores of each group (see Table Negative ICERs should be interpreted with caution. These can indicate both dominance (higher effectiveness ànd lower costs) and inferiority (lower effectiveness ànd higher costs) of PE strategy over RP treatment; therefore we refer to Figures Using health care costs the ICER for the VAS pain intensity at three months after baseline is about €161 per unit of improvement. This means that an additional amount of €161 is needed to achieve an improvement of one point on the VAS scale through Mensendieck/Cesar therapy as opposed to regular physiotherapy.Concerning the three-month follow-up effectiveness measures self-perceived WRULD and DASH; the PE treatment is both more expensive and less effective as the RP. This indicates that the PE treatment is inferior opposed to the RP when evaluated from the self-perceived WRULD and DASH at a three-month follow-up period.At one-year follow-up, the PE treatment is both more expensive  and less effective regarding pain (VAS) and disability (DASH), indicating that the PE treatment is inferior opposed to the RP.The ICER for self-perceived WRULD one year after baseline is about €286, meaning that an additional €286 is needed to achieve one more complaint-free patient through PE therapy.The societal costs of the PE therapy group are lower compared to the RP group, while the treatment is more effective in terms of QALYs during the one-year period. Consequently the PE treatment is considered dominant from this societal perspective. The gain of an additional QALY through PE treatment implicates a cost saving of about €33.773. =Figure The sensitivity analysis with maximized costs resulted in mean health care costs of €704 and societal costs of €1876 per RP patient compared to €697 respectively €1191 per PE therapy patient. In the sensitivity analysis with minimum costs, the mean health care costs decreased to €671 and societal costs decreased to €1507 per RP patient compared to €692 respectively €1073 per PE therapy patient. These sensitivity analyses only lead to small changes in the cost differences between both patient groups. The shape of the CEACs of the sensitivity analyses is comparable to the CEACs of the baseline analyses.Little research has been done regarding cost-effectiveness in WRULD-patients.One cost-effectiveness study  has beenThis cost-effectiveness study, comprising WRULD-related health care costs including costs for the patient and family and productivity costs resulting in societal costs, is as a randomized controlled trial in computer screen-workers with early stages of non-specific WRULD the first of its kind. There was only a small rate of missing values in this research making the chance of bias low. The low percentage of dropout (less than 10%) was the reason to perform only an intention-to-treat analysis and no per-protocol analysis. Moreover this patient group concerns patients with early non-specific WRULD complaints of which is assumed that their dropout would not imply a significant impact on the results.The results failed to show PE therapy according to Mensendieck/Cesar is more effective in computer screen-workers with early non-specific WRULD with respect to the effectiveness outcome measures Table . In bothMoreover, Figure Non-specific WRULD is 'work-related . HoweverRegarding cost-effectiveness, mean total health care costs including costs for the patient and family did not differ significantly between both groups at one year follow-up. This despite the fact that during the intervention period when most health care costs were made and mainly consisted of the costs of the treatments, these costs were higher in the PE group having one and a half hours more therapy compared to the RP group. On the other hand, productivity costs after one year were lower in the PE group. Productivity costs were based on 28.33 (28'20") contract hours divided over five working days a week . HoweverThe mean societal costs one year after baseline were in favor of the PE therapy group, mainly attributable to the higher productivity costs in the RP group.From the health care costs perspective at three months follow-up only with respect to the VAS the PE strategy had a high probability of acceptable cost-effectiveness. At one year follow-up the PE strategy had only regarding the self-perceived WRULD a- not the 60% exceeding- probability of acceptable cost-effectiveness.Considering societal costs in relation to QALYs, the PE treatment had a probability of over 80% to be cost-effective over a wide range of cost-effectiveness ceiling ratios.However, differences were marginal, possibly because this study only concerned computer screen-workers with early stages of non-specific WRULD and already no significant differences between the groups were found in the effectiveness of both therapies themselves.As our preference concerns the societal perspective and the QALYs we tend to prescribe postural exercise therapy according to Mensendieck/Cesar for computer screen-workers with early stages of non-specific WRULD.In conclusion, although our trial failed to find significant differences in VAS, QALYs and ICERs based on VAS and QALYs at one-year follow-up, CEACs suggest that the postural exercise therapy according to Mensendieck/Cesar has a higher probability of being cost-effective compared to regular physiotherapy; however further research is required.WRULD: work-related upper limb disorders; VAS: visual analogous scale; DASH-DLV: disabilities arm, shoulder and hand - Dutch language version; EQ-5D: Euro-Qol - 5 dimensions; QALY: quality adjusted life year; PE: postural exercise therapy; RP: regular physiotherapy; JSS: Job Stress Survey; MPS-F: Multidimensional Perfectionism Scale of Frost; STAI: State-Trait Anxiety Inventory; PCS: Pain Catastrophizing Scale; GP: general practitioner; LOC-F: Last Observation Carried Forward; ICER: incremental cost-effectiveness ratio; CEAC: cost-effectiveness acceptability curve; CBS: Centraal Bureau van de Statistiek ; CI: confidence interval.The authors declare that they have no competing interests.MVEB: first writer of all head chapters, interpretation of data. SG: acquisition of all the data for cost-effectiveness study and analyzing data. RdB: daily supervisor giving comments, interpretation of data. JS: final analyses and check of the completed article, final approval.


Electron tomographic analysis can be combined with the simple and rapid negative staining technique used in electron microscopy based virus diagnosis.Standard negative staining of representative examples of parapoxviruses and paramyxoviruses was combined with electron tomographic analysis.Digital sectioning of reconstructions of these viruses at a selected height demonstrated the viral ultrastructure in detail, including the characteristic diagnostic features like the surface threads on C-particles of a parapoxvirus and individual glycoproteins and the internal nucleoprotein strand of Newcastle disease virus. For both viruses, deformation and flattening were observed.The combination of negative staining of complex viruses with electron tomographic analysis, allows visualizing and measuring artifacts typical for negative staining. This approach allows sharp visualisation of structures in a subnanometer-thick plane, avoiding blurring due to superposition which is inherent to TEM. In selected examples, such analyses can improve diagnosis of viral agents. Negative staining has been a useful specimen preparation technique for biological and medical electron microscopists for almost 50 years since its introduction by Brenner and Horne as an established method . The tecVirus diagnosis based on morphology is hindered by the limitations of both negative staining and transmission electron microscopy (TEM). Limitations of negative staining, reviewed in detail in , includeAt least part of the 'hidden' three-dimensional (3D) information can be retrieved from two-dimensional (2D) projections, like micrographs, by tomographic reconstruction. The mathematical basis for tomographic imaging was already described and formulated in 1917  in the sIn this report, we aimed to demonstrate that electron tomographic analysis of negatively stained complex viruses, like paramyxoviruses and parapoxviruses, not only allows visualizing and measuring artifacts like deformation and flattening, but also yields additional and relevant structural information. In selected examples, such analyses can improve diagnosis of viral agents.The seed stock of the Newcastle disease virus (NDV) LaSota strain was obtained from Dr. D.J. Alexander  and propagated in the allantoic cavity of 9- to 11-day old SPF embryonated chicken eggs as described . ParapoxA standard protocol for negative staining was applied to these virus suspensions. Pioloform- and carbon-coated 400 mesh copper grids (Agar Scientific) were pre-treated with 1% Alcian Blue  to increase hydrophilicity and rinsed 5-times with water. The grids were deposited on a drop of viral suspension for 10 minutes and rinsed two times with water. For NDV, the virus-coated grids were incubated in addition for 1 minute with a suspension of 10-nm gold particles  as fiducial markers for image alignment. Afterwards, the grids were stained for 10 seconds on a drop of 2% uranyl acetate (Agar Scientific) or 1% potassium phosphotungstate .Specimens were mounted in a dedicated holder  and analyzed using a Technai Spirit TEM  with a BioTwin lens configuration and a Lab6-filament operating at an acceleration voltage of 120 kV.Series of micrographs (tilt-series) were recorded semi-automatically assisted by the Explore 3D tomography-module of the microscope control software over a tilt range of ± 70°, or highest angle possible, at intervals of 1.5°. Shift and focus changes were corrected at every interval. Images were acquired with a 4*4 k Eagle CCD-camera (FEI) at a magnification 49,000 times and a corresponding pixel sizes of 0.22 nm. The tilt series were aligned using the Inspect 3D software, version 2.5 (FEI). Tilt series of Orf virus were aligned by rounds of cross correlation. Tilt series of NDV were aligned by combining alignment based on two rounds of cross correlation with feature tracking based on 6 or more gold beads. Reconstructions using iterations of the SIRT algorithm were superior to reconstructions based on weighted back projection. Visualization was done using the AMIRA software . The processing of a sample by this method takes about 12 hours.The density map of the F-glycoprotein of Newcastle disease virus, as obtained by crystallography (RCSB protein databank entry 1G5G ), was reNegatively stained virions of Orf virus, representing the parapox genus, are ellipsoid-shaped and measure approximately 250 by 150 nm. A typical diagnostic feature are the surface threads that, because both sides of the virus are seen in the microscope, give a criss-cross appearance Figure . DependiFigure The silhouette of these glycoproteins strongly resembles the silhouette of the 3D model obtained from the density map of the F-glycoprotein of NDV. In digital sections of the central part of the virions Figure , and parFigure The electron tomographic reconstructions of negatively stained virions of parapoxviruses and paramyxoviruses have revealed ultrastructural details that in the standard micrographs can not or hardly be distinguished. Digital sectioning of the reconstructions at a selected height allowed sharp visualisation of structures in a subnanometer-thick plane, avoiding blurring due to superposition which is inherent to TEM.For Orf parapoxvirus the characteristic surface threads were clearly demonstrated in C-type particles where only surface undulation suggests the presence of threads seen in profile. Their semi-parallel orientation supports the suggestion of Madely and Field  that theFor NDV, individual glycoproteins were shown both on the periphery of virions and on the lower an upper parts of the viral envelope. Details of these glycoproteins like the cup-shaped head and a 2-nm-wide stalk were shown, and their sizes and silhouettes corresponded seamlessly with the sizes and silhouettes of the crystallography-based model of the NDV fusion protein . AlthougBecause of their pleiomorphic appearance and densely packed fringe, it can be difficult to distinguish paramyxoviruses from mycoplasms  and/or mThe rapid and simple negative staining procedure used here and in most diagnostic laboratories lacks the near-nature-state fixation of cryoelectron microscopy and results in structural perturbations. Electron tomographic reconstructions of both negatively paramyxoviruses and parapoxviruses demonstrated extensive flattening, while the fragmentation of the ribonucleoprotein strand of the paramyxovirus most likely results from shearing forces during drying. This approach can thus be used efficiently to objectify the artefacts induced by negative staining, reviewed in , which mDespite these shortcomings, the presented examples illustrate that the simple negative staining procedure shares certain advantages with cryonegative staining over unstained vitreous specimens. The combination of enhanced contrast and samples being less susceptible to radiation damage ,15 allowIn conclusion, the combination of negative staining of complex viruses with electron tomographic analysis, allows visualizing and measuring artifacts typical for negative staining. This approach allows sharp visualisation of structures in a subnanometer-thick plane, avoiding blurring due to superposition which is inherent to TEM. In selected examples, such analyses can improve diagnosis of viral agents.The authors declare that they have no competing interests.Both authors have contributed equally to the preparation of the manuscript. Both authors read and approved the final manuscript.Tomogram of a parapoxvirus. The video shows a negatively stained virion of the Orf parapoxvirus, a section of which is shown in Figure Click here for fileSurface rendered view of a parapoxvirus. The video shows a surface rendered view of the parapoxvirus particle seen in additional file Click here for fileFalse-colored 3D-rendering of a C-particle of a parapoxvirus. This figure shows snapshots of a false-colored 3D-rendering of the C-particles shown along the tomographic Y-axis (A) and Z-axis (B) se-colored rendering surface rendered view of the parapoxvirus particle seen in additional file Click here for fileTomogram of Newcastle disease virus. The video shows a negatively stained virion of the Newcastle disease LaSota strain, a section of which is shown in Figure Click here for file


Warburg Effect (increased aerobic glycolysis in cancer cells) is the basic principle underlying the value of FDG-PET imaging in neoplasms.[The high diagnostic accuracy of FDG-PET and PET/CT has been well documented for several neoplasms, with a consequent rapid increase in the utilization and acceptance of PET imaging in the clinical setting. As reported earlier, initial eoplasms. FDG-PET This article reviews the current clinical applications of FDG-PET and PET/CT in certain malignancies, specifically head and neck cancer, lung cancer, lymphoma and melanoma. Thyroid neoplasms are included in the head and neck cancer section and pleural malignancies are discussed in the lung cancer section.Anatomic imaging modalities like CT scan and MRI have superb structural resolution that is continuously improving with technological innovations. However, these imaging modalities rely significantly  on size and shape of lesions, in addition to indirect findings such as edema, to differentiate benign from malignant disease. Therefore, disease manifestations that occur at the molecular and cellular level, before manifesting as structural changes, are frequently not detected with these modalities. FDG-PET has the capability to depict abnormal metabolic activity before any anatomic change occurs. This is particularly important in patients who undergo therapeutic interventions such as radiotherapy and chemotherapy, after which structural changes often lag behind metabolic changes, for a significant interval of time. In fact, some lesions may never shrink to normal size or disappear completely in spite of adequate treatment. Functional or metabolic imaging with FDG-PET complements structural imaging and helps overcome some of these major shortcomings. As discussed in the previous article in this series, FDG accuMalignancies involving the head and neck region have generally been declining in incidence, but about 68,000 new cases are still estimated to occur in the US in 2007. These arThe majority of head and neck cancers tend to be squamous cell carcinomas (HNSCC) that show intense FDG uptake. FDG-PET in situ) are not readily visualized on FDG-PET imaging.[The diagnosis of head and neck cancer is usually established on the basis of directed biopsies during an endoscopic evaluation . Head and neck cancers are staged as per the Tumor, Node and Metastasis (TNM) staging criteria. Along with clinical evaluation with direct visualization, contrast-enhanced CT and MRI are extremely useful for optimal assessment of the ‘T’ stage as they provide further structural information as regards tumor extension and involvement of adjacent structures. FDG-PET imaging lacks the spatial resolution for providing such exquisite structural details. Moreover, small lesions with low tumor volumes, superficial lesions (<4 mm deep) and very low grade tumors  and normal-sized nodes may frequently be involved with metastatic disease, leading to inaccurate upstaging or downstaging of the disease with conventional imaging methods. FDG-PET  imaging has had a tremendous impact in improving the nodal staging of head and neck cancers. Numerous studies have documented the high sensitivity (87%), specificity (94-95%) and accuracy of FDG-PET imaging in assessing nodal involvement in head and neck cancers.–10 FDG-PIn addition to improved nodal staging, the most important added value of FDG-PET imaging is in the detection of unsuspected distant metastases  that canDue to its high sensitivity and the ability to perform whole-body scanning, FDG-PET is used in the evaluation of patients presenting with malignant cervical lymphadenopathy without evidence of a primary neoplastic lesion on conventional investigations, including CT scan / MRI and panendoscopy with directed biopsies. In such cases, FDG-PET and PET/CT can help identify the primary malignant lesion  in about13FDG-PET imaging is useful in therapy planning for patients undergoing radiation therapy with a curative or palliative intent or as neoadjuvant therapy. The increasing implementation of intensity-modulated radiation therapy (IMRT) is well complemented by the additional functional/metabolic information provided by the FDG imaging as it allows the delivery of maximal radiation dose to the most metabolically active areas of the tumor and more complete inclusion of loco-regional disease with sparing of the uninvolved areas. FDG-PET is very helpful in evaluating the cervical nodal chains in patients with advanced-stage head and neck cancer, since the presence of contralateral cervical lymphadenopathy will necessitate the inclusion of both sides of the neck in the radiation therapy field.In addition to the above, FDG-PET imaging is very useful in assessing response to therapy and in restaging of head and neck cancers.18 FollowThere are several limitations of FDG-PET imaging in the evaluation of head and neck cancer. Although it may detect tumors that may be missed by anatomic imaging , the suboptimal spatial resolution as compared to direct visualization with endoscopy and CT scan / MRI limits the evaluation of small and superficial lesions. Also, low-grade tumors may be missed on PET if there is significant intense physiologic FDG uptake in an adjacent structure. As described in the earlier article in this series, a numberApproximately 33,500 new thyroid cancer cases are estimated in the US in 2007, with about 1,500 deaths. Thyroid max: 125.1, SUVavg: 66.2) in the literature has been in an anaplastic thyroid cancer.[Generally, thyroid neoplasms are diagnosed after workup for a painless thyroid nodule/ mass. Most malignant thyroid nodules show increased FDG uptake. Usually,d cancer. Hurthle d cancer. False ned cancer.24 Sometid cancer., but stiExcept for anaplastic carcinomas, which are very aggressive rapidly progressing tumors, follicular thyroid neoplasms are treated with thyroidectomy followed by radio-iodine therapy with I-131. Follow-up usually consists of physical examination, serum thyroglobulin (Sr. Tg) levels and I-131 or I-123 whole-body scans to detect recurrence. Whole-body iodine imaging may be negative in 10-15% of patients with elevated Tg levels and clinical suspicion for disease recurrence, which can be a result of small volume disease or tumor dedifferentiation. In this MTCs behave differently from follicular thyroid carcinomas. Instead of secreting thyroid hormones, they secrete calcitonin as well as carcinoembryonic antigen (CEA). Primary treatment is surgical resection in the form of thyroidectomy. Radio-iodine therapy or radio-iodine scanning for detection of recurrence is of no use in these patients. MTCs are followed by assessing serum calcitonin and CEA levels. In the setting of elevated serum calcitonin and/ or CEA levels, FDG-PET imaging is probably the most accurate modality to detect recurrent disease with a sensitivity and specificity of around 80% (sensitivity ranging from 40-95% in various studies).–32 A recIn the United States, lung cancer remains the leading cause of cancer mortality for both men and women. Broadly,A solitary pulmonary nodule (SPN) is a frequent indication for a PET scan. Although many incidentally detected SPNs tend to be benign, in a high-risk patient , they are more likely to be malignant. As many as 20-30% of lung neoplasms initially present as SPNs and about 10-30% of all malignant SPNs are metastases from extrathoracic neoplasms . In viewIn general, any SPN with intense FDG uptake is considered to be of neoplastic etiology unless proved otherwise . AlthougSeveral studies have shown FDG-PET to be highly accurate in differentiating benign from malignant pulmonary lesions, with a sensitivity of greater than 95% and a specificity of 75-80%. The sensCertain PET imaging centers practice dual-time point imaging (at 1 and 1.5-2 h) to improve the specificity for malignant pulmonary lesions.–39 The hAn important caveat relates to the incidental detection of a focal intense lesion in the lung on FDG-PET imaging without a corresponding CT correlate . Since tFDG-PET imaging is an important component of the TNM staging workup of NSCLC. Anatomical imaging modalities such as CT scan or MRI are superior to FDG-PET and PET/CT  for evaluation of the ‘T’ stage. However, FDG-PET imaging is superior to CT imaging for ‘N’ and ‘M’ staging. FDG-PET has proven to be more accurate in assessing nodal involvement and it can accurately identify metastatic disease in normal-sized nodes on CT. It can also better differentiate lymph node metastases from reactive, but enlarged nodes as seen on CT. The sensitivity and specificity of FDG-PET imaging in N3 disease  is greater than 90% and its overall accuracy for assessing mediastinal nodal involvement is around 85%. However, caution is recommended in N0 disease per PET since micrometastases cannot be detected by FDG-PET imaging and invasive staging of intra-thoracic lymph nodes may yield positive nodal disease. The specFDG-PET imaging can also detect functional impairment of other structures due to lung carcinoma. Bulky metastatic mediastinal adenopathy can frequently compress/ involve the recurrent laryngeal and phrenic nerves. Elevation of the ipsilateral hemi-diaphragm or lack of FDG activity in the ipsilateral vocal cord with physiologic or compensatory increased FDG activity in the contralateral vocal cord indicates ipsilateral phrenic nerve or recurrent laryngeal nerve impairment respectively .FDG-PET imaging is presently considered the most accurate noninvasive means of staging a biopsy-proven NSCLC (excluding brain metastases). In addition to its high accuracy, FDG-PET imaging can also help make the diagnostic and staging workup of NSCLC more efficient and cost-effective. FDG-PET imaging can help to guide diagnostic biopsies and improve the yield from tissue sampling. We frequently see lung masses with intense FDG uptake but with a bronchoscopic workup negative for malignancy. These lesions are subsequently proven to be malignant on additional biopsies in a majority of such cases. Even CT-guided biopsies of lung masses may be falsely negative if a necrotic area is targeted during biopsy. FDG avidity of the lung lesion also provides prognostic information. Since FDG uptake correlates with tumor growth rate, doubling time and proliferation, intensely FDG-avid lesions tend to be more aggressive with poorer prognosis. SUV is considered an independent prognostic factor and tumors with SUV >10 have a median survival of less than a year.In patients undergoing curative, palliative or neoadjuvant radiation therapy, FDG-PET also helps in radiation therapy planning. Incorporation of the PET data in the process of determining the radiation therapy ports allows more accurate tumor localization, leading to tighter contouring and smaller biological volume calculation and sparing of uninvolved lung parenchyma. This mayFDG-PET imaging is also playing an increasing role in restaging lung cancer patients to assess response to therapy  and to detect recurrence. It is superior to CT scan for differentiating post-therapy changes  from residual or recurrent malignant disease  and the SCLC also shows intense FDG uptake. However, FDG-PET imaging is currently not approved by CMS for the workup of SCLC because it is frequently metastatic at the time of diagnosis and the addition of FDG-PET imaging is not felt to have a clinical impact on patient management and outcome. SCLC is generally staged as limited- and extensive-stage disease; and potentially, a small number of limited-stage SCLC patients  may be curable with aggressive surgical resection followed by chemotherapy. The major consideration in staging SCLC is to determine the need to add radiation with chemotherapy since limited-stage patients are considered for combined chemoradiation therapy with curative intent. FDG-PET has a potential role in SCLC to confirm limited-stage disease or upstage these patients to extensive-stage disease. However, this is all still under investigation.FDG-PET imaging is also useful in evaluating pleural malignancies like mesothelioma , which tvs. 2-3 days or more), higher sensitivity , better quantification potential (and hence better prognostic information) and superior assessment of response to therapy.[Lymphoma is the fifth most common cancer in the US and is one of the few malignancies whose incidence is rising. Lymphoma therapy.–51 FDG-Pperse  but rather images the intracellular accumulation of FDG-6-phosphate , the pattern of FDG-avid lesions may suggest a diagnosis of lymphoma when the scan is being performed for some other reason [Tissue diagnosis is the norm in lymphoma workup. Although FDG-PET does not image lymphoma cells r reason . In addir reason . It is ir reason 53 includr reason  a signifr reason 53ET alone for staging lymphoma and has been shown to be cost-effective and accurate when the initial scans have guided further conventional imaging.[The American Joint Committee on Cancer (AJCC) has based the staging of lymphoma on the Ann Arbor classification that considers the number of nodal regions involved, the extent of involvement and extranodal involvement along with modifiers according to the presence or absence of ‘B’ symptoms. FDG-PET imaging is crucial for staging lymphomas  and is c58 imaging.The intensity of FDG activity generally correlates with the grade of lymphoma on histology and provides prognostic information that may impact management decisions. AggressiGa-67 scintigraphy and CT scan have been the mainstay for assessing response to therapy in lymphoma patients; and the Chesson classification, based mainly on size criteria for scanning, has been in vogue since 1999. Based onFDG-PET imaging can correctly assess response to therapy as early as 7 days after the first chemotherapy cycle. Recent oncology practice management guidelines recommend an FDG-PET scan after two cycles of chemotherapy (around 3 weeks after the last treatment or just before the next cycle) to assess response and to then proceed with further therapy based on the results of this scan69 Figur. It is iFDG-PET imaging cannot assess microscopic disease and remission  is not equal to tumor eradication or cure. However,71FDG-PET also has proven value in assessing disease recurrence and residual disease after therapy. A mass m74Thus, FDG-PET imaging plays a crucial role in the workup and management of lymphoma. However, it does have certain limitations. Smaller Melanoma is the most serious of skin cancers and is much more frequent in white-skinned patients. In 2007, an estimated 60,000 new cases are expected in the US, with about 8,000 deaths. Diagnosi3).[Melanoma is considered one of the most FDG-avid tumors. Imaging 813). Moreover3). FrequentFDG-PET has a more established role in assessing response to therapy   and in dA key issue in scanning melanoma patients with FDG-PET is whether or not to perform a head-to-toes scan . Although no prospective studies have evaluated the value of including the brain and lower extremities in all patients, our current policy is to include the brain and lower extremities only if they are or have been previously, involved . In the absence of present or past lesions on the lower extremities, we also include them in the imaging field if the patient has proven or suspected disease in the inguino-femoral nodes. Literature review suggests that in the absence of the above factors, the likelihood of finding lesions in the lower extremities is very low (less than 1%) to warrant routine inclusion of these areas in imaging protocols.


As there is no optimal treatment of non small cell lung cancer due to its resistance to common chemotherapeutics, we investigated the effect of human placenta-conditioned medium on tumor tissue. The human placenta constitutes a mixture of maternal and fetal origin and displays a variety of immunomodulatory aspects.Freshly resected non small cell lung cancer tissues were incubated with placenta-conditioned medium in a short-term tissue culture model and A549 cells were challenged, respectively. Term placenta was used for producing conditioned medium and HOPE-fixed stimulated tumor tissue was analyzed for expression of caspase-3 and Ki67 via immunohistochemistry. The effects of conditioned medium on squamous cell carcinoma were further compared to physiological concentrations of Carboplat/Gemzar.Conditioned medium caused in 2 of 3 cases elevated expression of caspase-3 and reduced expression of Ki67 in 3 out of 3 cases, while the chemotherapeutic agents caused no comparable expression of caspase-3 or reduction of Ki67. In cell culture up to 50% of karyopyknosis was investigated and even sterile-filtrated medium caused widespread reduction of Ki67 on protein level.Human placenta releases substances that mediate apoptosis and reduce proliferation in tumor tissue and cell culture. As even sterile-filtrated medium caused the mentioned effects we hypothesize one or more soluble mediators. The detailed way of promoting apoptosis and nature of these mediators need to be elucidated in further studies. Although new treatment regimens are currently being developed, Non small cell lung cancer (NSCLC) still remains a fatal diagnosis and the success in its treatment up to date is not satisfying. Due to the poor response to common chemotherapeutics there are multifaceted endeavors to resolve these obstacles. This study focused on the effects of placenta-conditioned medium on human lung carcinoma.de novo vascularization, produce their own growth factors and invade adjacent tissue by the use of proteases like cathepsins or matrix-metalloproteinases (MMPs), one can speak of a transient pseudo-tumorigenesis [The human placenta as a unique structure of maternal and fetal origin has become a focus of interest in recent publications. The semi allograft fetus is a challenge for the maternal immune system and the exact way of avoiding its rejection is up to date not completely known. The implanting blastocyst adheres to the uterine wall and soon thereafter cytotrophoblast cells invade the endometrium to establish in cooperation with the maternal tissue a suitable habitat for the growing fetus. The processes after implantation resemble in many aspects some traits of neoplastic cells. As these cells have to gain access to the bloodstream via igenesis . As a stigenesis . A remarigenesis  and overigenesis .3, transferred into 6 ml RPMI-1640  and incubated for not more than 4 h at 37°C. For sterile filtration of media a Rotilabo®-syringe-filter  with 0.22 μm cut-off size was used. Filtrated and unfiltrated media were used for tissue culture and HOPE-fixation according to Lang et al. [As evidence of a possible effect of gene transcription and transformed cell growth suppression by placental extract is not new -7 we exag et al.  with freg et al. . The priImmunohistochemistry staining detected an elevated expression of caspase-3 (N = 2) and reduced expression of Ki67 (N = 3) after incubation of 16 h with conditioned medium : Non small cell lung cancer; (MMPs): Matrix-metalloproteinases; (PlGF): Placental growth factor; (STST): Short term stimulation of tissues; (HOPE): Hepes Glutamic Acid Buffer Mediated Organic Solvent Protection Effect; (TNF): Tumor Necrosis Factor.The authors declare that they have no competing interests.TG conceived of the study. SM conducted the experimental work, analyzed the sections and drafted the document with TG. TZ, HS and EV were involved in surgical and pathological parts, respectively. DK and MA took part in immunohistochemistry. HPH and PZ were involved in cell culture and pneumological aspects. All authors have read and approved the present manuscript.


Background: Although cytomegalovirus (CMV) is an uncommon cause of viral hepatitis during pregnancy, a definitive diagnosis is important because of the potential for congenital CMV. In the case reported here, a diagnosis of hepatitis caused by CMV was made after the more common viral pathogens had been ruled out.    Case: A 17-year-old, 12-week pregnant patient was evaluated for fever and right upper quadrant tenderness. A serologic evaluation revealed elevated liver function levels and a positive maternal serology for CMV IgM. A diagnosis of hepatitis caused by CMV was made after the more common viral pathogens and drug-induced hepatitis had been ruled out. She was counseled about the potential effects of CMV on her fetus.Conclusion: A step-wise approach to the diagnosis of viral hepatitis during pregnancy is needed to determine the etiology because a potential teratogenic virus may be involved.


The advances in modern radiation therapy with techniques such as intensity-modulated radiation therapy and image-guided radiation therapy (IMRT and IGRT) have been limited almost exclusively to linear accelerators. Investigations of modern Cobalt-60 (Co-60) radiation delivery in the context of IMRT and IGRT have been very sparse, and have been limited mainly to computer-modeling and treatment-planning exercises. In this paper, we report on the results of experiments using a tomotherapy benchtop apparatus attached to a conventional Co-60 unit. We show that conformal dose delivery is possible and also that Co-60 can be used as the radiation source in megavoltage computed tomography imaging. These results complement our modeling studies of Co-60 tomotherapy and provide a strong motivation for continuing development of modern Cobalt-60 treatment devices. Modern radiation therapy is moving towards advanced conformal techniques such as intensity-modulated radiation therapy (IMRT) in conjunction with image guidance, bringing about image-guided radiation therapy (IGRT), to ensure accurate patient treatment. The clinical application of these advanced techniques has been limited almost exclusively to linear accelerators. Investigations of conformal Cobalt-60 (Co-60) radiation delivery have been sparse, in part because of preconceived notions that the radiation beams from Co-60 do not have the properties required in modern radiation treatment. There ha6This report will review the results of experimental investigations of the potential of Co-60–based IGRT via tomotherapy — a rotational implement-ation of IMRT.5 Measure52 Co-60 pencil beam. Intensity-modulated radiation therapy is performed in a single slice by varying the velocity of the phantom as it moves through the pencil beam. By this simple approach, we are well able to imitate the beam delivery from a NOMOS MIMiC multileaf collimator . It is possible to extend this delivery to 3D by adding a translate capability to the height of the table. Quantitative 3D delivery has been achieved in our laboratory, but the quantitative work reported here will be limited to two dimensions only. The test irradiations were performed on a cylindrical phantom containing GafChromic film . Treatment planning was performed using an in-house developed inverse treatment planning system[All irradiations were performed on a Co-60 T780C unit  modified by the addition of a purpose-built computer-controlled rotate translate benchtop apparatus, as shown in ng system10 that ung system  and second-generation (using a fan beam and scanning diode) prototype Co-60 tomotherapy imaging systems. Recently our imaging studies have advanced with the development of a third-generation imaging system using a Varian PortalVision LC250 scanning liquid ionization chamber (SLIC) electronic portal imaging device (EPID) , shown in The findings to date confirm the viability of Co-60–based tomotherapy for conformal dose delivery. The ring in Various treatment plans of similar complexity have been achieved and validated with the GafChromic film measurements. These inImaging studies have been performed on a number of anthropomorphic phantoms using both a first-generation approach and a third-generation approach using the Varian PortalVision LC250 EPID imager . The reset al. incorporating MRI[In this paper, we demonstrate that Cobalt-60–based tomotherapy approach is capable of both highly conformal intensity-modulated dose deliveries and viable image guidance. While it may seem that the addition of more complex beam collimation and megavoltage imaging to cobalt may complicate a simple technology, and perhaps make the device too complicated or expensive for worldwide distribution, we believe that it is important to establish full modern radiation therapy with a cobalt unit. It is quite clear that in the coming decade, the standard of care for radiation therapy will require image guidance and that radiation units incapable of imaging at the time of delivery will no longer be used in clinics. The development of tomotherapy type approaches with Co-60 also indicates that the potential for use of more sophisticated machines as proposed by Kron ating MRI may alsoOur experiments have confirmed the results of our previous modeling studies that Cobalt-60–based tomotherapy is clinically viable. Two-dimensional conformal dose delivery in a single slice with Co-60 has been validated experimentally using radiochromic film dosimetry. We have successfully developed Co-60 MVCT imaging techniques which provide adequate image quality required for IGRT. We believe that these results clearly indicate that undertaking the task of further development and implementation of modern radiation techniques based on Co-60 units is fully warranted.