Despite the uniform selection criteria for the isolation of human mesenchymal stem cells (MSCs), considerable heterogeneity exists which reflects the distinct tissue origins and differences between individuals with respect to their genetic background and age. This heterogeneity is manifested by the variabilities seen in the transcriptomes, proteomes, secretomes, and epigenomes of tissue-specific MSCs. Here, we review literature on different aspects of MSC heterogeneity including the role of epigenetics and the impact of MSC heterogeneity on therapies. We then combine this with a meta-analysis of transcriptome data from distinct MSC subpopulations derived from bone marrow, adipose tissue, cruciate, tonsil, kidney, umbilical cord, fetus, and induced pluripotent stem cells derived MSCs (iMSCs). Beyond that, we investigate transcriptome differences between tissue-specific MSCs and pluripotent stem cells. Our meta-analysis of numerous MSC-related data sets revealed markers and associated biological processes characterizing the heterogeneity and the common features of MSCs from various tissues. We found that this heterogeneity is mainly related to the origin of the MSCs and infer that microenvironment and epigenetics are key drivers. The epigenomes of MSCs alter with age and this has a profound impact on their differentiation capabilities. Epigenetic modifications of MSCs are propagated during cell divisions and manifest in differentiated cells, thus contributing to diseased or healthy phenotypes of the respective tissue. An approach used to reduce heterogeneity caused by age- and tissue-related epigenetic and microenvironmental patterns is the iMSC concept: iMSCs are MSCs generated from induced pluripotent stem cells (iPSCs). During iMSC generation epigenetic and chromatin remodeling result in a gene expression pattern associated with rejuvenation thus allowing to overcome age-related shortcomings . The importance of the iMSC concept is underlined by multiple clinical trials. In conclusion, we propose the use of rejuvenated iMSCs to bypass tissue- and age-related heterogeneity which are associated with native MSCs. However, per definition in vitro cultured MSCs and their mesodermal differentiation potential are not comparable with the in vivo case  /medicinaivo case . Besidesivo case .MSCs have shown good therapeutic results in a plethora of studies and clinical trials , yet theMSCs are further characterized by predominantly beneficial immunomodulatory properties which is not in the focus of this review but described in more detail, e.g., by MSCs from distinct sources such as bone marrow or adipose tissue differ in certain aspects, e.g., Heterogeneity reflects predisposition for dedicated lineages as proposed by In this study we investigate the heterogeneity of MSCs from distinct tissues and individuals reflecting distinct genetic backgrounds and ages. We further compare MSCs to MSC populations of distinct tissue origins such as urine-derived renal progenitor cells UdRPCs;  and hepaGSE97311 with bone-marrow-derived aged and fetal MSCs and iMSCs  on two levels: first MSC gene expression microarray data from the same platform (Illumina Human HT-12) was downloaded to enable full comparison of expression signals. These datasets are shown in nd iMSCs , GSE1491ved MSCs , GSE7722al cells , GSE7727ved MSCs , GSE5966ved MSCs , and GSEved MSCs . Second, UdRPCs; , GSE1004ved MSCs , and GSEte cells .lumi  expression values of the comparable dataset with accession no. GSE52841 and applying this model to predict the detection p-value from the logarithmic expression values of dataset GSE149171. A threshold of det-p-value < 0.05 was employed to judge if a gene was expressed. Imported Illumina data was transformed to the log2 scale and normalized via quantile normalization. Hierarchical clustering analysis for the generation of the dendrogram was performed with the method hclust using Pearson correlation as similarity measure and complete linkage as agglomeration method and the package dendextend  and summarized Illumina next-generation sequencing (NGS) data listed in affy  for the lication . A detecsion 103 .p-value < 0.05 (microarrays) or FPKM > 1 (NGS data). Venn diagrams were generated with the R package gplots  and induced pluripotent stem cells (iPSCs) fall into the meta-group PSC, UC-derived, and fetal MSCs into the meta-group young as well as iMSCs for which we had shown the rejuvenated phenotype in a previous publication (adult. young MSCs (yellow color bar) cluster separately from adult MSCs (blue color bar). However, in a superior cluster, young and adult MSCs are joined and cluster separately from PSCs (red color bar) of embryonic or iPSC origin. This grouping of samples reflects the differences between pluripotent and multipotent cells as manifested in their transcriptome. Adult and young MSCs fall into the same group of multipotency but are distinguishable between each other pertaining to age-related heterogeneity within MSCs. Interestingly, kidney perivascular stromal cells cluster with adult MSCs thus confirming the common nature of MSCs and pericytes as suggested by In a meta-analysis of transcriptome data, we compared MSCs of distinct sources which includes bone marrow (BM), adipose tissue (AT), umbilical cord (UC), cruciate, tonsil, kidney, and iMSCs listed in lication . The remp-value < 0.05) on the same technical Illumina Human HT-12 microarray platform. The venn diagram in primary metabolic process (p = 6E-108) and cellular macromolecule localization (p = 1.1E-96).We set out to further investigate heterogeneity between distinct MSC types by comparing gene expression  in the specific subsets revealed GO terms of which the 20 most significant are listed here. These GO terms reflect the heterogeneity characterized by the specific original microenvironment of the MSCs: significant GO terms in fetal MSCs relate to developmental and morphogenetic processes , signifiIn a follow-up analysis we aimed at extending the transcriptome comparison of MSCs to MSC populations of distinct tissue origins measured on different microarray platforms and by RNAseq. in vitro . Several substances such as ascorbic acid or lactoferrin have been described to be able to reduce ROS levels and delay cellular senescence. Another relevant feature in aged cells is declining mitochondria function. Thus, several approaches target to improve mitochondria function in aged cells by upregulation of key proteins such as HSPA1L or FGF21. Also, the specific over-expression of key transcription factors such as FOXP1, YAP, or FOXD1 support the rejuvenation process. Another angle for attenuating aging is related with autophagy management. Inhibition of pathways such as mTOR can increase autophagy mechanisms and as such support protein homeostasis which is diminished in aged cells . A recenWe further aimed at identifying factors contributing toward the heterogeneity phenotype typical of MSCs and their reversion to the rejuvenated state associated with iMSCs. Epigenetic modifications are crucial for determining the gene expression profile of a given cell type. They are defined as mechanisms which enable or prevent access of the transcription machinery to genomic loci in a heritable and at the same time flexible manner . The mosEpigenetic factors have been described to regulate MSC heterogeneity in two distinct ways: (1) tissue- specific gene expression patterns and (2) age-specific gene expression patterns . Togetherunt-related transcription factor 2 (RUNX2) and peroxisome proliferator activated receptorγ (PPARγ) revealed an opposing profile with RUNX2 being demethylated in BM MSCs and methylated in AT MSCs while PPARγ was unmethylated in these cells and methylated in BM MSCs  favor adipogenesis of BM MSCs , while iWhen we analyzed the expression level of a panel of epigenetic factors described by With aging, the differentiation potential of BM MSCs changes from osteogenic to more adipogenic which is associated with impaired bone regeneration and a higher risk for osteoporosis . This isTo overcome age-induced impairments of MSCs, we and others have derived MSCs from iPSCs, so called iMSCs which have a rejuvenated transcriptome and epigenetic profile  and thusin vitro and over time the highly proliferative fibroblastoid colony forming units decline continuously from initially 20% to 1% after 2 months of cultivation  . Becauseer cells . MSCs crer cells . Their ser cells . By senser cells . A big aer cells .All these characteristics make MSCs very promising for the use in innovative therapeutic approaches. In line of this, MSCs are or have been used in over 1,000 clinical trials for versatile applications in the various organs and diseases.In total, MSC therapies have been shown to have an acceptable safety profile with parallel beneficial therapeutic effects in several clinical studies. The number of clinics offering stem cells therapy is constantly increasing; so is the number of companies working on MSC related products. But his also comes along with mal-practice and misuse of those cells . Althougin vivo , placenta, or adipose tissue . Allogenin vivo . The usuin vivo . The mos patient , in lineIn addition to the before mentioned use cases, other abilities of MSCs are under investigation. Since MSCs can evade the host immune system partially, MSCs have been employed as drug delivery vehicles for example in anti-cancer treatment since they are also able to home into tumor tissue . Many ofThe general problem of clinical trials is the long certification process which leads to the fact that the final end-therapeutic agent is 5–10 years behind the latest research advances. Although over 1,000 clinical trials using MSCs have been performed, many MSC therapies fail in the later clinical stages, which is reflected by the fact that so far only ten MSC-based products have reached  market approval. One of the main reasons for this are the many layers of heterogeneity associated with the use of MSCs: inter-cellular differences and various cell (sub)populations, inter-tissue differences and inter-donor or inter-recipient variations. Another layer of heterogeneity is introduced by the researchers themselves by the use of different protocols for cryopreservation, up-scaling and administration .in vitro function and the corresponding in vivo effect  animal models require a much lower cell number as compared to the human system where prolonged in vitro culture of MSCs are needed  has finished a phase I clinical trial investigating the safety, tolerability and efficacy using iMSCs which were derived from iPSCs treating steroid resistant acute GvHD in adults with preliminary promising results (NCT02923375). The company furthermore is just recruiting patients for a study to examine the early efficacy of intravenous administration of iMSCs derived from iPSCs in adults which have been brought to an intensive care unit with COVID-19 infection (NCT04537351). In a second phase of this study the effect of iMSCs on the Acute Respiratory Distress Syndrome will be assessed. The company has now announced a Phase 3 clinical trial of CYP-004, its Cymerus mesenchymal stem cell (MSC) product for osteoarthritis. Another study which is performed by the Tongji Hospital (China) in cooperation with the Chinese Academy of Sciences is investigating the safety of ESC-derived iMSCs in treating meniscus injury patients in a phase I clinical trial (NCT03839238). The Department of Urology of the Asan Medical Center in Seoul, South Korea is running a Phase 1 Study to investigate the safety of human ESC-derived iMSCs in the treatment of Interstitial Cystitis (NCT04610359). Another Phase 1 safety study is carried out by The First Affiliated Hospital of Zhengzhou University in China. Aim of this study is to evaluate the safety of ESC-derived iMSC for the treatment of Primary Ovarian Insufficiency (NCT03877471). The Tongji Hospital in China is evaluating the safe clinical use of intrauterine injected human ESC-derived iMSCs to treat moderate and severe intrauterine adhesion. In addition to safety this study should bring first insights for clinical effectiveness in supporting the regeneration and repair process within the endometrial region (NCT04232592)4 vials, each containing 1 × 106 iPSCs which in the end iMSCs stage have reached a number of 2.9 × 1015 iMSCs which represent 29 million therapeutic doses of 1 × 108 cells- all started with iPSC generation from one single blood donation  is the first completed clinical trial for the use of iMSCs . The clidonation . iPSC quanalysis . Based o product . These p product . The iMS product . The rou product .iMSCs as an off-the shelf product with high batch to batch consistency will result in more robust clinical measures . Furtherp-value < 0.05) between iMSCs fetal, MSCs, UC-MSCs, BM-MSCs, and AT-MSCs we found most genes (n = 9966) overlapping with smaller specific subsets of exclusively expressed genes (n between 29 and 558). Over-represented GOs in the exclusive subsets related to developmental processes in the young MSCs, to adipose-tissue-related  in AT-MSCs and bone-related  in BM-MSCs. We extended the comparison to MSCs of other distinct sources  generated on different technical platforms and again found the most genes in the overlap (n = 6637) and fewer genes in the exclusive subsets . Here, over-represented GOs in the exclusive subsets related to developmental processes in the AF-MSCs which may be assigned to the young meta-group, over-represented GO terms in UdRPCs included lipid transport and detoxification pointing at renal properties and over-represented GO terms in HSCs included metabolic, cristae, and mitochondrial processes characterizing liver.In this study on the heterogeneity of MSCs, we comparatively characterized the transcriptomes of MSC subpopulations and the relationships amongst them. We found large commonalities between them but also differences related to the tissue source of the MSCs. In a hierarchical clustering analysis we could stratify the analyzed stem cells into three meta-groups containing PSCs , MSCs of adult origin of various tissues and MSCs of young origin . Comparing the expressed genes  with the other young MSCs  and by clustering with the other young MSCs in heatmaps using our previous aging-rejuvenation gene signature . These aWe propose that the source of the MSCs characterizes the subpopulation by exchanging signals with the microenvironment and by epigenetic programs. We found that iMSCs derived from iPSCs resemble other MSCs of young age such as fetal MSCs and UC-MSCs and are determined by developmental biological processes. That confirms our earlier findings  and may An advantage of MSCs is their more differentiated state compared to PSCs what was considered to protect them from tumorigenicity . HoweverIn conclusion, in this review and meta-analysis comparing MSCs from multiple tissues and donor ages we have found predominant commonalities but also differences contributing to heterogeneity. Differences between subpopulations were related to the tissue source environment which also holds for MSC cells such as UdRPCs showing renal properties and HSCs showing hepatic properties but still bearing more commonalities than differences to other MSC types. iMSCs, MSCs derived from iPSCs, have overlapping developmental biological processes with other young MSCs of fetal or umbilical cord origin and thus confirmed our previous rejuvenation signature . We demoWW, NG, L-SS, and JA wrote the manuscript. WW performed the meta-analysis and follow-up analyses. JA initiated and supervised the study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


PIXE analysis revealed the restoration of near normal value of zinc, copper, and iron, which were substantially altered when compared to carcinogen treated groups. Supplementation of both vanadium and beta-carotene four weeks before DENA injection was found to offer significant  protection against generation of single-strand breaks when compared with the carcinogen control counter parts. A significant stabilization of hepatic architecture of the cells was observed as compared to carcinogen control in vanadium plus beta-carotene treated group. This study thus suggests that vanadium, a prooxidant but potential therapeutic agent yield safe and effective pharmacological formulation with beta-carotene, an antioxidant, in the inhibition of experimental rat hepatocarcinogenesis.Combined effect of vanadium and beta-carotene on rat liver DNA-chain break and Proton induced X-ray emission (PIXE) analysis was studied during a necrogenic dose (200 mg/kg of body weight) of Diethyl Nitrosamine (DENA) induced rat liver carcinogenesis. Morphological and histopathological changes were observed as an end point biomarker. Supplementation of vanadium (0.5 ppm  Trace element studies on cancer and normal tissues have interested groups of researchers for many years . AccumulV), which is an endogenous constituent of all or most mammalian tissues, is believed to have a regulatory role in biological system . Despite these protective enzymes, some 02- and H2O2 may escape and in the presence of decompartmentalized Fe, are catalyzed to more reaction reactive oxygen species (ROS) [V, BC and V plus BC respectively) could be explained by the ability of liver cells to form SOD-model compounds in vivo. These complexes scavenge super oxide radicals and account for the antitumor activity of vanadium and beta-carotene in rat hepatic neoplasia presumably by stabilizing the genome.The distribution of trace element levels in various organs indicates a significant differentiation between normal and malignant tissue, which is in good agreement with several previous findings . It is wl., 1984  reportedhemistry . H2O2 ises (ROS) . This coes (ROS) . In the In vitro and in vivo data suggest that Fe may be capable of mutagenic effects mediated through free radical generation or tumor promotion through nutritional mechanisms [V and BC to DEN-induced animals in Group E . At present it is doubtful that minor changes in mineral levels induced by V and BC combination played a major role in tumorigenesis in this experiment, but the possibility cannot be excluded.Several studies have shown augmentation of tumor growth in rodent models with Fe supplementation ,28. In vchanisms . As a trchanisms . This is E Table  could bein vivo could be used as an early biomarker for carcinogenic process. The results documented indicate that vanadium plus beta-carotene at a given dose can modulate other trace element concentration in serum by some unknown mechanism that is yet to be elucidated and in so doing preserve the genetic stability. The study remains noteworthy in the aspect that it records V and BC both-mediated suppression of DNA-strand break at the initiation stage in addition to essential / trace elemental concentrations alteration(s) in the liver and in the maintenance of the normal hepatic architecture during the preneoplastic stage of liver carcinogenesis.These studies suggest that trace element concentration Author 1 MBC carried out the rat hepatocarcinogenesis, morphology, morphometry and histopathological studies. Author 1 MBC and author 3 IK made DNA-chain break studies. Proton induced X-ray emission analysis and assessment was made by author 2 SM and author 4 VV and author 5 MD. Author 6 NBK, a senior medical specialist has done the expert consultant for the pathological aspects of the work. The planning, design and coordination of the experimental work was made by author 7 MC, the principal investigator.


Psychosocial factors are thought to influence risk and survival from cancer. We have previously studied specific behaviours in transgenic male CD-1 MT42 mice, which overexpress the gene encoding human transforming growth factor alpha  in multiple tissues, and which develop a high incidence of spontaneous hepatocellular carcinoma. The male TGF alpha mice spent a lengthened time immobile in the swim test, were highly aggressive, had increased plasma levels of 17 beta-estradiol (E2), and reduced natural killer (NK) cell activity. The female transgenic MT42 TGF alpha mice do not develop an increased rate of tumours at any site. We hypothesised that if the alterations in male TGF alpha mice are associated with their development of hepatocellular carcinomas, female TGF alpha should not show these alterations. The data in the present study indicate that female TGF alpha mice display shortened immobility in the swim test, suggesting an improved ability to cope with stress, and appear less aggressive in the resident-intruder test than non-transgenic female CD-1 mice. The female TGF alpha mice also exhibit a 3-fold increase in the plasma levels of E2, and a 3-fold increase in NK cell activity. These findings suggest that the elevated expression of TGF alpha in the transgenic mice is associated with gender-specific behavioural alterations, and the development of spontaneous hepatocellular tumours in the males. Furthermore, TGF alpha alters hormonal and immune parameters similarly in both sexes. It remains to be determined whether the development of hepatocarcinoma in the male TGF alpha animals is associated with an impaired ability to cope with stress and elevated aggressive tendencies and/or whether manipulations leading to an impaired ability to cope with stress will promote tumourigenesis in female TGF alpha mice.


Arabidopsis thaliana and rice (Oryza sativa) genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species.Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species . Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function.Despite the differences in genome sizes between The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species. The proper functioning of a cell is ensured by the precise regulation of protein levels that in turn are regulated by a balance between the rates of protein synthesis and degradation. Protein degradation is mediated by proteolysis, which paves the way for recycling of amino acids into the cellular pool. In addition to degradation, regulated proteolysis plays a major role in diverse cellular processes by regulating biochemical activities of proteins in concert with other cellular mechanisms such as post-translational modification,2. SerinArabidopsis thaliana, most of these studies are either restricted to specific families [Arabidopsis thaliana and Oryza sativa constitute model systems for Dicotyledons and Monocotyledons respectively, the major evolutionary lineages in angiosperms[Serine proteases appear to be the largest class of proteases in plants. Consistfamilies -13 or spgiosperms,15. The Arabidopsis and rice serine protease-like proteins, phylogenetic analysis was performed based at their protease domains. Cross genome comparison of the serine proteases across the two genomes has led to the identification of members likely to be conserved across the two species. Predictions of the sub-cellular localizations of serine protease-like proteins to obtain further insight into their possible functional associations were carried out using TargetP[Arabidopsis and rice serine proteases that are likely to be involved in basic functions associated with various physiological processes in plants would be useful in expanding the current understanding of these processes in regulating plant growth and development. This would prove beneficial in understanding protease processes in plants and application of this knowledge to important crop species.The current analysis aims at the identification and analysis of serine protease families encoded in the genomes of the two plant species. The domain organization of the putative serine proteases in the two plant genomes have been analysed employing several sensitive sequence search methods -18 that g TargetP. CompariArabidopsis and rice (125 Mb and 389 Mb respectively) and encoded number of genes  from rice are predicted to contain PDZ-like domains C-terminal to the protease domain, while the rest appear to be a single protease domain containing proteins. The absence of PDZ-like domains in majority of plant DegP-like proteins has been suggested to have as of yet unclear functional implications for these proteins[Arabidopsis DegP1 (At3g27925) was found to be chloroplast localised and was rapidly upregulated in response to elevated temperatures[16 DegP-like proteins could be identified in peratures. We idenArabidopsis and rice DegP-like proteins showed that a majority of sequences cluster into three major clades, supported by high bootstrap values, with additional members less related to the three clusters  with no other structural similarity posing aArabidopsis and rice are characterised by significantly larger number of subtilases in comparison to other organisms such as human, Caenorahabditis and Drosophila, suggesting that expansion of the subtilase gene family in the two plant species is due to multiple duplication events[A large number of subtilisin-like serine proteases have been identified in various plant species, where they have been implicated in diverse processes  domain, that occurs as a C-terminal insertion in the Peptidase_S8 domain of a majority of subtilisin-like proteins identified in these two plant species . However, the presence of gene products in both the proteomes with only the protease domain suggests that this domain alone is sufficient for the activity of these enzymes  family of serine peptidases belong to the α/β hydrolase fold grouped into SC clan of serine peptidases. The family includes members of different types and with distinct specificities. For example, prolyl oligopeptidase is an endopeptidase localised to the cytosol, while dipeptidyl peptidase IV (DPPIV) is a membrane bound exopeptidase. According to MEROPS database, POPs maPOPs have been implicated in degradation of biologically important peptides such as peptide hormones and neuropeptides associated with learning and memory and therefore have become significant targets for drug design. They haArabidopsis and 23 in rice. A majority of these either lack a predicted signal sequence or are predicted to localise to chloroplast and mitochondria. Only few gene products identified here are predicted to carry a β-propeller N-terminal to the protease domain ; At5g24260 ; At2g47390, LOC_Os03g19410, LOC_Os07g48970  [Serine carboxypeptidase-like proteins (SCPLs) have been identified in several plant species, where they form large and diverse gene families in contrast with prokaryotes and other eukaryotes. Plant SCPLs have been implicated in several biochemical processes including secondary metabolism  and SMT/SNG1 (At5g09640) have been shown to function as acyltransferases in phenylpropanoid metabolism[Arabidopsis SMT is a heavily glycosylated protein that localises to central vacoules of the leaf tissue, suggesting that some SCP-like proteins identified in the two plant species may also be vacoule localised[We identified 54 genes coding for serine-carboxypeptidase (SCP)-like proteins in CHC; Pfam accessioCHC; Pfam accessioetabolism,43. Studlocalised.Arabidopsis and rice SCPL proteins cluster into two major clades, but there are some additional SCPL proteins that are less closely related to members of the clades (See Arabidopsis SCPL proteins), Clade IB (includes only rice SCPL proteins) and Clade IC (includes a pair of Arabidopsis and rice SCPL proteins). Clade IA includes known SCPL acyltransferases such as SMT (At2g22990) and SCT (At5g09640). Clade IA and IB consist exclusively of Arabidopsis and rice sequences respectively that have no identifiable orthologues in either subcluster. Therefore these two subclades may represent early diversification of certain SCPL proteins followed by their lineage specific delineation and expansion subsequent to speciation  proteins were the first to be identified and are the most common beta-lactamases. All these proteins share sequence similarity within the class but not with members of other classes. However, proteins of different classes share significant structural similarity and are believed to be homologues that have undergone divergent evolution. Structure-based phylogeny studies suggest that beta-lactamases are ancient enzymes that probably evolved as means of protection against beta-lactam antibiotics. Site-diSerine beta-lactamases are widespread in bacterial genomes, and their corresponding genes may occur on bacterial chromosomes or on plasmids. This allows for their transfer to distant species and may account for their distribution and diversity. Serine Arabidopsis and rice proteomes, both of which are predicted to localise to mitochondria. An interesting feature is the presence of a single ABC1 domain . Though the protease domain is capable of proteolysis on its own, the ATPase subunits are essential for effective levels of proteolysis. Structural analysis  that share comparatively low pairwise sequence identity with each other, as opposed to the members within a cluster that share significant sequence similarity with each other  fall into a single gene cluster (Clade I) and share between 36 and 72% pairwise sequence identity with each other , a central ATPase domain (AAA+ module) and a C-terminal proteolytic domain (Lon_C). The N-terminal LON domain along with the AAA module is believed to impart substrate specificity to Lon proteases[Lon proteases are also a group of ATP-dependent serine proteases. Unlike the Clp proteases, the catalytic and ATPase domains of the Lon proteases reside in the same polypeptide. They are conserved in prokaryotes and eukaryotic organelles such as chloroplast and mitochondria. proteases,53. StruArabidopsis has been implicated in the control of cytoplasmic male sterility[Unlike Clp proteases, Lon proteases appear to be present in organisms from all forms of life. They are believed to play a significant role in removal of aberrant proteins and thereby maintain cellular stability. Interestingly, an archaeal membrane bound Lon protease was found to have both ATP-dependent and ATP-independent proteolytic activities towards folded and unfolded proteins respectively. Little sterility.Arabidopsis and an equal number in rice. In addition, we also identified five protein sequences containing only the Lon N-terminal domain in Arabidopsis and four in rice (data not shown). Of the Arabidopsis Lon protease-like proteins, At3g05790 and At5g26860 gene products are predicted to localise to chloroplast, while At3g05780 and At5g47040 gene products are predicted to be mitochondria localised. All four Lon protease-like proteins identified in rice are predicted to localise to chloroplasts. The Lon-protease like proteins identified in the two plant species share higher similarities with the LonA subfamily . The largest of these clusters consists of five sequences , all of which, except At3g05780 are predicted to localise to chloroplasts and share between 76 and 91% pairwise sequence identity with each other. The second cluster consists of two sequences (At5g47040 and LOC_Os09g36300) that are 82% identical to each other but predicted to have different subcellular localizations. At5g47040 is predicted to localise to mitochondria and LOC_Os09g36300 is predicted to localise to chloroplasts. The members of the two clusters display between 40 and 45% pairwise sequence identity with each other. A Lon protease like gene product from rice, LOC_Os06g05820, is less closely related and shares between 19 and 25% pairwise sequence identity with the rest of the Lon protease like gene products from the two species.Sequence analysis reveals that Signal peptidases (SPases or leader peptidases) are a diverse group of serine endopeptidases, that belong to MEROPS family SE.coli lepB) and homologues have subsequently been identified in archaea, mitochondrial inner membrane, the thylakoid membrane of chloroplasts and the endoplasmic reticulum membranes of yeast and higher eukaryotes[The Type I SPases are membrane-bound serine endopeptidases that were first identified in bacteria  has been found to be required for complete maturation of the plastid protein translocation channel Toc75. It is proposed that Plsp1 may have multiple functions and is believed to be essential for biogenesis of plastid internal membranes[In bacteria, Type I SPases appear to be essential for cell viability and their inactivation invariably leads to cell death. Therefore they have been attractive targets for antibacterial drugs,58. In henal side,60. An Amembranes.Arabidopsis and seven in rice. A majority of these gene products are predicted to localise to mitochondria, though some are predicted to be chloroplast localised, membrane bound and even secreted , though predicted by TargetP[We have identified nine genes coding for signal peptidase-like proteins in y TargetP to be miy TargetP, suggestArabidopsis and rice Type I SPase-like proteins cluster into two major clades supported by high bootstrap values and high pairwise sequence identity , dipeptidyl-peptidase II and thymus specific serine peptidase and is grouped in MEROPS clan SC Members of this family have been identified exclusively in eukaryotes and have been implicated in several processes  belong to MEROPS family SE. coli Tsp) are believed to play an important role in degradation of improperly synthesised proteins. For instance, proteins translated from damaged mRNA are modified by addition of a C-terminal tag, which enables their recognition and degradation by CPPs[In bacteria, CPPs  are predicted to be mitochondria localised, while a single gene product LOC_Os06g21380 is predicted to be translocated to chloroplasts , an inner membrane protein was the first protein identified with signal peptide peptidase activity  is a light-inducible membrane-bound protein with an unusual monotropic arrangement, associated with thylakoid membranes and has been proposed to be involved in light dependent degradation of photosystem II and LHCII antenna proteins[Protease IV family appears to be represented by single members in y TargetP to be mi proteins. A singls SppA Atg73390 isRhomboid proteins are a family of serine proteases that cleave substrates within transmembrane domains,70. RhomArabidopsis AtRBL2 was recently isolated and shown to have proteolytic activity and substrate specificity[Drosophila ligands Spitz and Keren but not similar proteins like TGFα, when expressed in mammalian cells, thereby releasing soluble ligands from the cell into the media. AtRBL2 along with AtRBL1, another Arabidopsis rhomboid homologue was found to localise to Golgi apparatus in plant cells consistent with previous observations where several eukaryotic rhomboid proteins have been predicted to localise within Golgi apparatus[Rhomboid like proteins have been identified in plants, however, there was no experimental information available about them till recently. A rhomboid homologue from ecificity. AtRBL2 apparatus,74. InteArabidopsis and 18 in rice. Subsequent to multiple sequence alignment, it became evident that several proteins appeared to be catalytically inactive, owing to loss or mutation(s) in residues believed to be essential for catalysis. While the role of these catalytically compromised proteins remains unclear, it is believed that they may function as regulators of active rhomboid proteases[Arabidopsis gene products  and two gene products from rice  are predicted to possess a ubiquitin associated (UBA) domain, C-terminal to the rhomboid protease domain. An Arabidopsis gene product At3g17611 is predicted to have a zinc finger-like module C-terminal to the predicted rhomboid protease domain  that were earlier identified as members of the PARL subfamily, the other major subfamily of rhomboid-like proteins[At1g18600, At1g74130, At1g74140 and LOC_Os01g55740; 29–56% pairwise sequence identity), Clade IV, consisting of two , Clade V, consisting of two  and Clade VI consisting of two  sequences respectively  were found to carry an additional UBA domain and At3g17611 was found to carry a zf-RAN domain C-terminal to the rhomboid protease domain  share high pairwise sequence identities with each other (see above) but share low sequence identity with other rhomboid-like proteins identified in the two plant genomes and are likely to have diverged early in evolution. Our results may in part be explained by previous observations by Koonin and coworkers that reveal a polyphyletic origin for eukaryotic rhomboids and the possibility of a bacterial origin for rhomboid-like proteins and their subsequent dissemination in other lineages through multiple horizontal gene transfer events followed by extensive gene duplication.HisPheSer motif and an intein-like mechanism for autoproteolysis - Domain architecture SN-S8-PA-zfCCHC - Domain architecture Ex2-S8-zfCCHC-rve - Domain architecture S8-zfCCHC-rve. The putative gene product carries a zhCCHC domain and an rve domain C-terminal to the protease domain - Domain architecture SN-S8-PA-Arf2-C2. The putative gene product carries an Arf2 and a C2 domain C-terminal to the subtilisin protease domain - Domain architecture S10-Transposase_21 - Domain architecture S10-Retrotrans_gag - Domain architecture ABC1-S12. The putative gene products are associated with an ABC1 domain N-terminal to the serine β-lactamase domain - Domain architecture S54-UBA. The putative gene products are associated with an UBA domain C-terminal to the rhomboid protease domain - Domain architecture S54-zfRanBP. The putative gene product is associated with a zf-RanBP domain C-terminal to the rhomboid protease domain , which has been associated with lateral root emergence[Arabidopsis counterpart and whether they display redundancy in function. Similarly, Arabidopsis ARA12 [Arabidopsis.When comparing multi-gene families between species it is frequently observed that several genes in one species may collectively be orthologues of a single gene in the other, indicating recent duplications exclusive to the former. In such cases, knowledge of gene function of certain members allows confirmation of orthologous and paralogous relationships. One such instance is emergence and has Arabidopsis and rice genomes and were able to identify instances of segmentally duplicated genes corresponding to certain families of serine proteases in Arabidopsis and rice genomes  proteomes were obtained from The Institute for Genomic research (TIGR[Sequences encoded by the complete rch (TIGR).Arabidopsis and rice proteomes, using Type sequences for each serine-protease family as classified by MEROPS[-3.a. A preliminary search for serine-proteases was performed using BLASTP on Arabiby MEROPS as queriby MEROPS employs b. The protein sequences thus obtained were scanned further against a dataset of hidden Markov models (HMMs) obtained from the Pfam A database, employiArabidopsis and rice genome sequence was matched to sensitive protein family profiles obtained from the PfamA database[-5 by IMPALA[c. A complimentary approach of matching sequences to a database of annotated profiles was also employed. Each  database, using I database. A sequeby IMPALA was consArabidopsis and rice genome sequences was scanned against protein family profiles obtained from the NCBI  conserved domain database (NCBI-CDD[d. A similar approach was carried out where each of (NCBI-CDD), a comp. The list includes gene identifiers, predicted subcellular localization, chromosome location, chromosomal nucleotide position and domain architectures of serine proteases identified in current analysisTable S1. An inventory of Click here for fileTable S2. An inventory of rice serine protease-like proteins. An inventory of rice serine protease-like proteins identified by multifold approach (see methods for details). The list includes gene identifiers, predicted subcellular localization, chromosome location, chromosomal nucleotide position and domain architectures of serine proteases identified in current analysisClick here for fileTable S3. Background information on serine proteases. Additional literature information on serine protease families taken up for study in current analysis. The information is categorized into three parts namely a brief structural overview, enzyme characteristics and functional information where known. Additional references for the material contained in the file have been provided at the end.Click here for fileArabidopsis thaliana serine protease-like proteins and their putative orthologues in rice genome (see methods for details). Functional information gathered from literature has been provided for Arabidopsis gene products where possibleTable S4. Orthologous serine protease-like proteins identified in the two plant species. A list of Click here for fileArabidopsis and rice DegP protease-like proteins. A: Multiple sequence alignment of Arabidopsis and rice DegP protease-like proteins. Multiple sequence alignment of the DegP domain region of the annotated Arabidopsis and rice DegP protease-like proteins. The catalytic triad residues are indicated. Gene names correspond to those in Additional files B: Phylogenetic analysis of Arabidopsis and rice DegP protease-like proteins. Unrooted N-J tree computed from multiple sequence alignments of Arabidopsis (red) and rice (blue) DegP protease domains. DegP protease-like protease domains were aligned using ClustalW[Figure SF1. Multiple sequence alignment and phylogenetic analysis of  ClustalW program  ClustalW for reprClick here for fileArabidopsis and rice subtilisin-like proteins. Multiple sequence alignment of the subtilisin domain region of the annotated Arabidopsis and rice subtilisin protease-like proteins. The catalytic triad residues are indicated. Gene names correspond to those in Additional files Figure SF2. Multiple sequence alignment of Click here for fileArabidopsis and rice prolyl oligopeptidase -like proteins Multiple sequence alignment of the Prolyl oligopeptidase domain region of the annotated Arabidopsis and rice prolyl oligopeptidase-like proteins. The catalytic triad residues are indicated. Gene names correspond to those in Additional files Figure SF3. Multiple sequence alignment of Click here for fileArabidopsis and rice serine carboxypeptidase-like proteins A: Multiple sequence alignment of Arabidopsis and rice serine carboxypeptidase-like proteins. Multiple sequence alignment of the serine carboxypeptidase domain region of the annotated Arabidopsis and rice serine carboxypeptidase-like proteins. The catalytic triad residues are indicated. Gene names correspond to those in Additional files B: Phylogenetic analysis of Arabidopsis and rice serine carboxypeptidase-like proteins. Unrooted N-J tree computed from multiple sequence alignments of Arabidopsis (red) and rice (blue) serine carboxypeptidase domains. Serine carboxypeptidase-like protease domains were aligned using ClustalW[Figure SF4. Multiple sequence alignment and phylogenetic analysis of  ClustalW program  ClustalW for reprClick here for fileArabidopsis and rice serine beta-lactamase -like proteins. Pairwise sequence alignment of the serine beta-lactamase domain region of the annotated Arabidopsis and rice serine beta-lactamase-like proteins. The catalytic residues are indicated. Gene names correspond to those in Additional files Figure SF5. Pairwise sequence alignment of Click here for fileArabidopsis and rice serine Lon protease-like proteins. Multiple sequence alignment of the Lon_C protease domain region of the annotated Arabidopsis and rice Lon protease-like proteins. The catalytic dyad residues are indicated. Gene names correspond to those in Additional files Figure SF6. Multiple sequence alignment of Click here for fileArabidopsis and rice serine Type I SPase-like proteins. Unrooted N-J tree computed from multiple sequence alignments of Arabidopsis (red) and rice (blue) Type I SPase domains. Type I SPase domains were aligned using ClustalW[Figure SF7. Multiple sequence alignment of  ClustalW program  ClustalW for reprClick here for fileArabidopsis and rice serine S28 protease-like proteins. Multiple sequence alignment of the family S28 protease domain region of the annotated Arabidopsis and rice family S28 protease-like proteins. The catalytic triad residues are indicated. Gene names correspond to those in Additional files Figure SF8. Multiple sequence alignment of Click here for fileArabidopsis and rice Ctpase-like proteins. Multiple sequence alignment of the Ctpase domain region of the annotated Arabidopsis and rice Ctpase-like proteins. The catalytic dyad residues are indicated. Gene names correspond to those in Additional files Figure SF9. Multiple sequence alignment of Click here for fileArabidopsis and rice protease IV-like proteins. Multiple sequence alignment of the protease IV domain region of the annotated Arabidopsis and rice protease IV-like proteins. The active site serine residue is indicated. Gene names are derived from those in Additional files Figure SF10. Multiple sequence alignment of Click here for fileArabidopsis and rice rhomboid-like proteins. Multiple sequence alignment of the rhomboid protease domain region of the annotated Arabidopsis and rice rhomboid protease-like proteins. The catalytic triad residues are indicated. Gene names correspond to those in Additional files Figure SF11. Multiple sequence alignment of Click here for fileArabidopsis and rice nucleoporin autopeptidase-like proteins. Multiple sequence alignment of the nucleoporin autopeptidase domain region of the annotated Arabidopsis and rice nucleoporin autopeptidase-like proteins. The catalytic residues are indicated. Gene names correspond to those in Additional files Figure SF12. Multiple sequence alignment of Click here for file


After surgical intervention with curative intention in specialised centres the five-year survival of patients with carcinoma of the exocrine pancreas is only 15%. The ESPAC-1 trial showed an increased five-year survival of 21% achieved with adjuvant chemotherapy. Investigators from the Virginia Mason Clinic have reported a 5-year survival rate of 55% in a phase II trial evaluating adjuvant chemotherapy, immunotherapy and external-beam radiation.The CapRI study is an open, controlled, prospective, randomised multi-centre phase III trial. Patients in study arm A will be treated as outpatients with 5-Fluorouracil; Cisplatin and 3 million units Interferon alpha-2b for 5 1/2 weeks combined with external beam radiation. After chemo-radiation the patients receive continuous 5-FU infusions for two more cycles. Patients in study arm B will be treated as outpatients with intravenous bolus injections of folinic acid, followed by intravenous bolus injections of 5-FU given on 5 consecutive days every 28 days for 6 cycles. A total of 110 patients with specimen-proven R0 or R1 resected pancreatic adenocarcinoma will be enrolled. An interim analysis for patient safety reasons will be done one year after start of recruitment. Evaluation of the primary endpoint will be performed two years after the last patients' enrolment.The aim of this study is to evaluate the overall survival period attained by chemo-radiotherapy including interferon alpha 2b administration with adjuvant chemotherapy. The influence of interferon alpha on the effectiveness of the patients' chemoradiation regimen, the toxicity, the disease-free interval and the quality of life are analysed. Different factors are tested in terms of their potential role as predictive markers. Only 10–20% of patients with pancreatic cancer can be resected with curative intent at the time of diagnosis. Loco-regional recurrence and/or metastatic disease develop in the majority of patients who undergo pancreatic resection. Relapse occurs within 9–15 months after initial presentation and patients have median life expectancies of only 12–15 months without adjuvant therapy. The 5-year survival rate of patients with resected pancreatic adenocarinoma is approximately 20%  or during chemotherapy [study arm B] patients will be assessed with laboratory evaluation: complete blood count and blood chemistries weekly. Laboratory parameters in study arm A will be evaluated before each dose of cisplatin. Creatinine will be determined also one day after administration of cisplatin. Blood from members of study arm A will be investigated weekly for immunological markers.Vital signs (blood pressure and pulse rate) and temperature are controlled daily during treatment. Patients are evaluated prior to receiving chemoradiation or chemotherapy. Patients enrolled in study arm A are evaluated weekly by the medical oncology and radiation oncology team during treatment. The team will check patients at each visit for symptoms due to therapy; a physical examination and complete safety labs should be performed. The patients' mental state will be investigated weekly. The questionnaire CES-D will be used as support. The three module questionnaire will be filled out during weeks 3 and 6 (study arm A) or week 3 and week 24 for study arm B.During post-chemoradiation, infusional 5-FU (Study Arm A) patients will be evaluated by a physician prior to treatment and every 2 to 3 weeks with clinical assessment and laboratory parameters including a CBC, electrolytes, BUN, and creatinine. If patients undergo post chemoradiotherapy infusional 5-FU coordinated by an outside medical oncologist, a study nurse will contact the patient at least once weekly by telephone.In the post-treatment period patients will be seen every 3 months by the surgical service for the first 2 years, every 4 months for the third year, and every 6 months during the 4th and 5th post-treatment years.The aggregate clinical, laboratory, and imaging evaluations required per protocol as well as the timing of the optional three module questionnaire are outlined in table 1.The follow-up will be continued for two years. Follow-up data of overall survival will be evaluated annually.Measurement of quality of life is one of the secondary objectives of the trial. Overall survival, return to previous employment as well as persistence of symptoms, the ability to perform appropriate activities and to care for oneself are criteria applied in the three questionnaires used in this study.EORTC QLQ-C30 is a general measure of qualitiy of life in cancer patients. It incorporates nine multi-item scales: five functional scales ; three symptom scales ; and a global health and quality-of-life scale . SpecifiTo assess disease-specific symptoms for patients with pancreatic cancer the pancreatic specific module (QLQ-PAN26)  that hasThe CES-D  is a 20-In addition to the Virginia Mason and the MD Anderson study, an investigation of the effects and mechanism of INTRON A will be performed. IFN alpha seems to have the greatest activity of all interferons in malignancies -23 but iIt probably consists of a combination of stimulation of cell-mediated cytotoxicity ,25, direIn view of the significant improvement in survival rates achieved by using interferon alpha in the Virginia Mason study, studies are now needed to define the molecular and immunologic mechanism(s) of this modality. If the mechanism of action of IFN-alpha is more clearly understood it can be applied more selectively and its therapeutic index will be enhanced.Patients in study arm A at the CapRI trial will receive a single low-dose injection of 1 million units (LDI) prior to therapy. Accompanying immunological analysis should help to define predictive response markers. During therapy patients blood samples of approx. 20 ml will be drawn once a week for immunological studies. The panel will include immunophenotypings, detection of cytokine mRNA in lymphocytes, determination of cytokine patterns in peripheral blood, cellular cytotoxicity assays against autologous tumor cells and determination of apoptotic effects of interferon against autologous tumor cells.group = -1 × ln/24) with a two-year survival rate of 35% in the control group [The primary endpoint in this study is the overall survival period, measured from the date of resection. The sample size calculation is based on the assumption of a constant monthly hazard rate of 0.044 in the standard group and a constant monthly hazard rate of 0.021 in the experimental group. The hazard values are derived from two-year survival rates in the study groups  is observed. TRD will be monitored using the design of Thall and Simon . A non-iIn view of the poor prognosis of the patient group, there will be no explicit stopping rules based on the overall number of toxicities, since even high rates of reversible toxicities seem acceptable if there is a large survival gain. Patients can withdraw from study participation at any time. Patients are taken off the study if unacceptable toxicity appears. Unacceptable toxicity is defined as unexpected serious side effects or irreversible Grade 4 toxicity. Patients who withdraw from the study may be treated with 5-FU and folinic acid or with gemcitabine. The decision will be based on the individual reasons for withdrawing from the study.One year after inclusion of the first patient an interim analysis will be done. If at this time point there already is a significant difference between both groups favouring the Virginia Mason scheme (p < 0.001) group B (5-FU/Folinic acid) will be stopped and replaced by group C .The role of adjuvant therapy in potentially curatively resected adenocarcinoma of the pancreas remains a matter of debate. As the first, large multi-centre randomised controlled trial (RCT), the ESPAC-1-Trial clearly favoured adjuvant chemotherapy over postoperative chemoradiation . HoweverThe CapRI study is an open, randomised controlled trial investigating the survival of patients after potentially curative resection of a pancreatic adenocarcinoma treated adjuvant with 5-FU, cisplatin, INTRON A combined with radiation or 5-FU plus folinic acid. The role and the mechanism of interferon alpha 2b in patient's chemoradiation regimen are evaluated. The toxicity, the disease-free interval and the quality of life are assessed. Different factors are tested for a potential role as predictive marker.The results of the CapRI trial will definitely advance clinical and scientific knowledge on the adjuvant treatment of pancreatic carcinoma as it may confirm or de-mystify the remarkable results from the Virginia Mason study group.CCC Comprehensive Cancer CentreCRF Case Report FormCRO Clinical Research OrganisationGCP Good Clinical PracticeICH International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals forHuman UseIEC Independent Ethics CommitteeITT Intention-to-treat-analysisNCT National Centre for TumourdiseasesRCT Randomised Controlled TrialSDGC Study Centre of the German Surgical SocietyThe author(s) declare that they have no competing interests.The first three authors contributed equally. AM, JS, MWB planned, coordinated and conducted the study. Medical care is covered by KL, KH, HSW, SF, HT and TH. HPK and CS recruited patients and provided randomization. HG, VD and DJ took part in conducting the study. Scientific program is planned by AM and carried out by AM and KH. All authors read and approved the final manuscript.Table 1: Investigation scheduleTable 2: Eligibility CriteriaThe pre-publication history for this paper can be accessed here:


The Viridiplantae  consist of two monophyletic lineages: the Chlorophyta and the Streptophyta. Most green algae belong to the Chlorophyta, while the Streptophyta include all land plants and a small group of freshwater algae known as Charophyceae. Eukaryotes attach a poly-A tail to the 3' ends of most nuclear-encoded mRNAs. In embryophytes, animals and fungi, the signal for polyadenylation contains an A-rich sequence (often AAUAAA or related sequence) 13 to 30 nucleotides upstream from the cleavage site, which is commonly referred to as the near upstream element (NUE). However, it has been reported that the pentanucleotide UGUAA is used as polyadenylation signal for some genes in volvocalean algae.Mesostigma viride, Klebsormidium subtile and Coleochaete scutata, and from two early-branching chlorophytes, Pyramimonas parkeae and Scherffelia dubia. In addition, to extend the database, our analyses included ESTs from six other chlorophytes  and one streptophyte (Closterium peracerosum). Our results indicate that polyadenylation signals in green algae vary widely. The UGUAA motif is confined to late-branching Chlorophyta. Most streptophyte algae do not have an A-rich sequence motif like that in embryophytes, animals and fungi. We observed polyadenylation signals similar to those of Arabidopsis and other land plants only in Mesostigma.We set out to investigate polyadenylation signal differences between streptophytes and chlorophytes that may have emerged shortly after the evolutionary split between Streptophyta and Chlorophyta. We therefore analyzed expressed genes (ESTs) from three streptophyte algae, Scherffelia (only NUE replaced) and Pyramimonas (UGUAA completely missing). The UGUAA element is completely absent from streptophytes. However, the structure of the poly(A) signal was often modified in streptophyte algae. In most species investigated, an A-rich NUE is missing; instead, these species seem to rely mainly on U-rich elements.Polyadenylation signals in green algae show considerable variation. A new NUE (UGUAA) was invented in derived chlorophytes and replaced not only the A-rich NUE but the complete poly(A) signal in all chlorophytes investigated except  In eukaryotes, a polyadenylate tail [poly(A)] is attached to the cleaved 3' end of the nuclear-encoded precursor mRNA of most genes . PolyadeArabidopsis . The FUE is generally U-rich. There are one or more NUEs, often AAUAAA or a related sequence 13 to 30 nt upstream of the CS , each peculiar to its own cleavage site (CS). Only in animals is there an additional DSE ). TherePyramimonas, UGUAA and U-rich elements are present in Scherffelia, and UGUAA is present and U-rich elements are absent in all the other late branching chlorophytes investigated. Once the UGA motif was established in late branching chlorophytes, it seems to have been under strong evolutionary pressure, as it has changed so little in the various chlorophyte lineages (compare the observed variation in streptophytes). The gain of the UGUAA-motif as poly(A) signal in Scherffelia correlates with major evolutionary transitions such as the conversion of an extracellular matrix consisting of scales into a cell wall, changes in mitosis and cytokinesis and other major cellular changes [Scherffelia , like the other late-branching chlorophyte classes Ulvophyceae, Trebouxiophyceae and Chlorophyceae (UTC clade), possesses a UGUAA-motif in the 3'UTR supports the sister relationship between the Chlorodendrales and the UTC clade as revealed my molecular phylogenetic analyses [If the first scenario outlined above is correct, the proposed replacement of AAUAAA with UGUAA in chlorophytes occurred stepwise and early during chlorophyte evolution. The A-rich NUE was lost in  changes ,13,35. OPyramimonas) the A-rich NUE was completely replaced by the UGUAA-motif, which represents a synampomorphic character of the Chlorodendrales and the UTC-clade. Also, the structure of the poly(A) signal was modified in many streptophytes. There is no A-rich NUE and the organisms seem to rely exclusively on U-rich elements.Our results show that putative poly(A) signals may vary considerably among organisms. In chlorophytes , Klebsormidium subtile  and Coleochaete scutata  as follows. Klebsormidium subtile and Coleochaete scutata were cultured in modified WARIS solution as described by Simon et al. [Pyramimonas parkeae was cultured in modified ASP medium [Coleochaete scutata), or 3'-RACE (Seegene) and C1 (5'-NNNNNNNNNNNNNNNATG-3') (Klebsormidium subtile and Pyramimonas parkeae). cDNA was size-fractioned by agarose gel electrophoresis and the smear between 500 bp-5000 bp was isolated and cloned into pGEM-T Easy vector (Promega). All libraries were transformed into ElectroMAX DH10B E. coli cells (Invitrogen) by electroporation.We retrieved all available EST sequences for r et al. . The seqn et al. . PyramimP medium . mRNA waIsolated plasmids were sequenced by the cycle sequencing method using an ABI3700 96 capillary sequencer. A minimal contig set was assembled using the phrap assembler and all contigs were manually curated.ESTs and cDNAs from public databases were checked for redundancy (within each organism specific data set) using the GCG software or assembled into contigs using the CAP-assembler of the Bioedit program. All non-redundant sequences from the different organisms were checked for the presence of a poly(A)-tail. Initially, sequences with at least 15 adenines in the 3'-end of the insert were assumed to include poly(A)-tails and were truncated to the base preceding the 5'-most adenine and trimmed to the 200 nt upstream the CS using the Bioedit editor. As we noticed that ESTs with fewer than 15 A at the 3'end contained a clear UGUAA about 20 nt upstream from the putative poly(A) in some species, we lowered the required number of As in the poly(A) tail to 5 to increase the number of sequences in the data sets. 1-nucleotide patterns were calculated for the 200 upstream from the CS using the Bioedit program.e(b) of oligonucleotide (b) was calculated using the collection of all 200 nt upstream regions for each organism. The expected oligonucleotide frequencies were then used to calculate the number of expected occurrences in a given organismal data set using the formula:The frequencies of all possible penta- and hexanucleotide patterns within the first 50 nt upstream from the CS were determined using a small Python script. The script is available from the authors upon request. To test whether the observed frequencies in penta- and hexanucleotide words were significantly different from chance we used the log-odds ratio (lnω) as described by Sokal and Rohlf . Brieflywheree(b) = number of expected occurrences of oligonucleotide (b) in the organismal data setOS = number of sequencesL = sequence lengthw = oligonucleotide lengthThe log-odds ratio lnω was calculated as followswhere1 = number of observed sequences containing oligonucleotide (b) as sequence motifq1 = number of observed sequences not containing oligonucleotide (b) as sequence motifp2 = number of sequences expected to contain oligonucleotide (b) as sequence motifq2 = number of sequences expected not to contain oligonucleotide (b) as sequence motifpAccording to Sokol and Rohlf, the log-odds ratios are approximately normally distributed . The staPyramimonas, Klebsormidium and Coleochaete data sets are presented as data set S1 [see Additional file The sequences  of the non-redundant Pyramimonas, Klebsormidium and Coleochaete cDNA libraries, and participated in sequence analysis and helped to draft the manuscript. GG performed EST sequencing and helped to draft the manuscript. BB conceived of the study and participated in its design, performed data analysis, and helped to draft the manuscript. All authors read and approved the final manuscript.AS prepared the Mesostigma cDNA library and participated in the sequence analysis. SW prepared the Figure S1: Single-nucleotide profiles of the 3'UTR in various green algae. Single-nucleotide frequencies within the 200 nt upstream from the CS are shown for the indicated organisms as point graphs.Click here for fileTable S1: The top 50 penta- and hexanucleotide words within 50 nt upstream from the CS in various chlorophyte and streptophyte algae.Click here for fileFigure S2: Distribution of various hexanucleotide words within 50 nt upstream from the CS in different chlorophyte and streptophyte algae. Chlorophyte sequence motifs are depicted on the left, streptophyte sequence motifs on the right.Click here for filePyramimonas, Klebsormidium and Coleochaete data sets.Data set S1: Sequences  of the non-redundant Click here for file


Protein sequence motifs are by definition short fragments of conserved amino acids, often associated with a specific function. Accordingly protein sequence profiles derived from multiple sequence alignments provide an alternative description of functional motifs characterizing families of related sequences. Such profiles conveniently reflect functional necessities by pointing out proximity at conserved sequence positions as well as depicting distances at variable positions. Discovering significant conservation characteristics within the variable positions of profiles mirrors group-specific and, in particular, evolutionary features of the underlying sequences.PROfile analysis based on Mutual Information (PROMI) that enables comparative analysis of user-classified protein sequences. PROMI is implemented as a web service using Perl and R as well as other publicly available packages and tools on the server-side. On the client-side platform-independence is achieved by generally applied internet delivery standards. As one possible application analysis of the zinc finger C2H2-type protein domain is introduced to illustrate the functionality of the tool.We describe the tool  where additional documentation can be found.The web service PROMI should assist researchers to detect evolutionary correlations in protein profiles of defined biological sequences. It is available at  Here we describe PROMI, a system to discover group-specific conservation characteristics in the amino acid distribution of profiles. For this we understand the sequences forming a general profile to be associated with a user-defined biological classification label, where the number of labels should be much smaller than the number of rows in the profile. In detail relations between profile columns and the applied group affiliation of the sequences forming the profile shall be investigated. The relations will be apparent by constituting significant amino-acid conservations, leading either to distinct amino acid consensus patterns in the analyzed groups or to knowledge about affinity between the groups .Xi and Y [Xi (in our case column of a profile X) and Y (here group affiliation) is understood as the knowledge one gains about Y if Xi is known and vice versa [k groups as well as all  pairwise profiles together with computed sequence logos finally allow conclusions regarding group-specific amino acid-positions where the distribution differ significantly and thus a group-discrimination on the basis of one profile-position is possible. Moreover the mean value of each pairwise MI-profile leads to formation of an elementary distance matrix D, where low MI-profile-mean-values state that the molecular similarity between groups of sequences is high opposed to higher MI-profile-mean-values with a higher molecular distance in the underlying groups. Further, by applying hierarchical clustering to D, a phylogenetic tree reflecting the distance between its constituents can be constructed.To tackle this aim the mutual information (MI) is used as an interdependence measure of random variables Xi and Y -5. The ice versa ,7. SmallIn the following we use "class" and "classification" synonymously with "group" and "group affiliation".PROMI is implemented in Perl as a web based service running on an apache web server and available for free use. Depicted in Figure 1 to n of the profile, as can be seen in Figure p-value for each mean and deviation is performed. Therefore the MI-profile resulting from the given (unshuffled) multiple alignment is compared against histograms of 100 profiles derived by shuffling each column of the underling multiple alignment. By observing separation of shuffled and unshuffeld values in both histograms  a positive supporting factor for significance is given.Sliding a window from column 2H2-type domain on several organism-specific "proteomes" using the tool PROMI. To take the structurally important linker region in zinc finger proteins of animals into consideration this pattern was enlarged at the N-and C-terminus by 7 amino acids. Each bar within the MI-profile corresponds to a specific column of the multiple alignment showing the obtained MI-value. As can be seen the MI-values differ over the alignment and the cysteines and histidines at positions 8, 13, 26, and 32 show the lowest MI-values (0.0) since these amino acid positions have to be conserved among all sequences. The blue line indicates the mean level, and two red lines correspond to the standard deviation. The row under the bar plot indicates the gaps' frequency according to the alignment . The histogram to the top right explicitly shows the MI standard deviation of 100 shuffled sample sets joined by the unshuffled standard deviation value marked in red whereas the histogram below (bottom right) shows the same for the mean (in blue). Thus, the histograms provide significance levels by showing the separation of shuffled and unshuffled MI-values. All MI-values larger or smaller than the marked red lines are therefore obvious significant positions with organism-specific differences within the amino acid distributions. At the bottom of Figure Figure k given groups . By setting up this structure n m-dimensional vectors Xi (with i = 1...n) are constituted, each consisting of characters from the amino acid one letter code plus one additional letter for gaps. All Xi combine as the matrix X =  reflecting the profile. Additionally an m-dimensional vector (Y) is formed from the classification consisting of a discrete set of group labels, where each label corresponds per row to the group affiliation of a sequence fragment in matrix X at the same row.Given a motif, describing sequence fragments of length Xi and Y is the mutual information I using the entropy and joint entropy respectivelyA convenient measure of correlation between two discrete random variables such as in our case and has been applied recently -5.H(Xi) is calculated using the number of occurrences of each character x within the vector Xi of length m. The same is used for H(Y). The joint entropy H is derived by concatenating the characters x and the group labels y and again calculating the likelihood of all possible combinations. Furthermore each MI value is multiplied by a factor giving the likelihood of gaps in each column. This heuristic approach was adapted from the C4.5 machine learning algorithm [i (i = 1...n) of the profile and calculating I a so-called mutual information profile (MI-profile) can be established, incorporating all MI-values.The entropy lgorithm . Iteratik groups,  pairwise orderless combinations of groups can be formed. By calculating MI-profiles for all pairwise combinations so called pairwise MI-profiles can be created. As one potential measure the (k*k)-dimensional distance matrix D is formed up by calculating the mean over all columns of each pairwise MI-profile, schematically shown in Figure r and s as one possible combination of group labels  can be calculated. r, s are all these sequence fragments belonging either to group r or to group s. Eventually, the phylogenetic tree reflecting the distances can be constructed by applying hierarchical clustering (complete linkage clustering) to D.Given sequences affiliated to • Project name: PROMI• Project home page: • Operating system: server side Linux; client side platform independent• Programming language: Perl, R• Other requirements: EXPASY ScanProsite tool, R, Bioperl, RSPerl, RSvgDevice, Berkeley weblogo software• License: GNU GPL• Any restrictions to use by non-academics: noThe source code of the web service script and the R script are available upon request. Other used software components are available at the according sources.2H2-type protein domain. Further he interpreted the results and participated in the design of the study and drafted the manuscript. JS contributed to the conception of this study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.JH assembled and developed the software components, conceived the idea of interpreting the MI as distance measure and participated in writing and drafting the manuscript. NK provided protein sequences, computed the biological results and drafted the manuscript. WW conceived the biological application of analysing the zinc finger C


The number of the indirect methods for LDL-C estimation is growing. Our result support the reliability of new Anandaraja's formula for low-density lipoprotein estimation from total cholesterol and triglycerides. Gazi et al  presenteIt is possible to supplement next calculation. Anandaraja et al  publisheOur team recently compared two methods of estimation of low-density lipoprotein cholesterol, the direct Wako method versus Friedewald's formula . We studWe re-counted our data with Anandaraja's formula. Main results are expressed in Table In each very low-density lipoprotein particle secreted from the liver, and therefore in each intermediate-density lipoprotein, low-density lipoprotein and lipoprotein (a) particles, a single molecule of apolipoprotein B (apo B) is present from the time of its assembly to its catabolism, which does not exchange between lipoproteins. More than 90% of apo B is in the low-density lipoprotein particles, and therefore, plasma total concentration of apo B can be considered as a measure of the number of atherogenic particles. New Anandaraja's equation takes into account only total cholesterol and triglycerides. Some authors recommend including apo B measurement as acomponent of the standard lipoprotein measurements performed to assess cardiovascular disease risk . ContribIn conclusion, LDL-C is the major indicator for initial classification of coronary heart disease risk status and lowering its level is a primary goal of therapy. The NCEP Working Group on Lipoprotein Measurement recommended the development of accurate direct low-density lipoprotein cholesterol methods. All available direct methods have yet limitations for general use. Our results support the reliability of Anandaraja's formula as indirect low-density lipoprotein cholesterol estimation – originally used in Indian population – in Brazil population, too.


Eukaryotic organisms contain mitochondria, organelles capable of producing large amounts of ATP by oxidative phosphorylation. Each cell contains many mitochondria with many copies of mitochondrial DNA in each organelle. The mitochondrial DNA encodes a small but functionally critical portion of the oxidative phosphorylation machinery, a few other species-specific proteins, and the rRNA and tRNA used for the translation of these transcripts. Because the microenvironment of the mitochondrion is unique, mitochondrial genes may be subject to different selectional pressures than those affecting nuclear genes.From an analysis of the mitochondrial genomes of a wide range of eukaryotic species we show that there are three simple rules for the pyrimidine and purine abundances in mitochondrial DNA transcripts. Mitochondrial membrane protein transcripts are pyrimidine rich, rRNA transcripts are purine-rich and the soluble protein transcripts are purine-rich. The transitions between pyrimidine and purine-rich regions of the genomes are rapid and are easily visible on a pyrimidine-purine walk graph. These rules are followed, with few exceptions, independent of which strand encodes the gene. Despite the robustness of these rules across a diverse set of species, the magnitude of the differences between the pyrimidine and purine content is fairly small. Typically, the mitochondrial membrane protein transcripts have a pyrimidine richness of 56%, the rRNA transcripts are 55% purine, and the soluble protein transcripts are only 53% purine.The pyrimidine richness of mitochondrial-encoded membrane protein transcripts is partly driven by U nucleotides in the second codon position in all species, which yields hydrophobic amino acids. The purine-richness of soluble protein transcripts is mainly driven by A nucleotides in the first codon position. The purine-richness of rRNA is also due to an abundance of A nucleotides. Possible mechanisms as to how these trends are maintained in mtDNA genomes of such diverse ancestry, size and variability of A-T richness are discussed. Mitochondria are the descendents of an early bacterium that developed a symbiotic relationship with another cell approximately 1.5 billion years ago . Featuren-dimensional DNA walks. One dimensional pyrimidine-purine walks were first used to find long-range correlations in nucleotide sequences [Because of the relatively small size of mitochondrial DNA, it is ideally suited for analysis by equences . Recentlequences . Combiniequences  gives a Using these techniques we demonstrate certain features of mtDNA sequences which have been preserved by evolution. Greater understanding of the evolutionary selection pressures on mtDNA will allow the construction of more accurate phylogenetic trees based upon mtDNA gene sequences ,7 as welL)  – purine (A and G) walk of the (+) strand of human mtDNA  is shown in Figure ynthesis ,9. HowevChondrus crispus (Irish Moss). The genes are color-coded based upon whether they encode membrane proteins, soluble proteins, or rRNA and upon the strand in which they are encoded. Figure Porphyra purpurea. The similar gene order in these two red algae species   Figure , are pyrDictyostelium discoideum  strand were purine-rich, just like the rest of its genome. To place the extreme purine richness of Dictyostelium in context, the human mtDNA genome (+) strand is 44 % purine while that of the plant Arabidopsis thaliana is 50 % purine. The purine abundance of the mitochondrial genome of other species is listed in Pedinomonas minor, contain the least amount of purine (44%).No matter which strand encodes the genes, or whether the entire mitochondrial genome is pyrimidine or purine-rich, there are highly conserved features in these walks. Places in the genome where the DNA walk went down were locations where rRNA or soluble proteins were encoded on the (-) (heavy) strand or where membrane proteins were encoded on the (+) (light) strand. Locations of membrane proteins on the (-) strand or rRNA or soluble proteins on the (+) strand were associated with an upward slope in the pyrimidine-purine walk. Exceptions to these rules are indicated on the figure with an asterisk. The most notable exception that we found was the mtDNA from the slime mold m Figure , which hWe analyzed the pyrimidine and purine content of mitochondrial transcripts from many diverse eukaryotic species Table . Unlike Rule 1) Oxidative phosphorylation complex and other membrane protein transcripts are pyrimidine-rich.Rule 2) Ribosomal RNA is purine-rich.Rule 3) Soluble protein transcripts are purine-rich.C. elegans, which has almost equal numbers of purines and pyrimidines. In other non-animal species the short 5S rRNA sometimes contains more pyrimidines than purines.Table We examined the mtDNA from 8 species that encode genes for both soluble and membrane proteins. In Figure P < 0.001) between the percent pyrimidine and the hydrophobicity of the encoded protein. This correlation has been shown previously for transcripts of nuclear-encoded proteins [Dictyostelium where almost all mitochondrial transcripts are purine-rich.To clearly illustrate the relationship between protein hydrophobicity and the pyrimidine content of the genes, we plot in Figure proteins  and for proteins . We showChondrus crispus S12 soluble ribosomal protein transcript. For comparison, the pyrimidine-purine walk of the human 16S ribosomal RNA is also shown. The base composition of mitochondrial genome sections encoding rRNA and tRNA from other species is given in Arabidopsis give a similar purine-rich signal in pyrimidine-purine walks  in the second codon position encode hydrophobic amino acids ,13 that Xenopus compared to 44 % in humans   may seleMitochondrial gene sequences, amino acid sequences and genomes were downloaded from the NCBI website. Java (JDK 1.50) programs were written to analyze the sequences. The programs or software details are available from the authors upon request. Other websites such as the OGRe database of mitochondrial genomes  also allPB generated the data, constructed Figures Arabidopsis thaliana. Figure S2 The 2-dimensional A-G and T-C walks of human mtDNA-encoded transcripts. Figure S3 Pyrimidine-purine walks of unspliced mitochondrial-encoded transcripts from Arabidopsis thaliana and Marchantia polymorpha. The introns in these transcripts do not encode known proteins.Table S1 Base percentages in the entire mitochondrial genome and in just protein-coding or RNA-coding sections. Figure S1 The pyrimidine-purine walk of each codon position of eight soluble mitochondrial-encoded protein transcripts from Click here for file


A comprehensive study of the gene expression profile of 96 mesenchymal tumors identifies molecular fingerprints for most tumors in this group. Bone and soft tissue tumors represent a diverse group of neoplasms thought to derive from cells of the mesenchyme or neural crest. Histological diagnosis is challenging due to the poor or heterogenous differentiation of many tumors, resulting in uncertainty over prognosis and appropriate therapy.We have undertaken a broad and comprehensive study of the gene expression profile of 96 tumors with representatives of all mesenchymal tissues, including several problem diagnostic groups. Using machine learning methods adapted to this problem we identify molecular fingerprints for most tumors, which are pathognomonic (decisive) and biologically revealing.We demonstrate the utility of gene expression profiles and machine learning for a complex clinical problem, and identify putative origins for certain mesenchymal tumors. Tumors of bone and soft tissue are a wide spectrum of benign and malignant neoplasms (sarcoma) derived from mesenchymal precursor cells  ,2. Many The discovery of definitive oncogenic gene fusions for certain mesenchymal tumors has aided pathologists greatly. These include the EWS-ERG or EWS-FLI1 fusion transcripts for Ewing's sarcoma (EWS) -5, or thThere are many gene expression microarray (GEM) studies covering a range of mesenchymal tumors -28. ThesUsing a machine learning model, we assess the overall utility of GEM for the diagnosis of 19 types of mesenchymal tumors. We find both expected and unexpected relationships between certain mesenchymal tumors, and ascertain expression fingerprints for decisive diagnostic or pathognomonic features for most tumor classes. These fingerprints give clues to the etiology of several mesenchymal tumors.Our machine learning model fits the data in two steps. The first step incorporates biological assumptions by merging related tumors into broader groups. Step two splits the broader groups into specific tumors. This latter process is derived by expert decision, rather than automated model selection. We consciously mirror the clinical diagnostic method of progressively resolving general differentiation types into specific tumors in a branch-wise (or tree-like) method. The use of a decision process structured by prior knowledge  gives our model an estimated error of about 0.1. Although further study and validation will be required to bring it to clinical use, we believe this method is a prototype for the extension of GEM and machine learning to other complex diagnostic problems. The GEM data from this study are available from the European Bioinformatics Institute (EBI) public repository ArrayExpress (accession no. E-MEXP-353) .®. A multi-dimensional scaling (MDS) of tumor samples using all genes reveals much structure in the data  and schwannoma (SWN), the alveolar (ARMS) and embryonal rhabdomyosarcoma (ERMS), and the well-differentiated liposarcoma (WLS) and lipoma (LMA). Similarities in the GEM profiles of malignant peripheral nerve sheath tumors (MPNST) and monophasic synovial sarcoma (MSS) were previously reported and this relationship is supported in our data . LikewisThe MDS is based on distances calculated from the whole molecular signature  Figure . Thus, cThe key to the success of supervised learning is to focus on a small set of strongly distinguishing features, thus ignoring confounding factors. Strong feature selection is appropriate in this study as we have sound histological evidence that there are underlying patterns to be uncovered (at least for most tumors). This reduced set of features (or genes) gives a molecular fingerprint of the tumors that is both diagnostic and descriptive. Our feature selection method is suited to this application as it selects ten genes evenly for each class. Using a simple k = nearest neighbors (k-NN) machine learning algorithm and the extended feature selection algorithm we attempted initially to create a model for all tumors simultaneously . The overall cross-validation error was 0.33 compared with a random guessing error of 0.96 +/- S.E. 0.01 . Therefore, even with this simplest approach, the machine learning model is fairly successful.The true error, and thus the generalization to other datasets, is likely to be underestimated (see Discussion). However, an important control shows that our method does not over-fit the data excessively. In simple terms, the method does not erroneously fit this particular dataset using random patterns. After randomly permuting all the labels to create a semi-random dataset we tried to cross-validate these false data . The error was 0.94 +/- S.E. 0.01 (n = 10 permutations) so was therefore not significantly better than random guessing , between MPNST and MSS, and between LMS, PMS and de-differentiated chondrosarcoma (DCS). By considering unsupervised learning methods  plus biological assumptions and trial-and-error, we arrived at an improved two-step method. This was designed, in part, to mirror the thought processes involved during clinical histopathological diagnosis. This two-step model is, thus, expert derived and not an automated procedure.In step one, we grouped tumors into composite classes assumed to reflect similar pathways or levels of differentiation; MPNST/MSS, PMS/LMS/DCS, ARMS/ERMS, NFB/SWN and LMA/WLS/MLS. In step two, we decomposed the composite classes into their sub-types. This decision process is illustrated in color code in Figure Using the same feature selection and learning algorithm as the initial method, the new model is remarkably effective in the first step with ~0.09 errors (9/96 errors). There were important limitations, however, as we could not decompose the adipocytic tumors fully in step two, yet recognized MLS as distinct. Moreover, after compositing the spindle-like tumors PMS/LMS/DCS in step one, we could not decompose this artificial grouping. Accepting these limitations, the sum error over both steps was 0.14 . Note that the samples wrongly classified in step two were classified correctly in step one thus having no carry-over error in this particular dataset. The types of error in step one are summarized in Table Feature selection of genes was carried out for each fold of our model, and while 237 distinct genes were encountered during cross-validation only 61 were selected in every fold . This robust 61-gene signature is illustrated in the gene-clustered heatmap of Figure From the molecular fingerprint used in step two we selected genes used in the majority of cross-validation folds for display in Figure We demonstrate here the feasibility of using GEM and machine learning to aid the diagnosis of mesenchymal tumors. In contrast to previous studies, our work encompasses a wide range of mesenchymal neoplasms and reflects the open-ended nature of histopathological diagnosis. It is clear from unsupervised methods such as MDS that there is information within the expression profile that can be further refined Figure . This coCross-validation of the first step of our model gives 0.09 errors. This is a useful guide but its accuracy should not be overstated. A reliable estimate of error usually requires hundreds of samples per class, and a leave-one-out estimation is likely to underestimate error . Moreoveperilipin (PLIN), lipoprotein lipase (LPL), and glycerol-3-phosphate dehydrogenase 1 (GPD1)  and receptor-associated protein of the synapse (RAPSN) associated with the musculoskeletal synaptic junction  Figure . Similartropomyosin-2 (TPM2), skeletal muscle actin (ACTA1) and myosin-light polypeptide-4 (MYL4), indicative of more maturity than the ERMS , a key molecule in the development of the neural crest. EDN3 promotes self-renewal of multi-potent neural crest precursors or de-differentiation of matured cell types including Schwann cells [Within these highly focused signatures there are clues to the origins of some of the poorly differentiated tumors. One striking similarity of gene expression is between the MPNST and the MSS . The MSS have a simple karyotype consisting of an aberrant SYT-SSX fusion. The MPNST have a complex karyotype and are believed to arise from Schwann cell precursors  derivednn cells -35.WNT5A recently linked to metastases of OS [Despite their similarity, the MPNST and MSS have distinct molecular signatures Figure  and 4d aes of OS  and melaes of OS . Yet SWNmyxoid liposarcoma-associated transcript-4 (MLAT4), which as its name suggests distinguishes MLS from WLS/LMA , and neural restricted genes . The profile also confirms previous reports of the expression of the immunotherapeutic targets PRAME and CTAG-1  [Some genes could make useful monoclonal antibodies for immunohistochemistry. Genes such as the A Figure  and 4h, Y-ESO-1) . These abrachyury (T) which is highly expressed in developing notochord strongly supports this theory [There is another clue to cellular origin within the CMA fingerprint. The overall expression pattern of CMA is closely related to the chondroblastic tumors CHS and chondroblastoma (CHB) Figure . Likewiss theory ,42.Not all tumors had distinctive markers. The LMS, PMS and DCS were not distinguishable from each other. Cross-validation of our model in step one was fairly successful as our algorithm collectively recognized these tumors through a lack of specific markers, perhaps analogous to the way in which histopathologists recognize them Figure . At leasThere are a number of extensions to the current study that may bring GEM technology closer to clinical use. Firstly, a larger study focusing on the poorly modeled cases (PMS/LMS/DCS) may help to elucidate categories supplementing current histological guidelines. Yet the pleomorphism and complex karyotypes of some mesenchymal tumors may confer a continuum of molecular pathology irreducible to simple categories. Such an analysis is unlikely to find adherents if it does not correspond with an improved interpretation of the histopathology or with appreciable clinical differences such as prognosis.Secondly, by sampling all groups more thoroughly we could manage uncertainty. Our model gives a simple prediction of tumor type appropriate for the broad base and low sample numbers in our dataset. A more nuanced approach would be to calculate a probability of tumor type. This could be achieved most simply using Bayesian classifiers that implicitly calculate class membership probability. Thus in a clinical setting a sarcoma could be identified to a histopathologist as a central or classical example of its type, or be highlighted as an uncertain outlier for further immunohistochemical examination.Thirdly, there is a large quantity of relevant molecular information that may transcend the diagnostic categories investigated here, yet may have prognostic significance. There is great interest, for instance, in both drug-resistance genes  and signTumor biopsies were obtained from 96 participants presenting at the London Bone and Soft Tissue Tumour Service , Great Ormond Street Hospital, London, or the Nuffield Orthopaedic Center, Headington, Oxford, in the UK. The diagnosis was determined by pathological examination using criteria established by the World Health Organization (WHO) tumor classification of soft tissue and bone tumors . Where nFrozen sections from each tumor sample (needle core or resection) were examined microscopically prior to RNA extraction to confirm that the sample was representative and contained more than 80% tumor cells. Total RNA was extracted from adjacent tissue sections with >80% tumor cell content, using TRIzol™ reagent  followed by purification with RNeasy columns  according to the manufacturer's instructions. RNA quality and quantity was assessed using RNA 6000 Nano chips on Agilent 2100 Bioanalyser according to manufacturer's instructions . Our RNA quality-control threshold for the rRNA peak ratio was 28s/18s ≤ 2.®, Santa Clara, CA, USA) according to manufacturer's recommendations. In cases where smaller amounts of total RNA were obtained , 150 ng of total RNA was subjected to two rounds of amplification to obtain a biotinylated cRNA yield of 20 μg according to Affymetrix recommendations . Parallel hybridizations of probe synthesized from 10 μg and 150 ng (via single and double amplification respectively) for the same tumor sample were found to have similar quality-control and expression profiles  was prepared from 10 μg of total RNA as previously described ,47. The Data analysis was carried out using the R statistical environment and programming language . We exteThe Cluster and Treeview software packages were used to produce the average linkage hierarchical clustering shown in Figure We chose simple k-NN pattern classification to model the data. This is a non-parametric algorithm, here based upon a table of inter-sample Euclidean distances . The ideAs most expression data are uninformative, or even confounding of pathological categories, feature selection is incorporated into each fold of cross-validation. For multi-class models we used a well-suited feature selection method to capture information from each of the 19 tumor classes (not to split classes in step two). The algorithm selects 10 genes for each class that are different in expression from all others. We tested 5, 10, 20 and 30 gene sets finding 10 to be sufficient (better than 5 or 30 and as good as 20). We used the 'limma' method for each comparison . Limma uThe following additional data are included with the online version of this article: an average linkage hierarchical clustering of the tumor samples using the same distance matrix as in Figure Average hierarchical clustering of tumor samples. The same set of inter-sample distances were used as in Figure Click here for fileSupplementary information on the genes shown in Figures Click here for fileClinical and pathological details of all samples used in this study.Click here for file


Mosquito resistance to the pyrethroid insecticides used to treat bednets threatens the sustainability of malaria control in sub-Saharan Africa. While the impact of target site insensitivity alleles is being widely discussed the implications of insecticide detoxification – though equally important – remains elusive. The successful development of new tools for malaria intervention and management requires a comprehensive understanding of insecticide resistance, including metabolic resistance mechanisms. Although three enzyme families  have been widely associated with insecticide detoxification the role of individual enzymes is largely unknown.Anopheles gambiae strain from Odumasy, Southern Ghana. RNA from the resistant strain and a standard laboratory susceptible strain, of both sexes was extracted, reverse transcribed and labelled with either Cy3- or Cy5-dye. Labelled cDNA was co-hybridised to the detox chip, a custom-made microarray containing over 230 A. gambiae gene fragments predominantly from enzyme families associated with insecticide resistance. After hybridisation, Cy3- and Cy5-signal intensities were measured and compared gene by gene. In both females and males of the resistant strain the cytochrome P450s CYP6Z2 and CYP6M2 are highly over-expressed along with a member of the superoxide dismutase (SOD) gene family.Here, constitutive expression patterns of genes putatively involved in conferring pyrethroid resistance was investigated in a recently colonised pyrethroid resistant A. gambiae and constitute a novel set of candidate genes implicated in insecticide detoxification. These data suggest that metabolic resistance may have multiple origins in A. gambiae, which has strong implications for the management of resistance.These genes differ from those found up-regulated in East African strains of pyrethroid resistant  Pyrethroid-treated bednets are the mainstay of malaria control programs in sub-Saharan Africa . PyrethrAnopheles gambiae is associated with a single point mutation, commonly referred to as knock-down resistance (kdr). The kdr allele leads to a modification of the voltage-gated sodium channel protein resulting in reduced sensitivity to both DDT and pyrethroids   which sePM conceived and carried out the microarray experiments, performed the statistical analysis and drafted the manuscript. MJD and HR participated in the study design and helped to draft the manuscript. All authors read and approved the final manuscript.Odumasy expression data ALL. This table contains a complete list of the current microarray analysis including fold expression and p-values for all genes represented on the detox chip.Click here for file