This finding indicates very strong selection during bursal lymphomagenesis. Therefore, in addition to providing a platform for gene copy number change, palindromes may alter microRNA genes in a fashion that can contribute to cancer development.Genetic instability plays a key role in the formation of naturally occurring cancer. The formation of long DNA palindromes is a rate-limiting step in gene amplification, a common form of tumor-associated genetic instability. Genome-wide analysis of palindrome formation (GAPF) has detected both extensive palindrome formation and gene amplification, beginning early in tumorigenesis, in an experimental Myc-induced model tumor system in the chicken bursa of Fabricius. We determined that GAPF-detected palindromes are abundant and distributed nonrandomly throughout the genome of bursal lymphoma cells, frequently at preexisting short inverted repeats. By combining GAPF with chromatin immunoprecipitation (ChIP), we found a significant association between occupancy of gene-proximal Myc binding sites and the formation of palindromes. Numbers of palindromic loci correlate with increases in both levels of Myc over-expression and ChIP-detected occupancy of Myc binding sites in bursal cells. However, clonal analysis of chick DF-1 fibroblasts suggests that palindrome formation is a stochastic process occurring in individual cells at a small number of loci relative to much larger numbers of susceptible loci in the cell population and that the induction of palindromes is not involved in Myc-induced acute fibroblast transformation. GAPF-detected palindromes at the highly oncogenic  bic/miR-155, beginning at an early stage of tumor development, and without significant further amplification of the locus. Thus, DNA palindromes themselves may directly influence tumorigenesis.Genetic instability is a key process in the development of naturally occurring cancer. Gene amplification is one important consequence of underlying oncogenic instability. Long DNA palindrome formation is a rate-limiting early step in gene amplification. The development of a new functional genomic tool called genome-wide analysis of palindrome formation (GAPF) led to the recent discovery of the widespread occurrence of such palindromes in both animal tumor models and human tumor cells. Using a Myc oncogene-induced lymphoma model system, this paper describes clustering of tumor-specific palindromes throughout the genome as well as an association of sites of palindrome formation with both preexisting short inverted DNA repeat sequences and occupied Myc DNA-binding sites. We discovered consistent palindrome formation at a cancer-associated microRNA gene called  By combining GAPF with chromatin immune precipitation (ChIP) we discovered an association between palindrome formation and the occupancy of nearby Myc binding sites in chromatin, and, finally, report a strong selection for palindrome formation at an oncogenic micro RNA gene, Conditions used for palindrome detection favor rapid formation of duplex fold back structure in denatured DNA followed by nuclease digestion of residual single strands, as described In Tetrahymena, yeast and mammalian cells short inverted repeats (IRs), adjacent to sites of double strand DNA cleavage, mediate long palindrome formation and subsequent gene amplification −5. A majority of these sites contained multiple IRs with a median loop size of 9 nucleotides, arm lengths between 10 and 20 nucleotides and a range of overall length between 22 and 45 nucleotides. These findings represent a minimum correspondence between long palindromes and preexisting short IRs because IR loop sizes could easily be larger, and we don't have complete sequences for cDNAs on the array.As summarized in In order to test for a relationship between Myc binding and palindrome formation we employed a chromatin immune precipitation assay for genome-wide analysis of Myc binding sites , and combined this approach with GAPF. We began by analyzing chromatin from DT40 bursal lymphoma cells, which express high levels of cMyc and then examined v-Rel transformed chicken B cell lines in which levels of Myc expression varied >50 fold. The results of the ChIP on chipAnalysis of DT40 bursal lymphoma cells is detailed in These results suggest a quantitative relationship between the level of deregulated Myc overexpression and palindrome formation. Transformation by vRel in the presence of normally low levels of cMyc in bursal cells produced only minimal increases in palindromes suggesting that neither B-cell transformation itself, nor all types of transcription factor activity itself, could induce large numbers of palindromes. Finally, DT40 cells are unusual among vertebrate cell lines in their exceptional capacity for homologous recombination in-vivo before establishment and those developing, perhaps quite slowly, after establishment in culture.Cultured primary chick embryo fibroblasts express low, barely detectable levels of c-myc RNA and protein. DF-1 is an immortalized morphologically normal chick embryo fibroblast cell line that can be transformed acutely by the oncogenes of a number of avian retroviruses We then rapidly transformed DF-1 cells by high multiplicity infection with a Myc-transducing retrovirus, HB1 Bic is a non-coding RNA gene discovered as a site of ALV insertional mutagenesis (in addition to activating insertions at c-myc ) selected during tumor development in a large proportion of ALV-induced bursal lymphomas bic was found to encode a highly conserved micro RNA, miR-155, shown to be essential for normal B-cell development bic overexpression was shown to accelerate the development of ALV induced bursal lymphomas bic was not activated by retroviral insertion in all ALV-induced bursal lymphomas bic RNA bic. In GAPF studies from all of our analyses of preneoplastic TF (three studies), bursal lymphomas (three experiments ) and multiple studies of derivative high Myc expressing transformed B- cell lines such as DT 40 or TF 26  indicates a strong selection in the development of these tumors for this otherwise uncommon change at the bic locus, and indicates a mechanistic alternative to retroviral insertional mutagenesis for alteration of bic/miR-155.or TF 26 . In contbic locus, and gain an initial insight into the structure of palindromes formed near bic, we carried out blot-hybridization studies of DNA from normal bursa, DT40 cells and bursal lymphoma from an in-vivo tumor before and after the snap-back procedure and digestion with S1 nuclease. bic exon 1 and a segment of exon 2 in DT40 and bursal tumor DNA, but not in normal chicken DNA, retains an S1 resistant duplex character after the snapback procedure, consistent with the GAPF results. It is difficult to assess the significance of the relatively weaker blot-hybridization signal of the S1 resistant bic DNA recovered from tumor cells, in comparison to that from the pre-snap back normal control DNA. Influences on signal strength arising from the amplification procedure, S1 digestion of one normal bic allele, and/or structural heterogeneity at the bic locus in DNA of the tumor cell population may all play a role in diminishing the signal. In any case we conclude that the S1 resistant segments detected are included in a complementary paired region of a palindrome at the bic locus of DT40 and bursal tumor cells that is not present in normal chicken DNA.In order to validate the GAPF results for the We concluded that the deregulated overexpression of Myc oncoproteins in the bursa results in the widespread accumulation of long DNA palindromes not normally present in chicken bursal B-cells. This response did not extend to constitutive overexpression of another oncogenic transcription factor, vRel, nor was it a consistent general property of the transformed state of B-cells or fibroblasts. Analysis of the genomic distribution of these structures with respect to clustering, the presence of pre-existing IRs and occupied high affinity Myc binding site indicated the existence of preferred sites for palindrome formation. However, the sub genomic target size for induction of palindromes still appears to be quite large in comparison to the numbers of palindromic sites found in individual tumors. As with our previous study of gene amplification in this system The GAPF studies of clones of untransformed DF-1 fibroblasts, in comparison with studies of parental mass cultures, suggests some important caveats in drawing conclusions about DNA palindromes in clonal neoplasms. The known genotoxic effects of long palindromes may dictate the instability of newly formed DNA palindromes in both germ line and normal somatic cells bic/microiRNA-155 locus during bursal lymphomagenesis. Why bursal target cells apparently differ from DF-1 fibroblasts with respect to palindrome formation and transformation in response to Myc is unknown. These bursal cells are exquisitely sensitive to radiation and other genotoxic agents The results of the effects of Myc overexpression in this set of experiments, however, are not explained simply by the clonal nature of tumor cells. For example low Myc expressing TF 32 is a clonal cell line derived from normal bursal cells with many fewer palindromes than the clonal TF 26 line derived from Myc-transformed TF cells. Furthermore non-clonal Myc-induced preneoplastic transformed bursal follicle populations show marked increases in palindrome formation over normal bursal DNAbic was not accompanied by gene amplification that was detectable by array CGH in our experiments bic RNA during tumor development. We have carried out PCR-based assays  of miR-155 in whole normal embryonic and post hatching bursa as well as from preneoplastic TF and bursal lymphomas(not shown). As might be anticipated from the complex regulatory role of this micro RNA in vertebrate B-cell development It would be desirable to know the molecular consequences of this change. Palindrome formation at There are a number of known mechanisms for Myc-induced genomic instability that could contribute to the formation of palindromes that we have detected in these studies. In principle any process that produces DNA double strand breaks could initiate the formation of long palindromes, for example at short IRs. As mentioned, cytogenetic abnormalities associated with Myc overexpression, which are known to result from such breaks, have been described in mammalian cell lines myc transducing retroviral vectors to generate preneoplastic transformed bursal follicles (TF), and metastatic bursal lymphomas, has been described previously The bursal transplantation model, employing defective c-2 transformed as before.These experiments employed a 13K chicken cDNA microarray cis-wise, self-complementary) sequences having arms of specified minimum length that are connected by loops of specified maximum length. Individual chromosomal maps were searched directly using an R-script Naturally-occurring DNA palindromes in the Gallus gallus genome were detected by scanning the UCSC chicken genome sequence database, May 2006, version 2.1 draft assembly CTGAGAAGTGAAGCTGCAGTCCTG, reverse: GATTACTCACCCG AGGTATGGCTC; for bic they were, forward: GCTGAGGTGCTCCAGTGGCAG, reverse: CTAGTCTTCTCTTTGTTGCAGGTC. PCR limit products were evaluated by electrophoresis on 3% agarose gels.These studies followed, essentially, the procedure of Ren et. al. bic cDNA probe used on the microarray.Ten µg of input normal chicken DNA was digested with restriction endonucleases pst1, cla 1+apa 1 or cla 1+bgl 1. One µg of normal and DT40 DNA was subjected to the same procedure as for the GAPF studies including rapid denaturation - renaturation, digestion with S1 nuclease and amplification. Ten µg of amplified snap-back normal and DT40 DNA was then digested with the same sets of restriction endonucleases. Blot-hybridization was carried out on 1% agarose gels and evaluated with the same Figure S1Frequency of IRs in the chicken genome with a loop length up to 25 nucleotides.(0.38 MB TIF)Click here for additional data file.Figure S2Clonal analysis of DNA palindromes in DF-1 fibroblasts before and after Myc transformation. Total numbers of palindromes detected by GAPF in immortal DF-1 fibroblasts in mass culture and multiple fresh clones before and two weeks after transformation by infection with HB1. The bars represent standard errors of the mean.(0.18 MB TIF)Click here for additional data file.Protocol S1Supplemental programming information. Code for R script for genomic scans of user-defined palindromes.(0.04 MB DOC)Click here for additional data file.


The association of paraneoplastic hypoglycemia [Doege-Potter syndrome] and finger clubbing [Pierre-Marie-Bamberg syndrome] with pleural solitary fibrous tumour is rare. We present a previously unpublished but typical example of this rare occurrence together with a detailed updated literature review of previously published cases of pleural SFT discussing the histopathology of SFT; pathophysiology of the hypoglycemia and finger clubbing; treatment and outcome of pleural SFT. The patient, a 57-year-old African male was admitted at our hospital with recurrent episodes of hypoglycemia. He was found to have digital clubbing and decreased breath sounds in the right lower chest but no other significant clinical findings. His insulin level measured during an episode of hypoglycemia was undetectable. Chest radiograph and CT-scan revealed a lobulated mass in the right chest which was diagnosed to be SFT on histology. Surgical excision of the mass resulted in cure of the hypoglycemic episodes and rapid regression of the clubbing. Less than 65 cases of pleural SFT manifesting with hypoglycemia with or without finger-clubbing have been published in the English literature. The mean diameter of these tumours manifesting with hypoglycemia is 20 cm, 54% being benign while 42% were malignant. They predominantly present in the 6th-8th decade, average age of 64 years and a slight male preponderance at 58%. Complete surgical resection remains the most important predictor of clinical outcome in terms of recurrence and metastases, while providing instant cure for the hypoglycemia and rapid resolution of the finger clubbing. The occurrence of hypoglycemia with an intrathoracic tumour was first reported by Doege and Potter independently in 1930 hence the eponym Doege-Potter syndrome [DPS] . HyperthA 57-year-old male was referred to the Chris Hani Baragwanath Hospital with recurrent episodes of symptoms of severe hypoglycemia including syncope. He had a 20 pack-year history of smoking. He was found on examination to have marked finger-clubbing and decreased breath sounds in the right lower chest, but no other significant clinical findings. Sputum cytology was negative for malignant cells. His blood glucose levels recorded over three consecutive days whilst remained low despite receiving continuous intravenous infusion of 10% dextrose table .A percutaneous needle biopsy of the mass showed morphological and immunohistochemical features of a benign SFT. The patient underwent thoracotomy with excision of the bulk of the mass. A small remnant attached to the diaphragm could not be removed. The tumour was large, well-circumscribed and encapsulated, with a tan-coloured whorled cut surface figure . It weigMicroscopic examination of the tumour revealed a bland spindle cell proliferation arranged in short intersecting fascicles with a patternless architecture, and hypo- and hypercellular areas separated by dense collagenous fibrous stroma figure . There wThe episodes of hypoglycemia abated postoperatively and the digital clubbing resolved within three months.et al which had analysed 360 cases in the literature and 8 additional cases from their series , high cellularity, nuclear pleomorphism and necrosis ,42. HoweHypoglycemia is rare in pleural SFT, occurring in approximately 5% of cases ,51. DoegParaneoplastic hypoglycemia results from secretion of an unprocessed or incomplete high molecular weight [HMW] form of insulin-like growth factor type II [IGF-II] . This HMRecently it has been shown that SFT cells have a markedly greater expression of IGF-II mRNA and a lesser expression of pro-hormone convertase 4 [PC 4] mRNA in the tumour tissue compared to normal placental tissue, raising the possibility that defective PC4 gene expression in these tumours may underlie the impaired processing of IGF-II . Pro-horThe detection of HMW IGF-II requires immunoblot analysis to distinguish it from normal IGF-II; unfortunately this technology was not available to us at the time and there was no pre-operative blood specimen preserved to enable IGF-II determination. Nevertheless all the features in this case fit with the diagnosis of DPS including severe intractable hypoglycemia with suppressed insulin secretion, decreased C-peptide and immediate resolution of the hypoglycemia following excision of the tumour.et al reported hypoglycemia in 12 out of 223 cases [5.2%] from a multi-institutional case series [3. Rena et al reported hypoglycemia in 3 out 21 cases [14.3%] [et al found only one case of hypoglycemia from a series of 60 cases [1.7%] [et al found one out of 14 cases [7.1%] in their series which at 33 cm also happened to be the largest tumour in that series  survival rates for pleural SFT vary from 75% to 100% in various series ,13,39,72Pleural SFT is an uncommon but not rare tumour that is now known to be of mesenchymal fibroblastic rather than mesothelial origin. Paraneoplastic manifestation of hypoglycemia and finger-clubbing in pleural SFT are exceedingly rare yet important clinical features in terms of diagnosis and follow-up. Hypoglycemia with or without finger-clubbing has been recorded in <80 cases in the English Literature. Both hypoglycemia and finger-clubbing resolve following excision of the tumour. In this updated review, hypoglycemia with and without finger-clubbing was found to associated with large tumours [>10 cm], but does not appear to be significantly associated with histological features of malignancy. The hypoglycemia is caused by HMW IGF-II which results from defective enzymes due to PC4 gene abnormalities in the tumour cells. The finger-clubbing results from abnormal hyaluronic acid deposition due to VEGF and PDGF cytokine expression. Histological features for malignancy and size, though important, are not truly predictive of local recurrence or worse clinical outcome. Complete surgical excision of the tumour remains the best treatment and most important predictor of clinical outcome. Long-term follow-up is recommended due to varyingly high recurrence rates particularly for tumours that were difficult to completely excise and have histological features for aggressive or malignant behaviour.CT: Computed tomography; DPS: Doege-Potter syndrome; IGF: Insulin-like growth factor; HMW: High molecular weight; HOA: Hypertrophic osteoarthropathy; MSA: Muscle-specific acting, PDGF: Platelet-derived growth factor; PMBS: Pierre Marie-Bamberg syndrome; SFT: Solitary fibrous tumour; SMA: Smooth muscle acting; VEGF: Vascular endothelial growth factor; WHO: World Health Organisation.The authors declare that they have no competing interests.All authors helped to draft the manuscript, read and approved the final manuscript. AK, MJH and JM were involved in making the pathological diagnosis. MW and TH were involved in the case report, clinical workup and management of the patient. AK and JM did the literature search and review. AK coordinated the drafting of the manuscript and its preparation for publication.Written informed consent was obtained through the University of the Witwatersrand Ethics Committee for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Colorectal tumors have characteristic genome-wide expression patterns that allow their distinction from normal colon epithelia and facilitate clinical prognosis. The expression heterogeneity within a primary colorectal tumor has not been studied on a genome scale yet. Here we investigated three compartments of colorectal tumors, the invasion front, the inner tumor mass, and surrounding normal epithelial tissue by microdissection and microarray-based expression profiling. In both tumor compartments many genes were differentially expressed when compared to normal epithelium. The sets of significantly deregulated genes in both compartments overlapped to a large extent and revealed various interesting known and novel pathways that could have contributed to tumorigenesis. Cells from the invasion front and inner tumor mass, however, did not show significant differences in their expression profile, neither on the single gene level nor on the pathway level. Instead, gene expression differences between individuals are more pronounced as all patient-matched tumor samples clustered in close proximity to each other. With respect to invasion front and inner tumor mass we conclude that the specific tumor cell micro-environment does not have a strong influence on expression patterns: largely similar genome-wide expression programs operate in the invasion front and interior compartment of a colorectal tumor. Recent reports have highlighted differences between the invasion front and interior compartments of colorectal tumors. Tumor cell budding at the invasion front is increasingly recognized as an adverse prognostic factor for colorectal tumors ,2. HypoxThis motivated us to study the expression of the invasion front, inner cells and surrounding normal epithelia of primary colorectal tumors by laser-capture microdissection and genome-wide microarray expression analysis. To our knowledge this is the first study of expression heterogeneity of a primary tumor on a genome scale. Cells from distinct tumor compartments were separated by laser-capture microdissection (documented electronic images are available upon request). RNA amplification was necessary to obtain sufficient starting material for hybridization of Affymetrix U133A DNA chips. For methodological details we refer to our previous work -13. We iMuch information about dominant trends in high-dimensional gene expression data can be discovered by the application of dimension reduction techniques. We applied principal component analysis on the covariance matrix to analyze major trends in our data  into two clusters containing 508 up-regulated and 415 down-regulated genes. Both sets of genes were tested separately for significant overlap with gene sets from public databases using Fisher's Exact test . In histograms showing the distribution of p values  buffer and 2% beta-mercaptoethanol. For molecular analysis, up to 100,000 cells were pooled. From each tumor we isolated cell populations of the invasion front, the inner cell mass and normal epithelium from the periphery.Preprocessing of frozen tissue blocks by laser-capture microdissection was essentially performed as described in our previous publications -13. BriePoly AC RNA from the microdissected specimens was prepared using the PolyA-tract 1000 kit  according to the manufacturer's recommendations. For each sample, the cDNA synthesis and repetitive in vitro transcription were performed three times. The total amount of prepared mRNA from each sample was used. First strand cDNA synthesis was initiated using the Affymetrix T7-oligo-dT promoter-primer combination at 0.1 mM. The second strand cDNA synthesis was generated by internal priming. In vitro transcription was performed using Ambion's Megascript kit  as recommended by the manufacturer. From the generated cRNA, a new first strand synthesis was initiated using 0.025 mM of a random hexamer as primer. After completion, the second strand synthesis was performed using the Affymetrix T7-oligo-dT promoter-primer combination. A second in vitro transcription was performed and then the procedure was repeated one additional time. During the last in vitro transcription, biotin-labelled ribonucleotides were incorporated into the aRNA, as recommended by the Affymetrix protocol. Hybridisation and detection of the labelled cRNA on the Affymetrix Chip were performed according to Affymetrix standard protocol.Raw expression data was condensed to probe set-wise intensity values using the GC-RMA algorithm. Present/absent calls for each data point were calculated with the Affymetrix standard algorithm. Only genes with significant expression signals  in a minimum of 30% of experiments were considered for subsequent analyses. For experiment normalization data were rescaled to have equal median expression intensities for all experiments, then intensities for each gene were divided by the average over all experiments with normal tissues and the logarithm of this ratio was calculated. Probe set annotation for U133A chips were retrieved from the Affymetrix web site (version 22). Gene-wise expression data were obtained by averaging intensities of all probe sets associated with a gene symbol, finally resulting in log ratio expression data for 7433 genes.The preprocessed data were subjected to a principal component analysis (PCA) of the covariance matrix of the data to investigate and visualize global trends in the data. Hierarchical clustering using the complete linkage algorithm and Euclidean distances was used as a complementary method to analyze the grouping of samples. These algorithms were used as implemented in the Expressionist Analyst software . Pairwise Welch t-tests were performed for each gene to compare expression levels in the the sample groups invasion front (IT), inner tumor mass (RT) and normal epithelial tissue (N). Histograms of all resulting p values for each comparison were plotted using the statistical software R. The p value distributions helped to assess whether there are differences in expression on the genome level between the three sample groups. If there are no significant differences in expression (comparable to a situation when the data are randomized) the p value distribution resembles a uniform distribution.The author(s) declare that they have no competing interests.ES performed data analysis and drafted the manuscript, JG and MH performed RNA sample preparation, microdissection and hybridization of chips, DM hybridized DNA chips and contributed to data analysis, SR was involved in data preprocessing and analysis, IK performed laser capture microdissection, ECV did laser capture microdissection, TB supervised chip hybridization and data preprocessing, BM and HJB were responsible for clinical part of the study including sample acquisition and patients' informed consent, CP and BW supervised chip hybridization, quality control and data preprocessing, AR conceived the study.Microarray expression data of 7433 genes. The pre-processed expression data for all 7433 genes that were considered for subsequent analysis. The data are logarithms (base e) of expression ratios between each measurement and the mean value over all experiments with normal epithelia. Therefore, these log ratios indicate relative changes compared to normal epithelia. Positive values indicate up-regulation compared to normal epithelia. For further preprocessing steps see legend of Figure Click here for file. All other gene groups were taken from the MSIGDB database .Results from statistical tests for differential expression. These tables comprise results from statistical testing for differential gene expression and the annotations for all 7433 genes. The gene-wise p-values and Q-values of unpaired Welch test or the paired t-test (Benjamini-Hochberg method) for each sample group comparison are listed. The gene group memberships of each gene are separated by a colon in the respective column. KEGG, GenMAPP and GO categories for all genes were taken from the Affymetrix U133A DNA chip annotation Click here for fileSignificantly deregulated gene groups. Here we show the results of Fisher's Exact test for the over-representation of functional gene groups among up- or down-regulated genes in colorectal tumors of our study. To correct for multiple testing Q-values were calculated using the Benjamini-Hochberg method.Click here for file


Analysis of the species distribution of members of these clusters has identified proteins that are specific to filamentous fungal species and a group of proteins found only in plant pathogens. By comparing the gene inventories of filamentous, ascomycetous phytopathogenic and free-living species of fungi, we have identified a set of gene families that appear to have expanded during the evolution of phytopathogens and may therefore serve important roles in plant disease. We have also characterised the predicted set of secreted proteins encoded by each genome and identified a set of protein families which are significantly over-represented in the secretomes of plant pathogenic fungi, including putative effector proteins that might perturb host cell biology during plant infection. The results demonstrate the potential of comparative genome analysis for exploring the evolution of eukaryotic microbial pathogenesis.Fungi and oomycetes are the causal agents of many of the most serious diseases of plants. Here we report a detailed comparative analysis of the genome sequences of thirty-six species of fungi and oomycetes, including seven plant pathogenic species, that aims to explore the common genetic features associated with plant disease-causing species. The predicted translational products of each genome have been clustered into groups of potential orthologues using Markov Chain Clustering and the data integrated into the  Phytophthora infestans and rice blast disease caused by the ascomycete fungus Magnaporthe grisea, both of which are responsible for very significant harvest losses each year. The enormous diversity of crop diseases caused by these eukaryotic micro-organisms poses a difficult challenge to the development of durable disease control strategies. Identifying common underlying molecular mechanisms necessary for pathogenesis in a wide range of pathogenic species is therefore a major goal of current research. Approximately 100,000 species of fungi have so far been described, but only a very small proportion of these are pathogenic Saccharomyces cerevisiae and trigger morphological and biochemical changes in response to external stimuli such as starvation stress or hyperosmotic conditions Magnaporthe grisea, stimulated by hard, hydrophobic surfaces is regulated by a MAP kinase cascade S. cerevisiaeM. griseaFungi and oomycetes are responsible for many of the world's most devastating plant diseases including late blight disease of potato, caused by the oomycete pathogen M. grisea and the corn smut Ustilago maydis and many of the advances in understanding the developmental biology of plant infection have occurred in these model pathogens M. grisea on the basis that a mutant strain containing a non-functional cutinase-encoding gene was still able to cause rice blast disease M. grisea genome has shown the presence of eight potential cutinase-encoding genes implicated in virulence Blumeria graminis, which cannot be cultured away from living host plants. Therefore, new approaches are needed to identify genes that are vital for the process of pathogenicity. These include high-throughput methods such as microarray analysis, serial analysis of gene expression (SAGE), insertional mutagenesis, proteomics and metabolomics Other pathogenicity factors identified to date have been shown to be involved in functions associated with host infection, such as plant cell wall degradation, toxin biosynthesis and protection against plant defences S. cerevisiae in 1996 M. griseaUstilago maydisGibberella zeaeStagonospora nodorumBotrytis cinerea and the white mould fungus Sclerotinia sclerotiorume-Fungi object-oriented data warehouse After the initial release of the genome of the budding yeast e-FungiEncephalitozoon cuniculi, a microsporidian fungus, has a reduced genome that codes only for 1,997 proteins and lacks genes encoding enzymes of many primary metabolic pathways such as the tricarboxylic acid cycle, fatty acid β-oxidation, biosynthetic enzymes of the vast majority of amino acids, fatty acids and nucleotides, as well as components of the respiratory electron transport chain and F1-F0 ATP synthase. It also lacks mitochondria and peroxisomes E. cuniculi, there are 377 clusters that contained proteins from 35 species of fungi and oomycetes , enzymes involved in haem biosynthesis, autophagy-related proteins, those involved in protein targeting to the peroxisome and vacuole and additional groups of proteins involved in signal transduction that are not present in E. cuniculi (including those involved in inosine triphosphate and leukotriene metabolism). The analysis also showed there were 105 clusters that contained proteins from 33 species of fungi (excluding E. cuniculi), but not from the two species of oomycete , it seems likely that these are required for the complex morphological changes that filamentous fungi undergo during their lifecycle and the production of differentiated cells, such as spores, fruiting bodies and infection structures. The results also suggest that filamentous fungal species make a greater use of lipids as signalling molecules than yeast species. For example, the occurrence of filamentous fungal-specific clusters representing two groups of lysophospholipases, as well as ceramidases that are involved in sphingolipid signalling Aspergillus nidulansSordaria macrosporaPodospora anserinaAspergillus nidulans, which is essential for nuclear migration and conidiophore development For the purposes of this study, the filamentous fungi were defined as the filamentous ascomycetes (subphylum Pezizomycotina), basidiomycetes and zygomycetes and the unicellular fungi were defined as the budding yeasts , the archiascomycete ar fungi . Interese-Fungi  but did not contain proteins from non-pathogenic species. There were, however, four clusters that were exclusive to filamentous ascomycete phytopathogens  M. griseaM. grisea, the metallothionein Mmt1 Pathogenicity factors have been defined as genes that are essential for successful completion of the pathogen lifecycle but dispensable for saprophytic growth  e-Fungi . Not surB. cinerea, G. zeae, M. grisea, S. sclerotiorum, S. nodorum with the non-pathogens Aspergillus nidulans, Chaetomium globosum, Neurospora crassa and Trichoderma reesei. Phylogenetic analysis suggests that the phytopathogenic species do not form a separate clade from the pathogenic species , found in 4 species of phytopathogen, including five copies in G. zeae, and the Yeast cell wall synthesis protein KRE9/KNH1 motif (PF05390), which was found in three species of phytopathogen. Cas1p is a membrane protein necessary for the O-acetylation of the capsular polysaccharide of the basidiomycete animal pathogen Cryptococcus neoformansS. cerevisiaeYDG/SRA domain motif (PF02182) is unknown, but is found in a novel mouse cell proliferation protein Np95, in which the domain is important both for the interaction with histones and for chromatin binding in vivoAllophanate hydrolase (PF02682) which is found in an enzyme involved in the ATP-dependent urea degradation pathway opioid growth receptor motif (PF04664) and Mnd1 (PF03962), which is involved in recombination and meiotic nuclear division A small number of Pfam motifs were not found in the proteomes of the filamentous ascomycete non-pathogens, but were found in the proteomes of at least three species of filamentous ascomycete phytopathogens . These icutinase (PF01083) and Glycosyl hydrolase family 53 (PF07745) which is found in arabinogalactan endo-1,4-beta-galactosidases that hydrolyze the galactan side chains that form part of the complex carbohydrate structure of pectin pectinesterase (PF01095) and Glycosyl hydrolases family 28 (PF00295) are both more than twice as common in the genomes of phytopathogens than saprotrophs. In contrast, domains found in cellulases have fairly equal distribution between the proteomes of phytopathogens and non-pathogens (data not shown). Therefore, for phytopathogens the most essential enzymes for pathogenesis may well be those that allow the fungus to penetrate the protective cutin layer of the plant epidermis and disrupt the pectin matrix of the plant cell wall in which cellulose fibrils are embedded. Pectin-degrading enzymes have already been shown to be pathogenicity factors in a number of fungi Phytophthora ramorum and 75 in Phytophthora sojae). Proteins containing the Chitin recognition protein domain (PF00187) are also very common in the proteomes of phytopathogens (18 in M. grisea and 16 in S. nodorum). A role for chitin-binding proteins has been proposed in protecting the fungal cell wall from chitinases produced by host plants arylesterase (PF01731) and EthD protein (PF07110) which breakdown organophosphorus esters To detect potential gene family expansion, we decided to identify Pfam motifs that were present in both phytopathogenic and non-pathogenic species of filamentous ascomycetes, but that were more common in the genomes of the former. The Pfam motifs were ranked on the ratio of the mean number of proteins containing each motif in phytopathogens, when compared to non-pathogens . The tabM. grisea using SignalP and ProtComp (www.Softberry.com) predicted only 739 secreted proteins  compared to our prediction of 1,546 secreted proteins  Studies in bacterial pathogens and oomycetes have shown that a range of secreted proteins known as effectors are important for establishing infection of the host plant Isochorismatase motif (PF00857) was found in the secretomes of all five species of phytopathogen. Isochorismatase catalyses the conversion of isochorismate to 2,3-dihydroxybenzoate and pyruvate. It has been implicated in the synthesis of the anti-microbial compound phenazine by Pseudomonas aeruginosaEscherichia coliS. nodorum) all show sequence similarity to ycaC from E. coli, an octameric hydrolase of unknown function Kelch domain (PF01344) shows the most striking difference in distribution between phytopathogenic and non-pathogenic genomes. This 50-residue domain is found in a number of actin-binding proteins 2O2) Phanerochaete chrysosporium2O2-producing copper oxidases have been shown to have roles in morphogenesis, in the corn-smut fungus Ustilago maydis for example, a glyoxal oxidase is required for filamentous growth and pathogenicity Stigmatella aurantiaca2O2-generating enzymes that catalyse the oxidative deamination of primary amines to the corresponding aldehydes Phanerochaete chrysosporiumXanthomonas. Many Kelch domain-containing proteins are involved in cytoskeletal rearrangement and cell morphology M. grisea co-opts plasmodesmata to move from cell to cell in infected rice leaves PAN domain (PF00024), that mediates protein-protein and protein-carbohydrate interactions F5/8 type C domain (PF00754), found in the discoidin family of proteins involved in cell-adhesion or developmental processes S. cerevisiae are responsible for responses to pheromones, nutritional starvation and osmotic stress S. cerevisiae, the receptors are often different, reflecting the different environmental cues to which the pathogen needs to respond One of the most fundamental aims in plant pathology research is to define precisely the difference between pathogenic and non-pathogenic microorganisms. The answer cannot be one of simple phylogeny, because phytopathogenic species are found in all taxonomic divisions of fungi and are often closely related to non-pathogenic species e-Fungi data warehouse provides a means to interrogate the vast amounts of genomic and functional data available in a simple integrated manner Analysis of all available genome sequences from a wider range of fungal species has for the first time allowed us to address the differences between phytopathogens and non-pathogens at a whole genome level. For this purpose, the S. cerevisiae has only three cytochrome P450-encoding enzymes. We have shown here that there are likely to be large differences in the gene inventories of filamentous fungi compared to unicellular yeasts.It has previously been speculated that the evolution of phytopathogenesis was associated with the expansion of certain gene families To study the differences between phytopathogenic and saprophytic fungi, we concentrated on the filamentous ascomycetes where there are a number of phytopathogenic species genomes have been sequenced along with closely related non-pathogens. Protein families were defined using Pfam motifs To identify potential fungal effectors, we compared Pfam motif frequency between the secretomes of phytopathogens and non-pathogens. This analysis identified potential effector-encoding genes, including secreted proteases, transcription factors and proteins that may be involved in cytoskeletal rearrangements (such as Kelch-domain containing proteins) and protein-protein interactions, as well as a group of pathogen-specific secreted isochorisimatases that potentially could suppress salicyclic acid-dependent host plant defences. Bacterial T3SEs are injected directly into the host cytoplasm via the type III secretion injection apparatus e-Fungi, has allowed us to pinpoint new gene families that may serve important roles in the virulence of phytopathogens, allowing their selection for gene functional studies, that are currently in progress. The analyses deployed here may also offer a blueprint for the types of larger, more comprehensive studies that will be necessary to interpret the large flow of genetic data that will result from next generation DNA sequence analysis utilizing both a much wider variety of fungal pathogen species and also large sets of individual isolates of existing species.Although the evolution of phytopathogenicity is likely to have happened several times and the lifestyles of these fungi are diverse, a comparison of gene inventories of a number of species using a powerful resource, such as −10 as an E-value cut-off, as described previously −20 using BLASTP), or Pfam motifs contained in proteins from the cluster in the absence of Swiss-Prot hits.Sets of predicted proteins were downloaded for each of the 36 genomes from respective sequencing project websites . Proteinhttp://www.sanger.ac.uk/Software/Pfam/). This library contains 7973 protein models constructed from manually curated multiple alignments and covers 75% of proteins in UniProt http://hmmer.wustl.edu/). Default thresholds were used, which are hand-curated for every family and designed to minimise false positives The Pfam-A library from release 18.0 of the Pfam database was downloaded from the Pfam website . Analyses described in this study were performed using the e-Fungi database.All the data produced, as described above, was stored in the Table S1(0.08 MB XLS)Click here for additional data file.Table S2(0.02 MB XLS)Click here for additional data file.Table S3(0.16 MB XLS)Click here for additional data file.Table S4(0.06 MB XLS)Click here for additional data file.Table S5(0.03 MB XLS)Click here for additional data file.Table S6(0.04 MB XLS)Click here for additional data file.


The to date evidence for a dose-response relationship between physical workload and the development of lumbar disc diseases is limited. We therefore investigated the possible etiologic relevance of cumulative occupational lumbar load to lumbar disc diseases in a multi-center case-control study.In four study regions in Germany , patients seeking medical care for pain associated with clinically and radiologically verified lumbar disc herniation  or symptomatic lumbar disc narrowing  were prospectively recruited. Population control subjects  were drawn from the regional population registers. Cases and control subjects were between 25 and 70 years of age. In a structured personal interview, a complete occupational history was elicited to identify subjects with certain minimum workloads. On the basis of job task-specific supplementary surveys performed by technical experts, the situational lumbar load represented by the compressive force at the lumbosacral disc was determined via biomechanical model calculations for any working situation with object handling and load-intensive postures during the total working life. For this analysis, all manual handling of objects of about 5 kilograms or more and postures with trunk inclination of 20 degrees or more are included in the calculation of cumulative lumbar load. Confounder selection was based on biologic plausibility and on the change-in-estimate criterion. Odds ratios (OR) and 95% confidence intervals (CI) were calculated separately for men and women using unconditional logistic regression analysis, adjusted for age, region, and unemployment as major life event  or psychosocial strain at work , respectively. To further elucidate the contribution of past physical workload to the development of lumbar disc diseases, we performed lag-time analyses.We found a positive dose-response relationship between cumulative occupational lumbar load and lumbar disc herniation as well as lumbar disc narrowing among men and women. Even past lumbar load seems to contribute to the risk of lumbar disc disease.According to our study, cumulative physical workload is related to lumbar disc diseases among men and women. B.1. is defined as follows: "Chronic low-back disease with pain ". In Denmark, the following exposures are required for recognition as an occupational disease: (a) Back-loading lifting work involving lifting/upward pulling of heavy objects and many tonnes of lifting per day for a considerable number of years; (b) Back-loading lifting work with generally occurring, extremely heavy and awkward single lifts and several tonnes of lifting per day for a considerable number of years; (c) Back-loading care work with many daily handlings of adults or older handicapped children for a considerable number of years; (d) Back-loading, daily exposure to whole-body vibrations from heavily vibrating vehicles for a considerable number of years. In France, according to table ("tableau") 98 only lumbar disc herniations associated with radicular symptoms can be recognized as an occupational disease . It is a precondition that heavy objects have been handled for at least five years in specified occupations.Several epidemiological studies show a relationship between lumbar disc diseases and physical workplace factors such as lifting or carrying of loads, forward bending, and whole body vibrations -16; cf. The considerable differences in recognition criteria might at least partly reflect the to date limited evidence for a dose-response relationship between physical workload and the development of lumbar disc diseases. We therefore conducted a population-based case-control study that deals with the following questions:- Does a positive dose-response relationship exist between occupational lumbar load and lumbar disc diseases (lumbar disc herniation or lumbar disc narrowing)?- Which type of exposure does contribute to the lumbar disc disease risk ?- Does past physical work play an etiologic role in the development of lumbar disc disease (in other words: Is a cumulative lumbar-load dose model appropriate)?Subject recruitment in our study was performed prospectively in four study regions in Germany: Frankfurt am Main, Freiburg, Halle/Saale, and Regensburg. In the mentioned regions, all hospitals or practices (n = 29) treating at least five patients with lumbar disc herniation per year as well as a random sample of orthopedic practices  were included. The corresponding physicians were asked to identify all patients between 25 and 70 years that belonged to one of the following case groups:Case group 1Males with outpatient or inpatient treatment in an included hospital because of lumbar disc herniation with sensitive and/or motor radix syndrome;Case group 2Females with lumbar disc herniation as defined under case group 1;Case group 3Males with outpatient or inpatient treatment in a hospital or outpatient treatment in an included orthopedic practice due to severe disc narrowing (of more than one third compared with adjacent unchanged discs) with sensitive and/or motor radix syndrome or with local lumbar syndrome with a finger floor distance of more than 25 cm;Case group 4Females with lumbar disc narrowing as defined under case group 3.The diagnosis of lumbar disc herniation had to have been confirmed by computerised tomography (CT) or by magnetic resonance imaging (MRI); the diagnosis of lumbar disc narrowing was primarily based on X-ray. Disc herniation and disc narrowing were defined as proposed by a German consensus group . AltogetControl subjects were randomly selected from a one percent random sample of residents aged 25 to 70 years drawn by the local population registration offices of the respective region. Of 1,687 population controls, 901 agreed to participate (53.4%). According to a non-responder analysis, the proportion of blue-collar workers was higher among non-responding cases as well as among non-responding control subjects. We found no evidence for a differential selection bias with respect to social status.The interviewers documented a complete (self-reported) occupational history for each participant, including every occupational period that lasted at least half a year. Interviewers were intensively trained in standardized interview techniques and a non-differential approach to cases and controls. Participants were not informed of the specific aims of the study. Actually, they were asked to participate in a study concerning the theme 'occupation and health'. A detailed computer-assisted personal interview was developed to elicit information about psychosocial workload ,20; leisWith those subjects who  exceeded relatively low "exposure thresholds" in lifting, carrying, pulling, pushing of objects, working postures or whole-body vibration, subsequently a semi-standardized personal comprehensive expert interview was performed by branch-orientated ergonomic experts of the institutions for statutory accident insurance and prevention. The experts were blinded for the case-control status. On the basis of job-task specific supplementary surveys, these experts documented the intensity, frequency and duration of all spine-related exposures induced by manual materials handling , trunk inclination and twisting, and whole-body vibration for the entire working life of each subject .A biomechanical analysis based on this expert assessment was conducted to determine the "situational lumbar load". As the characteristic indicator of lumbar load, the compressive force on the lumbosacral disc was calculated for each loading activity (range 0 to 8.4 Kilo-Newton). Quantification was based on biomechanical-model calculations applying the three-dimensional dynamic simulation tool "The Dortmunder" .[Comprehensive analyses were done to identify the "best" dose model  in terms of goodness of fit (estimated by the Akaike information criterion AIC), specifity, simplicity, and clarity of the dose-response relationship. First results of these analyses have been published elsewhere ,25. HoweTo evaluate the reliability of cumulative occupational lumbar load calculated on the basis of expert assessments, 40 expert interviews and subsequent expert assessments were repeated by the same experts (intra-rater reliability), and further 40 expert interviews and subsequent expert assessments were repeated by different experts (inter-rater reliability). We calculated Kappa values comparing the categorised cumulative exposures derived from the first expert interview with the corresponding exposure categories derived from the retest. Intra-rater agreement (Kappa = 0.64) as well as inter-rater agreement (Kappa = 0.46) were fair to good.As an a-priori defined procedure, all continuous variables were categorized in tertiles based on the distribution of the exposed control subjects. If less than 20% of the control subjects were "non-exposed", the reference category combined non-exposed subjects and subjects in the first exposure tertile. This was the case for all exposure variables in men, while in women, non-exposed persons and the first tertile remained in different categories.Odds ratios (OR) and 95% confidence intervals (CI) were calculated using unconditional logistic regression analysis. All statistical analyses were adjusted for age and place of residence, referred to as "region" in this text. As age is known to be strongly associated particularly with the occurrence of lumbar disc narrowing  and as cBesides the odds ratios solely adjusted for age and region, odds ratios for the fully adjusted "final model" are given. The final model should comprise all factors that might confound the relationship between cumulative lumbar load and lumbar disc herniation or lumbar disc narrowing, respectively. Therefore, selection of confounders was performed based on a list of biologically plausible factors in two steps: 1. Factors were considered as potential confounders if they were correlated with the cumulative lumbar load  among control subjects. For men, the following factors were identified: weight (body mass index), Scheuermann's disease, articular gout, and whole-body vibrations. In women, solely whole-body vibrations were correlated with the cumulative lumbar load. 2. Potential confounders were included in the final logistic regression model if they changed the odds ratio of cumulative lumbar load by more than 10% in at least one category. In the final model, the following confounders were included: age, region, distress by unemployment as major life event (solely for case group 1), and psychosocial stress (for case groups 2 and 4). No additional confounders were considered in case group 3. Missing values were analysed as a separate exposure category (results not shown here).To further elucidate the contribution of past physical workload to the development of lumbar disc herniation, we performed two lag-time analyses: First, we restricted our analysis to cumulative lumbar load that had occurred up to 10 years prior to diagnosis. Second, we performed a subanalysis for all subjects that were not exposed to materials handling or intensive-load postures during 10 years prior to diagnosis.Calculation of the statistical power of the study was based on an expected prevalence of heavy physical work of 21% among the male population and 15% among the female population according to Elsner and Seidler . To deteThe study was regarded as quality development work within the area of occupational health. The aims, methods, and procedures of the study were agreed by the Hesse Medical Association. The study was performed in compliance with the ethical principles of the Helsinki Declaration. The concept of data protection was developed in cooperation with and agreed by the Hesse Federal Commissioner for Data Protection and Freedom of Information Decline. All subjects gave their informed consent.Among men table , there wa; not adjusted for each other), both exposures were strongly associated with lumbar disc herniation as well as with lumbar disc narrowing among men and among women. When manual materials handling and intensive-load postures were adjusted for each other, handling of objects was more strongly associated with lumbar disc narrowing than with lumbar disc herniation . This was especially the case among women, who revealed a disc herniation risk of 3.2 (95% CI 1.6–6.3) in the highest category of cumulative lumbar load through intensive-load postures, but no elevated lumbar disc narrowing risk at all. However, in the control group materials handling is highly correlated with posture with a Kendall-Tau correlation coefficient of 0.61 for men and 0.78 for women. Therefore, collinearity could possibly explain the latter results.When manual handling of objects and intensive-load postures were separately included in the regression model , this had no substantial effect on the risk estimates for lumbar disc herniation or lumbar disc narrowing neither among men table  nor amonIn this study, we found a positive dose-response relationship between cumulative lumbar load and lumbar disc disease (disc herniation and disc narrowing) among men as well as among women. A distinction between the risks of manual materials handling and of intensive-load postures is difficult because of potential collinearity. Our lag-time subanalysis excluding subjects exposed during the last 10 years prior to diagnosis revealed elevated risks of lumbar disc diseases in men and women with high exposure to manual materials handling and working postures. These results point to a potential etiologic role of cumulative physical workload to lumbar disc disease even among persons with a long-term elimination of physical exposure. Strengths of our study include the extensive diagnostic procedure, the calculation of cumulative lumbar load based on expert assessment of occupational tasks and subsequent biomechanical analysis, and adjustment for multiple potential confounders according to an a-priori defined analytic concept.As a potential limitation of the study, the low participation rate  might have introduced selection bias. To further evaluate this potential bias, we asked non-participants by telephone about their longest held job. 57% of non-participating cases and 47% of non-participating control subjects gave their longest held occupation. According to this information, the proportion of blue-collar-workers was higher among non-participating cases and control subjects. Taking into account the higher response-rate among cases, the responder analysis does not point to substantial differential selection bias.Due to lack of radiographic examination, the frequency of lumbar disc disease is unknown among the population controls. A suspected prevalence of lumbar disc disease of up to 10% among population controls would result in a slight tendency to underestimate potential risk factors.Age – which is strongly associated with lumbar disc narrowing – was included in 10-years-categories, making residual confounding possible. When age – and additionally squared age – was included as a continuous variable in the regression model, this did not substantially alter the results. We therefore regard substantial residual confounding by age as improbable.Persons with high physical workload might seek medical advice more frequently than persons with low physical workload. To investigate this potential detection bias, we performed two subanalyses: Firstly, we restricted the case group to patients with severe lumbar disc disease being accompanied with paralysis . The results of this subanalysis did not substantially differ from the results of the main analysis. Secondly, we analysed the health seeking behavior of control subjects with low back pain (according to NORDIC questionnaire ). Among Several studies show an association between manual handling of objects and awkward working postures with the diagnosis of lumbar disc disease -16. HoweThe present study reveals evidence for a positive dose-response relationship between manual materials handling as well as intensive-load postures and care seeking with the diagnosis of lumbar disc disease (lumbar disc herniation as well as lumbar disc narrowing) among men and women. In the highest dose category, we partly see an unchanged or even a decreased risk; this phenomenon can be found even among men when the highest dose category is further divided according to the 95-percentile of the distribution of control subjects . The lack of a monotonous risk increase for very high cumulative lumbar load might be explained by a healthy worker survivor effect. However, we cannot exclude the possibility of effectively lacking further risk increase in highly exposed workers.In accordance with the biomechanical concept of lumbar-spine force as the pathogenic pathway from physical workload to lumbar disc disease, our study deals with the influence of lumbar compressive force from materials handling and intensive-load postures on lumbar disc disease. According to our study, even moderate lumbar load might contribute to the risk of lumbar disc disease. Several experimental studies suggest that compressive forces can lead to structural changes in intervertebral discs  as well as to changes in intervertebral disc cell metabolism -30. HuttBecause of the potential collinearity between object handling and posture, a definite conclusion concerning the separate effects of these exposures is not derivable. Exposure to manual handling of objects might play a role particularly in lumbar disc narrowing, while exposure to trunk inclination might be of particular importance for the development of lumbar disc herniation. This would be in accordance with Seidler et al. ,16, who In keeping with the pathogenic concept of a long-term increased biomechanical load of lumbar-spine structures being an important cause of lumbar disc disease, the present study uses cumulative occupational exposure as the independent variable. When all exposures which occurred during 10 years prior to diagnosis were neglected, this did not substantially alter the results. A further subanalysis of all subjects that were not exposed to manual handling of objects or trunk inclination during 10 years prior to diagnosis revealed elevated risks of lumbar disc disease. This suggests that past physical work plays an etiologic role in the development of lumbar disc herniation as well as lumbar disc narrowing. Our finding is in accordance with Seidler et al. , who as In conclusion, our results support a clear dose-response relationship between cumulative lumbar load and lumbar disc herniation as well as symptomatic lumbar disc narrowing.The authors declare that they have no competing interests.AS conceived the epidemiological analysis, performed the statistical analysis and drafted the manuscript, AB and JG developed the diagnostic procedure, MJ and AL performed the biomechanical lumbar-load analysis, RE and DD developed and coordinated the expert assessments of physical work, GE and FH participated in the interpretation of the study results, OL performed the verification of the clinical diagnosis as reference orthopedian, JH participated in the development of the epidemiological concept and in the interpretation of the study results, MM, GP, and BS developed the standardized interview and conducted plausibility checks, UB coordinated the study. All authors participated in the design and conduction of the study, read and approved the final manuscript.Dose models applied in the German multi-center case-control study EPILIFT*Dose models 1 and 2 represent the cumulative lumbar-load dose model actually used in worker's compensation procedures in Germany ("Mainz-Dortmund Dose Model" [- e Model" ); they cDose model 3 is comparable to dose model 2, but considers  all modes of manual material handling , not only lifting and carrying. Compressive force is weighted analogously to dose models 1 and 2; the shift-dose thresholds for men and women are chosen equally to those of dose models 1 and 2.- Dose model 4 provides the lowest exposure thresholds. For the calculation of cumulative lumbar load, in particular, manual handling of objects of about 5 kilograms or more and postures with trunk inclination of at least 20 degrees are considered. Lumbar-disc compressive force of 2.0 Kilo-Newtons or more is considered for day-related cumulation  and is weighted analogously to dose models 1 to 3. No shift-dose thresholds are considered as in all dose models 4 to 10.- Dose model 5 considers manual handlings of objects that involve a lumbosacral compressive force of 2 Kilo-Newton or more; furthermore, lumbar load through trunk inclination of 45 degrees or more is considered. Again, compressive force of at least 2.0 Kilo-Newtons is weighted overproportionally (squared) in relation to the respective action duration, i.e. analogously to dose models 1 to 4. No shift-dose thresholds are considered as in all dose models 4 to 10.- Dose model 6 is comparable to dose model 5, but considers only lumbar load through trunk inclination of 75 degrees or more. No shift-dose thresholds are considered as in all dose models 4 to 10.- Dose models 7 to 10 differ from dose model 5 only in the weighting of the compressive force in relation to the respective action duration. For example, in dose model 7 the lumbar-disc compressive force is equally weighted (linearly) as the respective action duration, and this weighting is varied . No shift-dose thresholds are considered as in all dose models 4 and 10.- *Remark: There is no dose model that shows the best goodness of fit in all four case groups. Altogether, the dose models 4, 6 and 7 show the best goodness of fit.The pre-publication history for this paper can be accessed here:


Limiting dilution experiments showed that there was no increase in the number of circulating osteoclast precursors in breast cancer patients with skeletal metastases  compared to controls. Osteoclast precursors in these patients also did not exhibit increased sensitivity to 1,25(OH)2D3 or M-CSF in terms of osteoclast formation. The addition of parathyroid hormone-related protein and interleukin-6 did not increase osteoclast formation. The addition of the supernatant of cultured breast cancer cell lines (MCF-7 and MDA-MB-435), however, significantly increased monocyte-osteoclast formation in a dose-dependent fashion. These results indicate that the increase in osteoclast formation in breast cancer is not due to an increase in the number/nature of circulating osteoclast precursors. They also suggest that tumour cells promote osteoclast formation in the bone microenvironment by secreting soluble osteoclastogenic factor(s). © 2001 Cancer Research Campaign http://www.bjcancer.comThe cellular mechanisms that account for the increase in osteoclast numbers and bone resorption in skeletal breast cancer metastasis are unclear. Osteoclasts are marrow-derived cells which form by fusion of mononuclear phagocyte precursors that circulate in the monocyte fraction. In this study we have determined whether circulating osteoclast precursors are increased in number or have an increased sensitivity to humoral factors for osteoclastogenesis in breast cancer patients with skeletal metastases  compared to patients with primary breast cancer and age-matched normal controls. Monocytes were isolated and cocultured with UMR 106 osteoblastic cells in the presence of 1,25 dihydroxyvitamin D


Activation of surfacebound plasminogen may localize protease activity in the pericellular microenvironmentand play a role in inducing both a conformational change and cell locomotion. Plasmin,however, can induce non-proteolytic effects on certain cell functions in a variety of celllineages. In the present study we examined the effects of plasmin on keratinocytes witha focus on its role in the process of re-epithelialization, which included studies of cellmigration, phagocytic-killing and cell proliferation. Migration of freshly isolated humanepidermal keratinocytes was analyzed utilizing the agarose gel assay in the presence of10% human serum. Plasmin at the concentration of 25 U/l induced a 160% increase inthe chemotactic migration of keratinocytes that was completely blocked by the plasmininhibitor α2-antiplasmin (Serpin). In the absence of serum, plasmin also induced a reversiblechemotactic migration of HaCaT keratinocytes as determined utilizing the microchemotaxisassay. Dose-response analysis showed a bi-phasic effect of plasmin with a maximumincrease of 52% in keratinocyte chemotaxis at a concentration of 25 U/l. HaCaT cells onthe other hand, showed no detectable in vitro chemokinesis byplasmin. Phagocytic-killingof Candida albicans by freshly isolated epidermal keratinocytes was enhanced in thepresence of 25 U/l plasmin which was also reversible by the addition of Serpin.Spontaneous proliferation of HaCaT keratinocytes as determined by 3H-Thymidine uptakeon the other hand, was reduced by 47 and 13% in cultures with 25 U/l plasmin for 24 and48 h respectively, in a Serpin reversible manner. These data suggest that plasmin-inducedchemotactic migration of epidermal keratinocytes is accompanied by enhancedphagocytic-killing coupled with suppression of proliferation of these cells which mayfacilitate re-epithelialization following skin injury.AbstractKeratinocytes were shown to induce the activation of plasminogen activator resulting inthe formation of plasmin and the initiation of proteolysis


FDK algorithm is a well-known 3D  approximate algorithm for CT (computed tomography) image reconstruction and is also known to suffer from considerable artifacts when the scanning cone angle is large. Recently, it has been improved by performing the ramp filtering along the tangential direction of the X-ray source helix for dealing with the large cone angle problem. In this paper, we present an FDK-type approximate reconstruction algorithm for gantry-tilted CT imaging. The proposed method improves the image reconstruction by filtering the projection data along a proper direction which is determined by CT parameters and gantry-tilted angle. As a result, the proposed algorithm for gantry-tilted CT reconstruction can provide more scanning flexibilities in clinical CT scanning and is efficient in computation. The performance of the proposed algorithm is evaluated with turbell clock phantom and thorax phantom and compared with FDK algorithm and a popular 2D  approximate algorithm. The results show that the proposed algorithm can achieve better image quality for gantry-tilted CT image reconstruction.


Briese et al.  of the first characterized HRV-A2 strain, HRV-QPM (It is an exciting time for those interested in rhinoviruses. With increased implementation of multiplexed screening approaches (such as the MassTag PCR), or by simply including a specific and sensitive PCR for all known strains ( We appreciate the enthusiasm for our recent publication highlighting the global distribution of a long-unrecognized third clade of rhinoviruses. Robust, sequence-based clock estimates with associated confidence limits indicate that these viruses have been circulating for hundreds of years (There has been discussion in the field as to whether the novel sequences represent a sublineage HRV-A2 of the classified species HRV-A (


Although epithelial cell adhesion/activating molecule (EpCAM/CD326) is one of the first tumour-associated antigens identified, it has never received the same level of attention as other target proteins for therapy of cancer. It is also striking that ever since its discovery in the late 1970s the actual contribution of EpCAM to carcinogenesis remained unexplored until very recently. With a First International Symposium on EpCAM Biology and Clinical Application this is now changing. Key topics discussed at the meeting were the frequency and level of EpCAM expression on various cancers and its prognostic potential, the role of EpCAM as an oncogenic signalling molecule for cancer cells, recent progress on EpCAM-directed immunotherapeutic approaches in clinical development and the interaction of EpCAM with other proteins, which may provide a basis for a therapeutic window and repression of its growth-promoting signalling in carcinoma. Future research on EpCAM may benefit from a unified nomenclature and more frequent exchange among those who have been working on this cancer target during the past 30 years and will do so in the future. The two-day meeting gathered participants from academic institutions, biotech, and pharmaceutical industry, and gave an excellent opportunity and framework to review the progress on epithelial cell adhesion and activating molecule (EpCAM) biology and the development of novel EpCAM-directed immunotherapies.In his introductory remarks, G Riethmueller  pointed out that the first human tumour-associated antigen identified with monoclonal antibodies (mAb) was in fact EpCAM or 17-1A antigen . In 1994EpCAM has meanwhile advanced to an established epithelial cell marker used in pathological examinations. Likewise, EpCAM can now be considered to be one of the most frequently and most intensely expressed tumour-associated antigens known. It is found expressed on a great variety of human adenocarcinoma and squamous cell carcinoma . These dAt least seven different immunotherapeutic approaches are currently in clinical trials that target EpCAM see . Some ofOnly recently, research on EpCAM started to focus on its role in carcinogenesis. In 2004, first evidence for a direct role of EpCAM in growth-promoting nuclear signalling was reported  as an abbreviation for the ability of the protein to act as an epithelial-specific cell adhesion molecule (EpCAM (CD326) is related to a protein called TROP-2 showing approximately 50% sequence identity. Because TROP-2 is much less investigated than EpCAM in terms of function and tissue distribution, the attendees agreed that, for now EpCAM should not be termed EpCAM-1, and TROP-2 not EpCAM-2, until this is justified by further data supporting a similar function and tissue tropism of the two proteins.epcam.Recently, the gene for EpCAM was renamed from GA733-2 to TACSTD1, the abbreviation for ‘tumour-associated calcium signal transducer protein 1-precursor’. It is also suggested to abandon this gene name as it does not properly reflect the function of the encoded protein and to either revert to the previous gene name, or adopt a new name, for example EpCAM is a type I membrane protein of 314 amino acids (aa) of which only 26 aa are facing the cytoplasm. The extracellular domain is believed to contain two epidermal growth factor (EGF)-like domains. P Baeuerle  presented results corroborating earlier reports showing that the second EGF-like repeat of EpCAM is in fact a thyroglobulin (TY) repeat domain  Figure . ThyroglN-glycosylation sites verified that all three sites in EpCAM are modified in human epithelial cells  reported on the glycosylation status of EpCAM. Point mutation analysis of potential ells see  in contr, EpCAM overexpression on breast cancer cell lines is mandatory for proliferation, migration, and invasiveness of tumour cells  that unravelled the entire pathway of EpCAM signalling from the cell membrane into the nucleus. Epithelial-specific cell adhesion activation molecule signalling was found induced either by soluble EpCAM or through zones of cell–cell contact, suggesting that oligomerisation is a trigger for EpCAM signalling. These signals led to cleavage of EpCAM by proteases, releasing an intracellular peptide termed EpIC. Involvement of proteases in EpCAM signalling was supported by both their co-precipitation with EpCAM and by the inhibitory effects of specific small molecule protease inhibitors, which also reduced EpCAM-mediated gene expression events.vs malignant cells.Two-hybrid screening identified adaptor proteins as intracellular receptors for EpIC allowing the formation of a multiprotein signalling complex shown to be involved in target gene induction and growth-promoting effects. Multicolour immunofluorescence analysis impressively demonstrated cytoplasmic/nuclear redistribution of all the involved proteins depending on EpCAM signalling. An antibody to EpIC was found to give a speckled staining inside the nucleus of stimulated cells, indicating an association of the EpCAM signalosome with specific chromatin sites. With specific inhibitors of EpCAM cleavage, a pharmacological means is given to interfere with EpCAM signalling in tumour cells. Future studies are needed to understand how EpCAM signalling is regulated in normal Support for a mitogenic signalling of EpCAM came from experiments in which EpCAM was overexpressed under control of the MMTV LTR in mammary glands of transgenic mice . Glands of virgin 1.5-year-old mice had hugely overgrown ducts that were dilated, showed extensive budding, and produced milk proteins. Reduced apoptosis and a high-level Bcl-2 expression, as well as increased proliferation (Ki67 marker), were noted.+ DTC in bone marrow of various cancers correlates with a poor survival prognosis of patients  has been established and is now being explored for therapy monitoring (Disseminated tumour cells (DTC) can be detected in bone marrow of cancer patients using a pan-cytokeratin (CK) antibody, as reviewed by K Pantel . Numerous studies have shown that the occurrence and number of CKpatients . A propobona fide tumour cells significantly increased with progression from M0 to BR, and further to M1 stages from 9 to 16–33%. Epithelial-specific cell adhesion activation molecule+ tumour cells had double the amount of chromosomal aberrations than CK+ tumour cells, and these aberrations affected different chromosome locations. There was only a small overlap between EpCAM+ and CK+ DTC populations of 9.5%. Epithelial-specific cell adhesion activation molecule thus defined a subpopulation of DTC in prostate cancer patients that – unlike CK+ DTC – already expanded during biochemical relapse and had a phenotype different from that of CK+ tumour cells.C Klein , a pioneer in the study of single DTC for genomic aberrations and transcriptional patterns , investiExpression of EpCAM on DTC and CTC, which are suspected to include early progenitor cells for metastases, is consistent with a role of EpCAM in tumour growth and progression, and stem cell fate. Future studies need to investigate and corroborate whether EpCAM is a marker for highly tumorigenic cancer stem cells, as has recently been suggested (P=0.015), and that there is a decrease in EpCAM expression with increasing Gleason score (P=0.011). In the subgroup of pT2 adenocarcinoma of the lung, a high EpCAM expression was correlated with a better prognosis of patients.Another topic of the symposium was the question why EpCAM expression had in some tumour types a negative, in most a seemingly neutral and in other tumour types, a positive prognostic impact on the overall survival of patients . P Went G Gastl  reviewed the expression of EpCAM in human breast cancer and its strong negative correlation with survival parameters in node-positive patients (P=0.0002)  reported a negative correlation between EpCAM expression and overall survival in patients with squamous cell carcinoma of the oesophagus, a very deadly disease with no established systemic treatment option. Although normal epithelium showed no EpCAM expression, up to 41% of patients showed high-level expression with a strong negative prognostic impact  and analysis of the methylation and amplification status of the EpCAM gene . No differences in promoter methylation could be found in breast cancer M Mitas  presented data from screening for genes commonly expressed in metastatic lymph nodes from lung, breast, and pancreas cancers but not in normal lymph nodes. Epithelial-specific cell adhesion molecule and activation molecule, TROP-2, CK8, CK19, and claudin-3 were among the few commonly expressed genes, which fell into two clusters. Upregulation of Ets family transcription factor Esx/Elf3 in metastatic lymph nodes correlated well with expression of EpCAM, and is being investigated as potential transcriptional regulator.G Schlimok  reviewed the clinical development of the anti-EpCAM mAb edrecolomab . This well-tolerated murine IgG2a antibody, which was temporarily marketed in Germany, showed a significant increase in overall survival of colorectal cancer patients in the adjuvant setting in two out of four trials  and complement-dependent cytotoxicity (CDC), and shows high antitumour activity in a lung metastasis mouse model when equipped with a murine Fc portion of the γ2A subtype, which is better compatible with the murine immune system than the human γ1 subtype. The speaker also presented preclinical data on MT110, a single-chain EpCAM/CD3-bispecific antibody construct of the BiTE class  described adecatumumab (MT201), a fully human anti-EpCAM antibody  reviewed preclinical and clinical data of catumaxomab, a hybrid mouse IgG2a/rat IgG2b antibody that is trispecific for EpCAM, CD3 and -via its Fc portion- for antigen-presenting as well as a variety of other cytotoxic immune cells. In mouse tumour models the trioma format shows high antitumour activity and survival of which part is due to a vaccination effect . Hybrid antibodies containing one half of the EpCAM-specific antibody HEA125 and one half of the anti-CD3 antibody OKT3 were generated by the hybrid-hybridoma technique. Preclinical experiments showed that T cells isolated from malignant ascites support a redirected lysis of tumour cells Pseudomonas exotoxin. The linker contains a furin cleavage site that allows for release within the endosome of the toxin after EpCAM binding and endocytosis. A conformational change of the cleaved exotoxin enables its cytosolic entry and highly efficient inhibition of the cell's protein synthesis. A novel EpCAM-directed immunotoxin is under development that has the furin cleavage site replaced by a site cleaved through matrix metalloproteinases-2 and -9, as are selectively expressed by tumour cells. This enables a dual targeting that may increase the immunotoxin's therapeutic window. In vitro data support that cell lines lacking MMP−9 and −2 are much less vulnerable to the immunotoxin, and that specific MMP inhibitors partially protect cells expressing the proteases from the immunotoxin. Another EpCAM-directed therapy presented by the speaker uses liposomes with single-chain anti-EpCAM antibodies linked via polyethylene glycol moieties. These long-lived liposomes are being loaded with a mix of anti-apoptotic antisense molecules specific for Bcl-2 and Bcl-XL or with doxorubicin. Xenotransplant mouse models designed for studying the targeting of [3H]-labelled liposomes and antitumour activity support the usefulness and potency of this approach and a combination of liposomes with anti-apoptotic and chemotherapeutic payloads.U Zangemeister-Wittke  reported on the development of an EpCAM-specific immunotoxin called Proxinium (4D5MOC31-ETA) that currently is in a pivotal phase II/III trial for local treatment of head & neck cancer. An earlier phase I/II study has shown a tumour control rate of 88% in which 25% of injected lesions showed a complete response. Median survival of treated patients was 301 days compared with 125 days for a historic control group. The same antibody construct is now being developed for local treatment of bladder cancer . The immunotoxin uses a stability-engineered single-chain humanised anti-EpCAM antibody fused to a subunit of the bacterial D Herlyn  reviewed progress on using EpCAM as a vaccine to elicit tumour-specific T-cell and humoral immune responses. A variety of approaches were tested in small un-controlled clinical trials that use for vaccination an anti-idiotypic antibody (BR3E4), the extracellular domain of EpCAM or EpCAM encoded by an adenovirus. Evidence for specific T-cell responses and antigen spreading could be obtained.In summary, a fair number of EpCAM-directed immunotherapies are under development as therapeutics exploiting the frequent expression of the antigen on adeno and squamous cell carcinoma . Althougde novo in certain squamous cell carcinoma relative to their respective normal epithelia, EpCAM expression on colon carcinoma just remains at the high level of normal colonic tissue. The basis for a therapeutic window of EpCAM-directed therapies was thus lively debated at the symposium.Already 20 years ago, it had been realized that EpCAM is also expressed on a wide range of normal epithelia . With increasing dose, the human EpCAM-specific IgG1 antibody USB54 stained an increasing number of epithelial tissues in transgenic mice. At the low 10 μg dose, the staining of pancreas, kidney and small intestine by the antibody was most prominent. Apparently, animals showed no adverse events even at the 1-mg dose despite the apparent binding of a potentially cytotoxic human IgG1 antibody to epithelial cells of many organs. It is unclear whether the co-expressed murine EpCAM in the transgenic model could prevent cytotoxic effects of the antibody. Animal models of this kind will in the future be helpful in deciphering the basis for a differential effect of antibodies on normal vs tumour tissue.C Potten  set the stage for this discussion by reviewing in a very comprehensive manner the epithelial architecture, cellular dynamics, and fates of epithelial and stem cells in small intestine. P McLaughlin  investigated the consequences of targeting monoclonal antibodies to normal and tumour tissue in a transgenic murine model expressing human EpCAM. Antibodies were administered i.p. and murine tissue staining for bound antibodies performed 24–48 h later. Doses of 1, 10, 100, and 1000 vs tumour cells may come from recent studies identifying a number of other proteins found in close association with EpCAM within the cell membrane. F Le Naour  presented evidence on the interaction of EpCAM with tetraspanins, a family of 4-transmembrane domain proteins with diverse biological functions. One of them, CD9, was shown by crosslinking experiments to directly bind EpCAM whereas other proteins including ADAM10, integrins, G proteins, and a novel CD9 binding protein (CD9P-1) were indirectly associated. Intriguingly, loss of CD9 expression correlates with metastasis  reported an association of EpCAM with CD44 splice variants v4–v7 and tetraspanin D6.1A. Partial cholesterol depletion destroyed these complexes, indicating their co-localisation within membrane microdomains. A novel interaction of EpCAM was found with the serine-phosphorylated version of claudin-7, a protein found in tight junctions and the basolateral side of epithelia. Crosslinking experiments implied that interaction with EpCAM was direct and does not require the EGF-like and TY domain for binding see . Interesvs tumour tissue and the basis for a therapeutic window of some but not all EpCAM-directed immunotherapies. Certain antibody epitopes on EpCAM may only become accessible when accessory proteins are reduced on malignant cells or when EpCAM upon high-level overexpression outnumbers its protein partners. Subsequent oligomerisation of EpCAM may then provide a constitutive proliferation signal for tumour cells.The new insights into membrane protein complexes containing EpCAM may be the key to understand both the control of EpCAM signalling in normal This meeting has provided major insights into the role of EpCAM in cancer, and ongoing research promises to broaden this understanding. A focus of future research will be a more detailed understanding of EpCAM signalling in the nucleus, the regulation of EpCAM signalling as may be controlled via interaction of EpCAM with itself and other proteins in the plasma membrane, the expression and role of EpCAM on normal and cancer stem cells, and the study of clinical efficacy of a number of EpCAM-directed immunotherapies. It is hoped that this meeting review will stipulate more research on EpCAM and that a common name will be adopted by the scientific community.