Electron microscopy analyses of replicating chloroplast molecules earlier predicted bidirectional Cairns replication as the prevalent mechanism, perhaps followed by rounds of a rolling circle mechanism. This standard model is being challenged by the recent proposition of homologous recombination-mediated replication in chloroplasts.Small Single Copy (SSC) and the Large Single Copy (LSC) regions between inverted repeats. These gradients increase bi-directionally from the center of each region towards the respective ends, suggesting that both the strands were left single-stranded during replication.We address this issue in our current study by analyzing nucleotide composition in genome regions between known replication origins, with an aim to reveal any adenine to guanine deamination gradients. These gradual linear gradients typically result from the accumulation of deaminations over the time spent single-stranded by one of the strands of the circular molecule during replication and can, therefore, be used to model the course of replication. Our linear regression analyses on the nucleotide compositions of the non-coding regions and the synonymous third codon position of coding regions, between pairs of replication origins, reveal the existence of significant adenine to guanine deamination gradients in portions overlapping the Single-stranded regions of the genome and gradients in time that these regions are left single-stranded, as revealed by our nucleotide composition analyses, appear to converge with the original bi-directional dual displacement loop model and restore evidence for its existence as the primary mechanism. Other proposed faster modes such as homologous recombination and rolling circle initiation could exist in addition to this primary mechanism to facilitate homoplasmy among the intra-cellular chloroplast population Euglena gracilis [Nicotiana tabacum [Chlamydomonas reinhardtii [Oenothera [Zea mays [The chloroplast is a vital organelle, responsible for photosynthetic metabolism in plants. Their replication is crucial in ensuring the cellular maintenance and working within plants . Understgracilis ; single  tabacum ; Chlamydnhardtii ; Oenotheenothera ; Zea mayZea mays ).in vitro tobacco chloroplast replication intermediates also revealed Y-arc patterns, indicative of rolling circle replication[The rolling circle mechanism could be initiated after one round of Cairns type of replication, so as to generate multiple copies of the chloroplast genome even though replication is initiated only once . Electrplication.Bendich and colleagues recently countered the entire proposition of bi-directional replication mediated by two D-loops  in NCBI reveals four origins on each of the two inverted repeats: A1 (35 nucleotides), A2 (82 nucleotides), and two copies of B (243 nucleotides), one on each strand. Formation of D-loops were observed only in those tobacco clones that contained all the origins, and these replication origins were also found to be the minimal sequences to ensure the completion of replication [Small Single Copy (SSC) and Large Single Copy (LSC) regions. The single-strandedness window experienced during homologous recombination is too small to result in such nucleotide composition patterns. Secondly, the single-stranded bubble moves as strand invasion progresses during recombination and does not expand like in the case of conventional replication mechanism, thus preventing accumulation of deaminations. On the other hand, the dual displacement loop model [The tobacco chloroplast genome is best-documented in terms of replication origins . Annotatlication . Replica as of September 2008. The names of these plant species, their abbreviations and locus IDs are presented in a table , A2 82 bp), B 243 bp) and R (243 bp), known for the tobacco chloroplast genome  to guanine (G) deaminations were estimated as the ratio: A/(A+G), where the nucleotide 'A' at each position was assigned a binary value of 0 and likewise, 'G; was assigned a value of 1. Linear regressions were performed for each extracted region, where the X axes were the positions of nucleotides on the genome, and Y axes values were the estimated site-specific A/A+G ratios. Significance was determined according to a two-tailed t-test (P < 0.05). Similar regression analyses were also performed after splitting the region of interest mid-way into two regions, for each sub-region.Chlamydomonas reinhardtii, Chlorella vulgaris, Helicosporidium sp ex Simulium jonesii, Nephroselmis olivacea, Oltmannsiellopsis viridis, Oedogonium cardiacum, Pseudendoclonium akinetum and a blue spike-moss: Selaginella uncinata), which lack similarity to any of the tobacco replication origin sequences.We found varying numbers of sequence homologues to tobacco chloroplast replication origins in 95 out of 115 other chloroplast genomes Table , 7 and 8Medicago truncatula which bears only one inverted repeat; see Additional File Zea mays chloroplast genome  in genome regions interspersed between replication origin homologues. These regions comprised of non-coding and synonymous third codon positions of coding genes. We consistently find significant negative deamination gradients in the region (A2 → A2-C) containing LSC and tail ends spanning ~15kb each of the two inverted repeats for all species belonging to the first six categories  and its derivatives in E.coli, carrying a similar arrangement of vegetative replication origins [We also find symmetric A → G gradients in opposite directions in regions between replication origins for an  origins .Borrelia burgdorferi and Borrelia hermsii were observed to be different from that of the E. coli nucleoid [Arabidopsis [M. truncatula [Ethidium bromide stained fluorescence microscopy images of the nucleoids of nucleoid ,50, and nucleoid , Arabidobidopsis , pea, touncatula , especiauncatula . These ouncatula . Since rThe relative abundance of oligomeric forms observed for chloroplast DNA stays constant through all stages of leaf development, as found in the case of spinach , triggerHomologous recombination cannot explain the generation of nearly symmetric consensus gradients in A → G deaminations in the SSC and LSC regions of the chloroplast genomes as observed by our approach, since the single-strandedness generated during this process is small and non-cumulative. Nevertheless, mixed modes of traditional Cairn's replication via origin firing as well as replication slippages  followinWith advent of approaches that monitor DNA dynamics in living cells, animal mitochondria were found to not exist as autonomous individual organelles but instead form a highly dynamic semi-tubular network. It is possible that the arrangement of ethidium-stained chloroplast DNA as clots and comets with extended fibers as observed by fluorescence microscopy visualization  resemblein vitro, whether the D-loop mode or rolling circle mode of replication is adopted as the predominant mechanism in tobacco chloroplast DNA [The minimalist model of chloroplast replication presented here Figure , excludelast DNA . It is pModel of chloroplast replication as inferred by analyzing local deamination gradients in regions between replication origins conforms to the bi-directional replication model put forth by the Kolodner group. Homologous recombination could exist as an alternate or additional mechanism.NMK and BJR conceived the idea of exploring chloroplast genome nucleotide composition. NMK devised the comparative approach to identify consensus nucleotide composition patterns in chloroplast genomes that could result from single-strandedness during replication, and subsequently put forth a model of single-strandedness based on consensus deamination gradient patterns. NMK further shortly extended this approach to bacterial plasmid DNA. NMK and BJR have read and given final approval of the version to be published.List of Complete Chloroplast Genomes. Shown in this table are the names, abbreviations and locus IDs of 116 species for which complete chloroplast genomes were available in NCBI GenBank as of September 2008.Click here for fileLocations of complete and partial sequence homologues to the tobacco chloroplast replication origin sequences. Begin and end limits are shown in this table for replication origin homologues of each sub-category of genomes, for the replication origin as the query (Q) sequence and the complete chloroplast genome as the subject (S) sequence. Reverse complementary sequence homologues to any of the replication origins  are indicated as '-C'. Partial homologues to any replication origin sequence are represented as the abbreviation for that origin, superscripted with P. Blast analyses were performed using NCBI's pairwise blast tool .Click here for filet-statistics of the regression slopes between (A/A+G) ratios at each site and their relative positions on the genome, in regions between homologues to tobacco chloroplast replication origin sequences. The t-statistics of the A → G (i.e. A/(A+G)) deamination trends (i.e. of regression slopes between A/A+G ratios at each site and the relative position of this site on the genome) are depicted for all sub-categories of genomes in this table, for the complete regions between homologues to tobacco chloroplast replication origins (first row for each species appended with '-C'), and after dividing these regions mid-way, for the first half (second row for each species appended with '-I') and second half (third row for each species appended with '-II') respectively for these regions. Significant values  are emboldened.Click here for fileE. coli plasmidSymmetric A → G deamination gradients in . The replication origins  are mapped on the circular pOLA52 plasmid of E. coli (NC_010378) using SimVector 4.22, and the regions between these origins are annotated as R1, R2 and R3, respectively (see Additional File Click here for file


Physcomitrella patens is a model for the study of plant cell biology and, by virtue of its basal position in land plant phylogeny, for comparative analysis of the evolution of plant gene function and development. It is ideally suited for ‘reverse genetic’ analysis by virtue of its outstanding ability to undertake targeted transgene integration by homologous recombination. However, gene identification through mutagenesis and map-based cloning has hitherto not been possible, due to the lack of a genetic linkage map. Using molecular markers [amplified fragment length polymorphisms (AFLP) and simple sequence repeats (SSR)] we have generated genetic linkage maps for Physcomitrella. One hundred and seventy-nine gene-specific SSR markers were mapped in 46 linkage groups, and 1574 polymorphic AFLP markers were identified. Integrating the SSR- and AFLP-based maps generated 31 linkage groups comprising 1420 markers. Anchorage of the integrated linkage map with gene-specific SSR markers coupled with computational prediction of AFLP loci has enabled its correspondence with the newly sequenced Physcomitrella genome. The generation of a linkage map densely populated with molecular markers and anchored to the genome sequence now provides a resource for forward genetic interrogation of the organism and for the development of a pipeline for the map-based cloning of Physcomitrella genes. This will radically enhance the potential of Physcomitrella for determining how gene function has evolved for the acquisition of complex developmental strategies within the plant kingdom.The moss  Physcomitrella patens is the first non-flowering land plant for which an annotated genome sequence has been determined (http://genome.jgi-psf.org/Phypa1_1/Phypa1_1.home.html). A member of the bryophytes, the first group of plants to diverge from the modern land plant lineage, Physcomitrella retains many features typical of ancestral land plants  stage of the life cycle.Reverse genetics permits the analysis of genes whose function may be postulated by virtue of their homology with genes previously identified in other organisms. However, the majority (63%) of predicted Physcomitrella mutants have been isolated and described, with phenotypes associated with metabolism, cellular differentiation and hormone responses, and cellular-level growth responses such as gravi- and phototropisms  . The forPhyscomitrella accessions were first screened for their relative genetic diversity, using gene-linked SSR markers identified following a computational analysis of the large Physcomitrella EST database, as well as by analysis of ribosomal DNA internal transcribed spacer sequences . This analysis identified strains that could contribute the genetically distinct parental genotypes necessary for the generation of a mapping population. Because the object of the programme was to provide linkage between the genetic and physical maps, we chose the sequenced genotype (Gransden2004).A number of independently isolated and geographically diverse nicB5/ylo6 male-sterile Gransden mutant  and Australia (P. patens ssp. readeri) failed to produce reasonable numbers of viable progeny. Consequently the Villersexel K3 strain was selected as the most suitable choice for the second parent.The French accession Villersexel K3 exhibited the greatest genetic distance from Gransden among all tested European lines. The ability to hybridise and produce viable progeny with a n mutant  was evalnptII selection cassette to the PpLea-1 (AY870926) locus  locus . When crPhyscomitrella is the haploid gametophyte. Gametes are produced by mitosis, and their fusion (the outcome of fertilization of a single egg cell within an archegonium by a spermatozoid) results in the formation of a diploid sporophyte that remains dependent on the gametophyte for its nourishment and development. Within the sporophyte, meiotic spore mother cells generate the haploid spores   . RecombiPhyscomitrella EST database and their utility as molecular markers was tested. F1 progeny selected from six independent spore capsules resulting from Villersexel K3 × Gransden crosses were chosen as the mapping population. Initially, 1238 microsatellite loci were examined for polymorphisms by PCR. Subsequently, the allelic status of 256 SSR markers was scored in a mapping population of 94 F1 recombinant haploid lines. Computational linkage analysis and map construction was performed with MapMaker (The SSR identification (Freiburg) was carried out as previously described , of which 1574 were polymorphic (We determined the utility of AFLP markers (Leeds) to discriminate between the Gransden and Villersexel genotypes. We used genomic DNA digested with ymorphic .1 recombinant haploid lines and entered into JoinMap3.0 , with an overall map length of 4410–4418 cM. Three pairs of linkage groups that were separate at LOD = 10 coalesced at lower LOD thresholds. . The AFLPs comprise sequence-uncharacterised bands on gels. To be useful, it is essential to identify the underlying polymorphic DNA sequence. Typically, this is achieved by physical excision of a band from a gel, reamplification and sequence analysis , SCALPHunter identifies all possible length variants within the range ±4 bp. The sequence scaffolds containing mapped SSRs were therefore interrogated by SCALPHunter to identify candidate AFLP sequences corresponding to the Gransden-specific markers with which the mapped SSRs co-segregated. In many cases, this identified several candidate genomic loci corresponding to a cluster of AFLP markers, linked to the SSR locus, enabling an initial integration between genetic linkage and underlying DNA sequence. The candidate AFLP sequences identified are listed in We developed a Perl program, ‘SCALPHPhyscomitrella gene. The G418-resistant phenotype in the Gransden parent used to construct the Leeds mapping lines results from the targeted integration of a selection cassette into the PpLEA-1 locus. This locus is present in sequence scaffold 12 of the v1.1 genome assembly. Segregation of the G418-resistant phenotype indicated that the gene was located at 106 cM on linkage group 11 of the integrated map . Interrogation of these sequence scaffolds by SCALPHunter proposed candidate sequences for 21 of the 29 the Gransden-specific AFLP loci within the integrated LG11 . We designed +6 primers (Eco-ACAATT/Taq-GAGGAG) to amplify the allelic pair. Amplification generated a fragment of about 500 bp from each parental genotype  built in to SCALPHunter predictions.In many cases, the order in which candidate AFLP sequences occur in the genome sequence scaffolds is consistent with the order of these markers in the linkage map, but there are also several instances where marker position appears to be inverted, or intercalated markers are observed. This might arise either through incorrect assembly of the sequence scaffolds, through the identification of an incorrect sequence by SCALPH in LG11 ]. Where  in LG11 ], then tunter. Currently, only 94 of the 2106 sequence scaffolds comprising the Physcomitrella version 1.1 sequence assembly are represented by mapped markers. These comprise about 143 Mbp, approximately 30% of the estimated complete genome sequence  B.S.G. plants were vegetatively propagated on agar medium as previously described  and L = Σ[(linkage group length) × (no. markers + 1)/(no. markers − 1)]. Similarly, genome coverage (c) was calculated as c = 1 − exp(2dn/L) where d is distance in cM, n is the number of markers and L is map length.We followed the approach of unter is a Perl script that, given a list of AFLPs, identifies the possible candidate sequences within a given set of scaffolds. It can run 800 AFLP sequences across the whole Physcomitrella genome in about 30 min . The basic process is as follows:SCALPHunter reads in a list of codes describing AFLPs (length after subtraction of primers and end sequence), which provides the information for pattern matching against the scaffolds. The forward- and reverse-complement regular expressions are precompiled and placed upon a hash (using precompiled regular expressions greatly speeds up the process of searching for the existence of a list of AFLPs amongst a set of scaffolds). In addition, the regular expressions include a user-defined length variation (e.g. ±4 bp) to allow for the variable accuracy of fragment length determination.SCALPHEach scaffold is searched in turn using all of the precompiled AFLP regular expressions and all matches are stored.The complete set of matches is sorted by size  and output in three different forms. Firstly, a set of matches-per-scaffold, showing all of the AFLPs found to match a scaffold given the pattern/length-variation criteria. In addition, a set of matches-per-AFLP, showing all of the scaffolds that an AFLP is found to match, and the corresponding sequences within those scaffolds. Finally, a list of the matches-per-length-variation, showing all of the AFLPs that matched scaffolds for a given sequence-length variation.last search of the Physcomitrella genome.To identify candidate AFLPs within a scaffold identified as carrying an SSR, the matches-per-scaffold file was interrogated with each AFLP within the corresponding linkage group. The sequence of each candidate was obtained from the matches-per-AFLP file and its position was located within the scaffold by B


Identification of epitopes that invoke strong responses from B-cells is one of the key steps in designing effective vaccines against pathogens. Because experimental determination of epitopes is expensive in terms of cost, time, and effort involved, there is an urgent need for computational methods for reliable identification of B-cell epitopes. Although several computational tools for predicting B-cell epitopes have become available in recent years, the predictive performance of existing tools remains far from ideal. We review recent advances in computational methods for B-cell epitope prediction, identify some gaps in the current state of the art, and outline some promising directions for improving the reliability of such methods. Antigen-antibody interactions play a pivotal role in the humoral immune response. Antibodies bind to antigens at specific sites which correspond to the antigenic determinants or B-cell epitopes. Identification and characterization of B-cell epitopes in target antigens is one of the key steps in epitope-driven vaccine design, immunodiagnostic tests, and antibody production. B-cell epitopes typically belong to one of two classes: linear  epitopes or conformational (discontinuous) epitopes. Linear epitopes are short peptides that correspond to a contiguous amino acid sequence fragment of a protein ,2. LineaSeveral experimental techniques are currently available for experimental mapping of B-cell epitopes . HoweverSeveral computational methods for B-cell epitope prediction have been developed in recent years e.g., -14). How. How14])Against this background, we review recent advances in computational methods for B-cell epitope prediction, identify some gaps in the current state of the art, and outline some promising directions for improving the reliability of such methods.Although it is believed that the majority of B-cell epitopes are conformational epitopes , experim propensity value to each amino acid which measures the tendency of an amino acid to be part of a B-cell epitope (as compared to the background). To reduce fluctuations, the score for each target amino acid residue in a query sequence is computed as the average of the propensity values of the amino acids in a sliding window centered at the target residue. The propensity scores are then used as a basis of predicting whether a given amino acid sequence residue is likely to be part of a linear B-cell epitope. Propensity scale based methods rely on the observed correlations between specific physico-chemical properties of amino acids and the antigenic determinants in protein sequences to identify the location of the linear B-cell epitopes in the query protein sequence.Propensity scale methods -26 assigThe first propensity scale method for predicting linear B-cell epitopes was introduced by Hopp and Woods  and utilSubsequently, several other propensity scales have been proposed for predicting linear B-cell epitopes. For example, Parker et al. , KarplusRecently, Blythe and Flower  have perSeveral authors have explored methods for improving the predictive performance of propensity scale methods in predicting linear B-cell epitopes. BepiPred  combinesMotivated by the findings of Blythe and Flower  and the Although more than 90% of B-cell epitopes are estimated to be conformational in nature , most exSequence-based methods in predicting conformational B-cell epitopes have the advantage that they do not require the structure of the target antigen to be known. The amino acid propensity scale methods that assign a prediction score to each residue in the antigen sequence can in principle be used to predict conformational B-cell epitopes . Such meA large body of work using machine learning methods for predicting protein-protein ,36, protThe most accurate experimental method for identifying conformational B-cell epitopes relies on determination of the structure of antigen-antibody complexes using X-ray crystallography ,42. BecaOne of the first conformational B-cell epitope predictors is the conformational epitope predictor (CEP) . Given aDiscoTope, a method developed by Andersen et al. , uses a Å) of at least 20% in the complex are predicted as belonging to a discontinuous epitope. This method was shown to outperform the CEP method [The B-cell conformational epitope predictor proposed by Rapberger et al.  works asP method  using a P method .Ponomarenko et al.  introducEllipro, a conformational B-cell epitope predictor developed by Ponomarenko et al. , implemePEPITO, a method for predicting conformational B-cell epitopes introduced by Sweredoski and Baldi , uses a Rubinstein et al.  exploredPizzi et al.  have proIn general, mimotope analysis methods available in the literature differ from each other in terms of: i) how they represent the antigen structure/sequence; ii) how they align mimotopes with the target antigen structure/sequence; iii) how they cluster the mimotopes and rank the predicted epitopes. For example, PepSurf ,64 repreIn this section, we highlight recent developments, ongoing efforts, and some promising directions for developing reliable B-cell epitope prediction methods.Söllner et al.  have recS, into a single consensus or meta predictor exist: (i) majority voting: the score for consensus prediction is obtained by the averaging the predicted scores of the individual predictors in S; (ii) weighted linear combination: the consensus prediction is obtained via a weighted sum of the predictions obtained from the predictors in S. The weights can be assigned based on the estimated performance of the predictors on a training dataset, or optimized to minimize the prediction error of the combined predictors on a training dataset; (iii) meta-learning: A meta-classifier is trained on a training dataset using the outputs of the predictors in S on each input sample as input to the classifier and the corresponding class label as the desired output of the classifier.Ensemble methods that combine the predictions of several predictors often outperform individual predictors in many biomolecular sequence and structure classification tasks -71. SeveRecently, Söllner  introducAntigen-antibody interactions constitute a subtype of protein-protein interactions. Therefore, the development of improved conformational B-cell epitope prediction tools may benefit from recent advances in developing protein-protein interface residue prediction methods. Hence, it would be interesting to explore the development of conformational B-cell epitope predictors that utilize or adapt sophisticated protein-protein interface predictors e.g., those that make use of sequence and structure-derived features, analyses of surface patches ,73,74 shThe immune epitope database (IEDB) ,79 is peGiven the large number of B-cell epitope prediction methods available, there is an urgent need for systematic assessment of different methods on standard benchmark datasets . In pracRigorous comparative analyses of alternative methods are indispensible for improving our understanding of the strengths and weaknesses of different B-cell epitope prediction methods and for expediting the development of improved methods. Such critical assessment of alternative methods has proven quite valuable in other tasks e.g., protein structure prediction , proteinThe development of standardized data representations that would allow different prediction methods to be evaluated on standardized benchmark datasets would be extremely useful not only for comparing the methods but also for developing meta-servers combining the predictions of several prediction tools .The Epitopes Toolkit (EpiT)  represen model builder, an application for building and evaluating epitope predictors and serializing these models in a binary format (model files). This application is an extension of WEKA [ predictor, an application for applying a model to test data .EpiT consists of two main components: i) of WEKA , a widelhttp://ailab.cs.iastate.edu/epit. In addition, the project web site offers a rich resource for the developers of epitope prediction tools and for EpiT users. Some examples of the useful resources available at the EpiT project web site include:EpiT is implemented in Java . EpiT can be freely downloaded from the project web site at ● EpiT documentation: A tutorial and an API documentation of EpiT components that includes several examples of how to build an epitope predictor and how to build an ensemble or a consensus predictor.● An expanding Repository of Epitope Predictors (REP): Ready to use models for predicting linear B-cell epitopes using BCPred , FBCPred● A Repository of Epitope Datasets (RED): A repository of epitope benchmark datasets made available by the authors as well as several other publicly available datasets (in WEKA format) that can be used by EpiT users to build their own customized epitope prediction tools. Researchers can contribute additional benchmark datasets to the repository.EpiT toolkit is available under the GNU General Public License (GPL) which allows others to freely extend or modify the software so long as the modified software is also made available under the GNU GPL.Developing improved methods for predicting B-cell epitopes requires large datasets of experimentally well-characterized B-cell epitopes, and especially, antigen-antibody complexes and protective epitopes. Special care must be exercised to ensure that the datasets used to train and evaluate the predictors are of high quality: unique epitopes. However, the uniqueness of epitope sequences is not a sufficient condition for non-redundancy of the dataset because a pair of unique linear B-cell epitopes can share a high degree of pairwise sequence similarity. Unless additional similarity reduction steps are taken to eliminate similar epitopes from the dataset, the predictive performance of B-cell epitope predictors estimated using cross-validation on such datasets can be overly optimistic; and in some cases, lead to false conclusions regarding the performance of different predictors  relative to each other [● Constructing Non-Redundant datasets: Redundant antibody-antigen complexes should be eliminated from the datasets of B-cell epitopes used to train and evaluate B-cell epitope predictors. Otherwise, the estimated performance of predictors can be overly optimistic due to presence of similar complexes in both the training and test datasets. Most of existing linear B-cell epitope datasets ,13,87 coch other . reported epitope are treated as “non-epitope” residues. Because the experimental data available are necessarily incomplete, some of these “non-epitope” residues might in fact be epitope residues. Training an epitope predictor using a dataset in which some epitope residues are incorrectly labeled as non-epitope residues is tantamount to training the predictor on a noisy dataset. Performance estimates of the predictors on such a dataset tend to exaggerate the number of false positives . Some authors [● Identifying Non-epitope data: In the currently available conformational B-cell epitope datasets ,17, the  authors ,10,12,13 authors . However authors  has show threshold-dependent because it describes the performance of the predictor for a given choice of the classification threshold. Moreover, it is possible to trade off one measure  against another . To get a more comprehensive picture of the performance of the predictor, it is useful to consider the Receiver Operating Characteristic (ROC) curve which describes the performance of the classifier over all possible choices of the classification threshold. The ROC curve is obtained by plotting the true positive rate as a function of the false positive rate or, equivalently, sensitivity versus (1-specificity) as the discrimination threshold of the binary classifier is varied. Each point on the ROC curve corresponds to a specific choice of the classification threshold, and hence a particular choice of the tradeoff between true positive rate and false positive rate. The area under ROC curve (AUC) is a useful threshold-independent summary statistic for comparing two ROC curves. The AUC is defined as the probability that a randomly chosen positive sample will be ranked higher than a randomly chosen negative sample. An AUC = 1 corresponds to a perfect predictor, whereas an AUC = 0.5 corresponds to a predictor that classifies each input sample by randomly guessing its class label. Any predictor with a performance that is better than random will have an AUC value that lies between 0.5 and 1.0. AUC is one of the most widely used metrics for comparing the performance of different B-cell epitope predictors. It is also the recommended metric for assessing the performance of B-cell epitope predictors [The use of large, non-redundant, and experimentally well-characterized datasets can help increase the accuracy of the cross-validation based estimates of the performance of B-cell epitope predictors trained on such datasets. However, it does not necessarily inform the choice of performance measures to use for comparing different predictors . Accuracy, sensitivity, specificity, and correlation coefficients are widely used metrics for evaluating the performance of prediction methods . None ofedictors . Howeveredictors  has showAAP: Amino Acid Pair; AUC: Area Uunder ROC Curve; CEP: Conformational Epitope Predictor; EpiT: Epitopes Toolkit; FADE: Fast Atomic Density Evaluation; GPL: General Public License; HMM: Hidden Markov Model; IEDB: Immune Epitope Database; NIAID: National Institute of Allergy and Infectious Diseases: PDB: Protein Data Bank; PSSM: Position Specific Scoring Matrix; RED: Repository of Epitope Datasets; REP: Repository of Epitope Predictors; ROC: Receiver Operating Characteristic; SSP: Statistically Significant Pair; SVM: Support Vector Machine.The author declare no competing interests.YE and VH conceived and wrote this review article.


Type 2 diabetes mellitus is a worldwide epidemic with considerable health and economic consequences. Diabetes is an important risk factor for cardiovascular disease, which is the leading cause of death in diabetic patients, and decreasing the incidence of diabetes may potentially reduce the burden of cardiovascular disease. This article discusses the clinical trial evidence for modalities associated with a reduction in the risk of new-onset diabetes, with a focus on the role of antihypertensive agents that block the renin–angiotensin system. Lifestyle interventions and the use of antidiabetic, anti-obesity, and lipid-lowering drugs are also reviewed. An unresolved question is whether decreasing the incidence of new-onset diabetes with non-pharmacologic or pharmacologic intervention will also lower the risk of cardiovascular disease. A large ongoing study is investigating whether the treatment with an oral antidiabetic drug or an angiotensin-receptor blocker will reduce the incidence of new-onset diabetes and cardiovascular disease in patients at high risk for developing diabetes. The information used to prepare this manuscript was gathered by reviewing guidelines for treatment of prediabetes and from a PubMed search using the following keywords: ‘antihypertensive therapy’, ‘cardiovascular disease’, ‘impaired fasting glucose’, ‘impaired glucose tolerance’, ‘renin–angiotensin system’ and ‘type 2 diabetes mellitus’. The major randomised, controlled clinical trials evaluating the capacity of lifestyle interventions, antidiabetic drugs, lipid-lowering drugs, and antihypertensive drugs on the delay of new-onset diabetes and cardiovascular disease are reviewed herein.Evidence suggests that lifestyle modifications aimed at weight reduction and increased physical activity and antidiabetic pharmacologic interventions reduce the risk of new-onset diabetes. Although there is ample evidence that antihypertensive therapy with RAS inhibitors is associated with a reduced risk of new-onset diabetes compared with other classes of antihypertensive drugs, the prognostic significance of this differential effect remains controversial. For now, lifestyle measures and the reduction of global cardiovascular disease risk may be a more effective strategy to improve vascular health and limit insulin resistance in patients with hypertension than restricting the use of any particular antihypertensive agent.The prevalence of type 2 diabetes mellitus is increasing worldwide. The number of people living with diabetes is expected to more than double from 171 million in 2000 to 366 million by 2030   and the The current criteria for diagnosis of diabetes are a fasting plasma glucose level ≥ 126 mg/dl, symptoms of hyperglycaemia, and a casual plasma glucose level ≥ 200 mg/dl, or a 2-h postchallenge plasma glucose level ≥ 200 mg/dl during an oral glucose tolerance test; fasting plasma glucose < 100 mg/dl and 2-h plasma glucose < 140 mg/dl are considered ‘normal’ . The intA comparison of recommendations for treatment of prediabetes by the American Diabetes Association, American Heart Association and American College of Endocrinology is attached . For theLifestyle modification aimed at weight loss has been shown to be highly effective in preventing or delaying the development of diabetes in high-risk subjects . In the Although neither the Finnish Diabetes Prevention Study nor the DPP was designed to assess cardiovascular disease benefit, cardiovascular disease risk factors were reduced in both trials. In the Finnish study, at the end of 1 year, patients in the intervention group had significantly greater reductions in weight (p< 0.001), systolic (p= 0.007) and diastolic (p= 0.02) blood pressure, serum triglycerides (p= 0.001), and fasting plasma glucose levels (p< 0.001) . In the A number of studies have assessed the impact of different classes of antidiabetic drugs on prevention of diabetes  26–28).–28.26–28Similar results were achieved in the glycaemic arm of the Diabetes REduction Assessment with ramipril and rosiglitazone Medication (DREAM) trial, in which 5269 patients with impaired fasting glucose and/or impaired glucose tolerance and no previous cardiovascular disease or significant renal disease were randomised to treatment with rosiglitazone or placebo (and ramipril or placebo) in a 2 × 2 factorial design . During The Outcome Reduction with an Initial Glargine Intervention (ORIGIN) may help to clarify the effect of glycaemic control on cardiovascular disease risk. In ORIGIN, 12,612 people aged 50 years or older with evidence of cardiovascular disease and impaired fasting glucose, impaired glucose tolerance or diabetes were randomised to treatment with insulin glargine or standard glycaemic care (and long-chain ω-3 polyunsaturated fatty acids or placebo) to determine the effect of these treatments on cardiovascular disease risk . The ant2) individuals with normal or impaired glucose tolerance, orlistat plus lifestyle interventions reduced the incidence of new-onset diabetes by 37.3% (p= 0.0032) compared with placebo plus lifestyle intervention  were evident in the Anglo-Scandinavian Cardiac Outcomes Trial-Blood Pressure Lowering Arm (ASCOT-BPLA) . In the Analyses of several clinical trials using antihypertensive agents in patients with and without hypertension have demonstrated that RAS blockade significantly reduces the risk of new-onset diabetes in patients treated with ACE inhibitors –44 or ARSeveral recently completed trials using ACE inhibitors or ARBs included new-onset diabetes as a prespecified outcome measure. In the hypertension arm of the 3-year DREAM trial, the incidence of the primary composite end-point of new-onset diabetes or death was similar in the ramipril and the placebo groups  . There wpost hoc analysis of the VALUE trial indicated that the risk of cardiac morbidity  in patients who developed diabetes during the 4.2-year followup was intermediate between that of those who had diabetes at baseline and those who did not develop diabetes at any point , which randomised 15,245 patients aged 50 years or older with hypertension and high risk of cardiovascular disease events to a mean of 4.2 years of valsartan- or amlodipine-based therapy . Althougny point . Diabeteny point ,55.New-onset diabetes was a secondary end-point in both the Ongoing Telmisartan Alone and in Combination with Ramipril Global Endpoint Trial (ONTARGET)  and the Recent analyses of metabolic and clinical outcomes in non-diabetic patients in the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) confirm the metabolic benefits of RAS inhibitors compared with diuretics but fail to demonstrate a commensurate reduction in cardiovascular disease events. ALLHAT, which did not include new-onset diabetes as a prespecified outcome, randomised patients aged 55 years or older with stage 1 or stage 2 hypertension and at least one additional cardiovascular disease risk factor to chlorthalidone, amlodipine or lisinopril for a mean of 4.9 years . Among pThese analyses have not resolved the controversy regarding the relationship between new-onset diabetes and cardiovascular disease risk and have led some investigators to question whether diuretic- or β-blocker-associated diabetes confers the same clinical implications as new-onset diabetes that develops outside the setting of antihypertensive therapy .Further clarification of the comparative value of antihypertensive combinations comparing the use of a glucose neutral agent such as a CCB with a thiazide-type diuretic will come from the Avoiding Cardiovascular Events Through Combination Therapy in Patients Living With Systolic Hypertension trial. In this trial, the high-risk hypertensive patients were initially randomised to a fixed-dose combination of either an ACE inhibitor/thiazide-type diuretic or an ACE inhibitor/CCB. New onset diabetes is a prespecified secondary end-point .The question of whether preventing new-onset diabetes is associated with a reduction in cardiovascular disease events may be resolved with the results of two ongoing large-scale clinical trials – Nateglinide And Valsartan in Impaired Glucose Tolerance Outcomes Research (NAVIGATOR)  and ACE post hoc analyses of ASCOT-BPLA (In the interim, the effects of antihypertensive therapy and the presence of comorbid risk factors on the risk of new-onset diabetes and cardiovascular disease should be carefully evaluated for each patient. Indeed, in COT-BPLA  and VALUCOT-BPLA , baselinCOT-BPLA , highligThe mechanisms by which RAS inhibition reduces the development of diabetes remain to be established. ACE inhibitors and ARBs improve insulin sensitivity, possibly caused by their vasodilatory effects, which result in increased blood flow and increased insulin delivery to peripheral skeletal muscles –67. ACE Diabetes is a worldwide epidemic with the substantial health ramifications. Because cardiovascular disease is the leading cause of death in patients with diabetes, the prevention of diabetes has the potential to reduce the burden of cardiovascular disease. Evidence suggests that lifestyle modifications aimed at weight reduction and increased physical activity and antidiabetic pharmacologic interventions reduce the risk of new-onset diabetes. Although there is ample evidence that antihypertensive therapy with RAS inhibitors is associated with a reduced risk of new-onset diabetes compared with other classes of antihypertensive drugs, the prognostic significance of this differential effect remains controversial. No study using antihypertensive therapy has yet demonstrated a commensurate reduction in cardiovascular disease risk, and secondary analyses of trials of RAS blockers have provided conflicting results. It appears reasonable to avoid traditional β-blocker and thiazide-type diuretic therapy in the minority of patients who have impaired fasting glucose or impaired glucose tolerance and whose blood pressure can be controlled on single-agent antihypertensive therapy. In these patients, the use of ACE inhibitor and ARB therapy, like in those with the metabolic syndrome, may have an advantage in decreasing the subsequent risk for new-onset diabetes. When blood pressure is not effectively controlled with ACE inhibitor or ARB therapy, the addition of a thiazide-type diuretic is clearly indicated as the benefits of achieving blood pressure control appear to outweigh any negative effects on glucose metabolism. Whether a CCB should be added before a thiazide-type diuretic remains uncertain. The results of the large-scale NAVIGATOR study may help resolve the issue of whether treatment of high-risk patients with an antidiabetic drug or an ARB reduces the incidence of new-onset diabetes and cardiovascular disease.For now, lifestyle measures including weight loss and exercise as well as the importance of addressing global cardiovascular disease risk through the use of statin and aspirin therapy may be a more effective strategy to improve vascular health and limit insulin resistance in patients with hypertension. Additionally, achieving blood pressure control with agents that do not worsen insulin resistance should be attempted; failing that, vasodilating β-blockers and low-dose diuretics must be used to reduce risk. Physicians should be aware that in most cases multiple antihypertensive agents will be needed. While keeping serum potassium levels normal, diuretics should be considered when blood pressure goals have not been achieved in patients with impaired fasting glucose. When used in this setting, although new-onset diabetes may be more likely to occur, cardiovascular outcomes are still improved by achieving additional blood pressure reduction.


Papain-solubilized tumour-specific antigens from the aminoazo dye-induced rat hepatoma D23 were purified by a combination of lectin affinity and immunoadsorbent column chromatography. Isolated antigens were radio-iodinated using three procedures and analysed for their reaction with specific antibodies in syngeneic immune sera by double-antibody co-precipitation tests and by the rebinding of labelled antigens to specific and non-relevant antibodies immobilized on Sepharose-4B. Soluble hepatoma D23-specific antigens were labile to radiolabelling, and for optimal retention of serological reactivity it was necessary to protect the antigenic determinant by performing the chloramine T method of iodination with antigen bound to the immunoadsorbent followed by elution from the solid phase with 3M NaSCN. Immunoadsorption chromatography indicated that one consequence of radiolabelling hepatoma D23-specific antigen with 125I was a reduction in the affinity of the labelled antigen for its syngeneic specific antibody.


Pre-operative X-irradiation of s.c. implanted first-generation mammary tumours in C3H mice, using either 500 rad or two fractions of 350 rad, produced no improvement in the success of surgery in causing local control or in reduction of distant metastases. The metastasis rate was just significantly higher after the two-fraction treatment of the implanted tumour than after surgical removal alone. The results are in agreement with previously published results on carcinomas and a sarcoma but contrast with those for murine lymphomas.


The mechanism by which the arthropod Oskar and vertebrate TDRD5/TDRD7 proteins nucleate or organize structurally related ribonucleoprotein (RNP) complexes, the polar granule and nuage, is poorly understood. Using sequence profile searches we identify a novel domain in these proteins that is widely conserved across eukaryotes and bacteria.Using contextual information from domain architectures, sequence-structure superpositions and available functional information we predict that this domain is likely to adopt the winged helix-turn-helix fold and bind RNA with a potential specificity for dsRNA. We show that in eukaryotes this domain is often combined in the same polypeptide with protein-protein- or lipid- interaction domains that might play a role in anchoring these proteins to specific cytoskeletal structures.Thus, proteins with this domain might have a key role in the recognition and localization of dsRNA, including miRNAs, rasiRNAs and piRNAs hybridized to their targets. In other cases, this domain is fused to ubiquitin-binding, E3 ligase and ubiquitin-like domains indicating a previously under-appreciated role for ubiquitination in regulating the assembly and stability of nuage-like RNP complexes. Both bacteria and eukaryotes encode a conserved family of proteins that combines this predicted RNA-binding domain with a previously uncharacterized domain (DUF88). We present evidence that it is an RNAse belonging to the superfamily that includes the 5'->3' nucleases, PIN and NYN domains and might be recruited to degrade certain RNAs.This article was reviewed by Sandor Pongor and Arcady Mushegian. Animal germ cells possess specialized ribonucleoprotein (RNP) granules termed nuage or germinal granules that appear as electron-dense fibrous and motile bodies localized to the cytoplasm -3. In seDrosophila, where the RNA-binding protein Oskar has been found to be a critical player in initiating the formation of polar granules [Nucleation of such RNP complexes has been extensively studied in granules ,8,9. In Drosophila polar granule protein Oskar showed that it contains a C-terminal catalytically inactive SGNH hydrolase domain [Drosophila protein CG8920 , vertebrate protein limkain b1 , the Arabidopsis protein AT2G15560 , proteins typified by TTHERM_00129230 from ciliates  and cgd2_2940 and its orthologs from apicomplexans . Some of these proteins, such as TDRD5, TDRD7 and Limkain b1 from vertebrates showed multiple tandem repeats of the homologous region that enabled us to precisely determine its boundaries  in a profile-profile comparison using the HHSEARCH program. PSI-BLAST and JACKHMMER searches recovered a bacterial representative NE0665 (gi: 30248674) from Nitrosomonas europaea, whose NMR structure has been determined as part of the structural genomics initiative  domain.Examination of this structure showed that the domain adopts a winged helix-turn-helix (wHTH) fold, which is characterized by a core 3-stranded HTH with a C-terminal extension of two strands. Structure prediction of just the eukaryotic versions using the JPRED program, which combines information from residue frequencies in columns, a HMM and a PSSM derived from the alignment, also predicted a secondary structure progression completely congruent with that observed in the above bacterial version. Based on these observations we named this novel conserved domain the e.g. Mbur_0520 Methanococcoides burtonii) that appear to be relatively late lateral transfers of bacterial versions. The bacterial and the eukaryotic versions can be distinguished based on their sequence conservation patterns  showed that it is a member of the 5'->3' nuclease domain superfamily that includes RNAses such as the PIN, NYN, and phage T4-type viral RNAse H domains [The OST-HTH superfamily is primarily found in bacteria and eukaryotes with very rare occurrences in archaea  [Eukaryotic members of the OST-HTH superfamily, other than those in Limkain b1, show a remarkable diversity of architectures Fig. . Versionlipases) ; howeverlipases) ,16.We represented the domain architectures of the OST-HTH as an ordered network where each node is a domain and each edge represents co-occurrence of two domains as immediate neighbors in the same polypeptide Fig. . This neNitrosomonas europaea NE0665) as a query. The best hit in this search was the wHTH domain of the archaeal replication protein CDC6 that binds dsDNA [Others and we have formerly characterized several other RNA-binding domains with the classical and circularly permuted versions of the wHTH fold . Exampleds dsDNA . Both thds dsDNA ,10, suggThalassiosira). These findings indicate the presence of robust RNAi machinery that might be specifically involved in chromatin level silencing as in other eukaryotes. Identification of several OST-HTH superfamily members in these eukaryotic lineages raises the possibility that they participate in the formation of RNP structures that are critical for counter-transposon defense. As in the case of the animal nuage/germinal granules these RNPs could also participate in sub-cellular localization of various RNAs in various eukaryotes. For example, in photosynthetic stramenopiles such as Aureococcus anophagefferens there are giant proteins in which the OST-HTH domain occurs with multiple distinct RNA-binding and ubiquitin E3 ligase domains in the same polypeptide and could function as large scaffolds for assembly of RNP complexes .Sequence profile searches were performed against the NCBI non-redundant (NR) database of protein sequences , and a locally compiled database of proteins from eukaryotes with completely or near-completely sequenced genomes. PSI-BLAST searches  were perThe authors declare that they have no competing interests.VA and LA were made the discovery and wrote the paper. DZ was performed the analysis on the LK-nuclease (DUF88) domain. All authors read and approved the final manuscript.Sandor Pongor, International Centre for Genetic Engineering and Biotechnology, Trieste, ItalyThe discovery of RNA regulatory mechanisms is one of the most fascinating insights of modern biology. RNP granules are especially difficult targets within this widespread and varied set of molecular scenarios because of their complex and presumably loosely defined molecular architecture is not easily amenable to structural studies. The mechanism by which RNP granules self-organize at specific cellular locations is currently poorly understood. The authors present a predictive bioinformatics analysis of the proteins of the polar granule and nuage complexes, based on sequence alignment, domain architecture analysis and sequence-structure superpositions and suggest that a hitherto unknown domain-type, OST-HTH, with a potential specificity for dsRNA may play a key role in the recruitment process that anchores nucleoprotein granules including miRNAs, rasiRNAs and piRNAs to specific cellular structures. The paper is clearly and carefully presented and the conclusion represents an important advance.1. The Materials and Methods section could be more detailed and more understandable for a wide audience. Minor: I did not find reference to JACKHMMER,Response: A detailed Materials and Methods in available as a part of Additional File 1Arcady Mushegian, Stowers Institute, Kansas City, United StatesI have no concerns about the validity of the sequence relationships and structural predictions described in this work. Several queries to the authors:"It is also possible that the nuage complex is related to other major RNP granules such as those found in neurons ." -- relResponse: The two RNPs indeed share several common proteins or those with homologous RNA-binding domains and similar architectures. Additionally, both neuronal RNPs and nuage-like complexes share other distinctive features: 1) A similar ultrastructure; 2) Presence of dsRNA; 3) intracellular motility. The motility of both these granules marks them as specialized elements required for active sub-cellular RNA localization as against passive aggregations of RNAs complexed with proteins.p.6 last paragraph: I do not think that the network shown in Fig. This point is in fact not so minor, as it concerns the explanatory and predictive utility of networks in biology.Response: The network in Fig. This file contains additional material and methods, data for phyletic patterns, domain architectures and alignments of OST-HTH and associated conserved domains.Click here for file


These data suggest that aging diminishes the emigration of IgA immunoblasts from these lymphoid aggregates, as well as their migration to the intestinal LP. Flow cytometry and lymphocyte adoptive transfer studies showed 3- to 4-fold age-related declines in the homing of antibody-containing cells and mesenteric lymph node lymphocytes to the small intestines of rhesus macaques and rats, respectively. The number of peripheral blood IgA immunoblasts expressing the homing molecule α4β7 declined 30% in senescent rats. This was accompanied by a >17% decrease in the areal density of LP blood vessels staining positive for the cell adhesion molecule MAdCAM-1. Cumulatively, declines in expression of these homing molecules constitute a substantial age-related diminution of IgA immunoblast homing potential. In vitro antibody secretion by LP plasma cells, i.e. antibody secreted per antibody-positive cell, remains unchanged as a function of donor age. Intestinal mucosal immunosenescence is a consequence of reduced homing of IgA plasma cells to the intestinal LP as a result of declines in homing molecule expression.The elderly are characterized by mucosal immunosenescence and high rates of morbidity and mortality associated with infectious diseases of the intestinal tract. Little is known about how the differentiation of immunoglobulin A (IgA) plasma cells in Peyer's patches (PPs) and their subsequent homing to the small intestinal lamina propria (LP) is affected by aging. Quantitative immunohistochemical analyses demonstrated a 2-fold increase in the number of IgA


Rituximab has also shown activity in other B-cell disorders such as chronic lymphocytic leukaemia. The full potential of this immunotherapeutic agent remains to be defined in ongoing and future clinical trials.Rituximab is a chimeric human/mouse monoclonal antibody that is approved for the treatment of relapsed and refractory non-Hodgkin's lymphoma (NHL) and in combination with CHOP  chemotherapy as first-line therapy for diffuse large B-cell NHL, where it has shown the first survival advantage over CHOP alone in more than 20 years. Strategies to help define the optimal therapeutic usage of rituximab are being assessed, including first-line and maintenance or extended therapy, and the combination of rituximab with chemotherapy in indolent NHL. Emerging data suggest that earlier use may yield higher response rates, extended therapy can prolong remission, and the addition of rituximab to chemotherapy can increase clinical and molecular remission rates when compared with those achieved using chemotherapy alone. Studies in the peritransplant setting suggest a role for rituximab  Combination chemotherapy has markedly improved outcome in aggressive non-Hodgkin's lymphoma (NHL) since its introduction in the 1970s, but is curative in less than half of the patients. In indolent NHL, the impact of chemotherapy has been much lower; while response rates and progression-free survival (PFS) may be improved, overall survival (OS) has not increased and advanced indolent NHL remains essentially incurable without allogeneic bone marrow transplant.A number of newer approaches to improve outcome are under investigation, including the use of monoclonal antibodies targeted against specific antigens expressed by lymphoma cells. The cell surface antigen CD20 is in many ways an ideal target: it is expressed by 95% of B-cell NHL cases but not on haematopoietic stem cells; it has a functional role in B-cell growth and also does not normally circulate as a free antigen in the plasma, so free antigen does not compete for antibody binding.in vitro studies. These include complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and induction of apoptosis , with no increase in treatment-related adverse events. In the previously untreated patients, the difference in EFS was even greater .Several studies have examined rituximab as a first-line therapy in patients with indolent NHL . This trTable 1ituximab  showed hThe efficacy and tolerability of rituximab suggest that it may be particularly useful as a first-line single-agent therapy in elderly patients, and also in young women who want to preserve their fertility. It may also have a role as a first-line therapy in asymptomatic patients with advanced disease (stages 3 or 4), aiming to prolong the asymptomatic period. This will need to be assessed in clinical trials, however.bcl-2, an antiapoptotic gene that may serve as a survival factor for lymphoma cells and can be detected by polymerase chain reaction (PCR). Achievement of molecular remission post-therapy (clearance of bcl-2 positive cells) appears to be associated with prolonged PFS and may even predict longer OS  has demonstrated high activity, achieving a response rate of around 97% (57% CR) in 82 patients with previously untreated follicular lymphoma  chemotherapy has shown high rates of molecular remission in the peripheral blood (90%) and bone marrow (76%) of patients with previously untreated follicular lymphoma at a 6-month follow-up  after CHOP or fludarabine/mitoxantrone (FM) chemotherapy .Rituximab has also been assessed as consolidation treatment in patients with minimal residual disease (MRD)  with rituximab plus FCM in patients with relapsed follicular and mantle cell lymphoma (MCL), and interim analyses have reported a response rate of 83% for the immunochemotherapy arm compared with 53% for FCM alone , demonstrated a response rate of 34% with a median PFS of 14 months  with autologous stem cell transplantation (ASCT) is being increasingly considered for patients achieving their first complete remission. Addition of rituximab to HDT/ASCT has been evaluated by The activity of single-agent rituximab in relapsed aggressive NHL has been demonstrated, but the strongest data in aggressive NHL have come from studies of combination immunochemotherapy, particularly rituximab plus CHOP (R-CHOP). vs 63%, P=0.005), improved PFS  and increased EFS and OS . No significant difference in treatment-related toxicity was seen between the groups. In an interim subset analysis of 328 patients, it was shown that R-CHOP was superior to CHOP alone in both low-risk and high-risk patients, and also in patients aged 60–70 or 70–80 years  with previously untreated diffuse large B-cell lymphoma (DLCL) demonstrated a significant advantage for R-CHOP over CHOP alone  in patients treated with a rituximab-containing salvage regimen which appear superior to those achieved with chemotherapy alone .While HDT/ASCT may be considered a potentially curative therapy for aggressive NHL, approximately 40–60% of patients with aggressive NHL, and most patients with indolent NHL, will eventually relapse following transplant. Relapse post-transplant is due either to contamination of the stem cell harvest or persistence of MRD post-HDT. A number of strategies have therefore been employed in attempts to prevent/reduce stem cell contamination (stem cell purging) and/or eradicate lymphoma cells that survived HDT (post-transplant ‘mopping up’).bcl-2-positive cells have increased relapse-free survival compared with patients whose bone marrow harvests remain positive  is limited (up to 58% of harvests remain bcl-2 positive), in vivo purging with rituximab has produced bcl-2-negative harvests in 69–90% of stem cell harvests without compromising stem cell yield or function  and small lymphocytic lymphoma (SLL), the lymphomatous equivalent of CLL. However, single-agent rituximab has yielded low response rates (approximately 15%) in patients with relapsed CLL/SLL . Given tituximab . The impRituximab has been evaluated in a range of other B-cell malignancies, including post-transplant lymphoproliferative disorder (PTLD), Waldenström's disease and HIV-associated lymphoma. Data from two studies indicate that approximately 50% of the patients with PTLD may respond to rituximab monotherapy, and CRs of 33 and 52% have been reported , single-agent rituximab has demonstrated responses in up to 72% of patients, with CR in 32% and a response duration of 12+ months for patients in CR  or human anti-chimeric antibody (HACA) are available (Rituximab Summary of Product Characteristics). Of 67 patients evaluated for HAMA, no responses were observed; of 356 evaluated for HACA, 1.1% (four patients) were positive. These data indicate that retreatment or maintenance therapy with rituximab remains feasible: patients may continue responding to repeated rituximab exposure without developing immune resistance.in vivo purge and as maintenance therapy. A BNLI trial of rituximab vs watchful waiting in patients with advanced-stage asymptomatic indolent NHL is planned.Several trials are ongoing or planned in order to better define the role of rituximab in therapy for indolent  for elderly patients (ECOG 4494) or younger patients (MInt) with untreated DLCL . Severalin vivo purging is active and does not compromise ASCT although the role of both in vivo purging and post-transplant maintenance remains to be defined. One characteristic central to the integration of rituximab in these diverse clinical settings is its ability to achieve not only clinical but molecular responses–the mechanisms of action of this monoclonal antibody underpin its potential and utility in the treatment of CD20-positive disorders. An improved understanding of these mechanisms and the molecular basis of NHL will help the full therapeutic potential of this agent to be realised.In summary, rituximab has shown efficacy and tolerability as monotherapy in indolent NHL. The data suggest that patients treated earlier in the course of their disease may respond better to therapy and that maintenance therapy may provide additional benefit. In both indolent and aggressive NHL, the use of rituximab with chemotherapy may provide an advantage over chemotherapy alone and this is reflected in the number of current and planned studies of immunochemotherapy. The survival benefit for rituximab plus CHOP over CHOP alone demonstrated in the GELA study is awaiting full confirmation by the MInT and ECOG trials in elderly and younger patients with untreated DLCL. The general trend seen in NHL is for improved response rates and quality of response when rituximab is added to chemotherapy: whether this will translate into improved survival will be determined by longer-term follow-up of completed studies. Transplantation studies indicate that rituximab


Collectively, the observations indicate that the EGF receptor and the ERK MAP kinase pathway mediate autocrine signals. In contrast to previous reports, the GRP and IGF-I receptors were shown not to be required for autocrine effects on pancreatic cancer cell proliferation. Autocrine stimulation of the EGF receptor can contribute to sustained mitogenic activity and proliferation of pancreatic cancer cells. © 2001 Cancer Research Campaign http://www.bjcancer.comIn-vitro and in-vivo studies have shown that autocrine growth factors and receptors are frequently expressed in human malignancies. Few of these studies, however, provide evidence that the identified autocrine pathway is functional. In this study, a functional autocrine growth pathway in pancreatic cancer has been identified using an in-vitro cell culture system. When pancreatic cancer cells were grown without change of medium, proliferation was greater than when either medium was replaced frequently  or cells were grown in the presence of the EGF receptor tyrosine kinase inhibitor AG1478 or the MEK inhibitor PD098059 (HPAF or CAPAN-2). Activity of extracellular-regulated kinases (ERK) 1 and 2 and c-


Myocardial infarction causes significant mortality and morbidity. Timely diagnosis allows clinicians to risk stratify their patients and select appropriate treatment. Biomarkers have been used to assist with timely diagnosis, while an increasing number of novel markers have been identified to predict outcome following an acute myocardial infarction or acute coronary syndrome. This may facilitate tailoring of appropriate therapy to high-risk patients. This review focuses on a variety of promising biomarkers which provide diagnostic and prognostic information.Heart-type Fatty Acid Binding Protein and copeptin in combination with cardiac troponin help diagnose myocardial infarction or acute coronary syndrome in the early hours following symptoms. An elevated N-Terminal Pro-B-type Natriuretic Peptide has been well validated to predict death and heart failure following a myocardial infarction. Similarly other biomarkers such as Mid-regional pro-Atrial Natriuretic Peptide, ST2, C-Terminal pro-endothelin 1, Mid-regional pro-Adrenomedullin and copeptin all provide incremental information in predicting death and heart failure. Growth differentiation factor-15 and high-sensitivity C-reactive protein predict death following an acute coronary syndrome. Pregnancy associated plasma protein A levels following chest pain predicts risk of myocardial infarction and revascularisation. Some biomarkers such as myeloperoxidase and high-sensitivity C-reactive protein in an apparently healthy population predicts risk of coronary disease and allows clinicians to initiate early preventative treatment. In addition to biomarkers, various well-validated scoring systems based on clinical characteristics are available to help clinicians predict mortality risk, such as the Thrombolysis In Myocardial Infarction score and Global Registry of Acute Coronary Events score. A multimarker approach incorporating biomarkers and clinical scores will increase the prognostic accuracy. However, it is important to note that only troponin has been used to direct therapeutic intervention and none of the new prognostic biomarkers have been tested and proven to alter outcome of therapeutic intervention.Novel biomarkers have improved prediction of outcome in acute myocardial infarction, but none have been demonstrated to alter the outcome of a particular therapy or management strategy. Randomised trials are urgently needed to address this translational gap before the use of novel biomarkers becomes common practice to facilitate tailored treatment following an acute coronary event. Coronary artery disease (CAD) and its end result, myocardial infarction (MI) continue to be a significant cause of mortality and morbidity in the western world. Over the past 50 years, it has become clear that the cascade of thrombotic events following atherosclerotic plaque rupture causes occlusion of the coronary artery, interrupting blood supply and oxygen to myocardium thus resulting in infarction. Myocardial necrosis following infarction is followed by heart failure, myocardial rupture or arrhythmias. Early treatment of myocardial ischaemia to prevent necrosis with treatments such as fibrinolysis, coronary artery bypass grafting and percutaneous coronary intervention have improved outcome .Over time it has become clear that in order for such treatments to be of maximal benefit, timely diagnosis is important. Here, biomarkers become important, to help us improve our diagnostic accuracy of the disease, as treatments are not without risk. Furthermore, biomarkers also provide prognostic information about the disease, which then aids clinicians in deciding how aggressively they need to treat the disease.th percentile of the upper reference limit) and one of 1) symptoms of ischaemia, 2) electrocardiogram (ECG) changes of new ischaemia, 3) new pathological Q waves or 4) imaging evidence of new loss of viable myocardium.Biomarkers are measurable and quantifiable biological parameters which serve as indices for health and physiology assessments . This inBoth the ECG and cardiac troponin are biomarkers, but the focus of this review will be on serum proteins/markers which have become increasingly important to improve our diagnosis of myocardial infarction, in some cases identifying people at risk of having an infarct and in others to predict long term prognosis following an actual event.A good biomarker is something that is easily measured and can be used as a surrogate marker for disease and its severity . For insSome of these newer biomarkers and their relationship to various pathophysiological processes are depicted in Figure Two well known biomarkers in use for diagnosis of acute myocardial infarction are Creatine-Kinase-MB isoform and Cardiac Troponin. In 2000, Cardiac Troponin replaced CK-MB as the biomarker of choice for diagnosing a myocardial infarction . Troponiwashout phenomenon. Troponin levels peak at 12 hours, and stay elevated for 10 days or more. Whilst the use of Troponin for diagnosing AMI and risk stratification to aid decision making has revolutionised the management of patients presenting with chest pain, the 12-hour wait for the levels to peak remains the Achilles heel of this biomarker. Newer, more sensitive troponin assays [P = 0.003). In addition, the following two biomarkers may help facilitate early diagnosis of AMI, although neither has been compared with the newer high sensitivity troponin assays.Cardiac troponin level is dependent on infarct size , thus gin assays  have beeCopeptin is the more stable surrogate of arginine vasopressin (AVP), with well-known effects on osmoregulation and cardiovascular homeostasis . Post AMCopeptin can rule out MI earlier in addition to a negative Troponin T test . At the H-FABP is a low molecular weight protein involved in myocardial fatty-acid metabolism . It is aBefore broaching the subject of biomarkers it is important to note that as a result of various randomized control trials and registry studies, various risk factors have been identified and entered into scoring systems that allow a clinician to risk stratify disease . PopularNewly introduced biomarkers should complement and have incremental prognostic value over and above these simple risk scores. It is therefore no surprise that biomarkers providing prognostic information following an acute coronary syndrome reflect the various physiological pathways described in the GRACE score . Currently, the only accepted biomarker affecting a change in management of a patient with an acute coronary syndrome is the cardiac troponin.One of the best known biomarkers of biomechanical stress is the B-type Natriuretic Peptide (BNP). Secreted by the ventricles in response to cardiomyocytes under tension , BNP binNTproBNP/BNP provides incremental information on cardiovascular death at one year in the older population above and beyond GRACE score . On its The TACTICS-TIMI 18 study  randomizLike BNP, ANP has similar neurohormonal effects and has a similar secretory profile post AMI. Prior studies have attempted to accurately measure levels of ANP and N-ANP, with limited success ,40. N-ANGDF-15 is a member of the Transforming Growth Factor Beta cytokine superfamily. It is not normally expressed in the heart, but under episodes of stress  its levels go up in a variety of tissues, including cardiomyocytes. It has an antihypertrophic effect, demonstrated in knockout mice which develop early cardiac hypertrophic growth following pressure overload . GDF-15 These findings were validated in ST-elevation MI (STEMIs)  and prosHowever, GDF-15 is not specific for cardiovascular disorders and has been found to be elevated in a variety of malignancies .ST2 is an IL1-receptor-like protein which was found to be elevated in serum of hearts under mechanical stress . ST2 preP= 0.002). This variable is independent of age, Killip class and past medical history. Plasma concentration of CTproET-1 peaks at Day 2 [Endothelin-1 or the more stable C-Terminal portion of pro-Endothelin-1(CTproET1) has also been found to be predictive of death or heart failure following an AMI . ET1 is at Day 2 .Adrenomedullin was first identified in human phaeochromocytoma cells . It is hThe same study group also investigated Copeptin as a prognostic biomarker. They found that Copeptin predicted mortality or heart failure at 60 days post AMI . In addiFigure vulnerable plaques. It is thought that one of the driving forces causing atheromatous plaques to rupture or erode, causing a cascade of events leading to coronary artery occlusion, is inflammation in the plaques. An elevated C-reactive protein measured in seemingly healthy adults was associated with increased cardiovascular risk [Acute coronary syndromes are caused by lar risk ,82. CRP lar risk -87. Thislar risk . While tP = 0.04) and death  post-MI [One study found that CRP measurements (taken between 12 and 24 hours post event) predicted occurrence of heart failure . A new assay for Human Pentraxin 3 is now available. Human Pentraxin 3 is an isoform which is secreted exclusively in vascular endothelium and therefore may be more specific to the vascular plaque inflammatory activity . It remaLeucocytes play a central role in atherosclerotic plaque rupture -96. MyelOne trial showed that post-acute coronary syndrome MPO levels higher than median predicted future death and MI at one year . It alsoTwo population studies show that MPO (and CRP) in healthy individuals are both associated with future development of CAD ,102.Collectively the current evidence supports the need for further studies into the actual role of MPO, and whether elevated MPO levels in the serum directly correlates with MPO released from circulating neutrophils.PaPPA is a proatherosclerotic metalloproteinase  which isLike CRP, PaPPA is expressed when there is a heavy burden of unstable atheromatous plaque, including in carotid arteries . It alsoThe structural integrity of myocardial Extracellular Matrix (ECM) is dependent on endogenous zinc-dependent endopeptidases known as matrix metalloproteinases (MMP). These enzymes are regulated by tissue inhibitors of metalloproteinases (TIMPs). MMPs may degrade myocardial ECM leading to the development of LV dilatation and heart failure and their inhibition in experimental models of AMI has been associated with reduced LV dilatation and wall stress. Although NTproBNP, TIMP1 and MMP 9 were associated with cardiovascular death, heart failure or both, they were not associated with re-infarction . MMP2 isin vitro, so that preanalytical contributions to variation are minimised to some extent. For example, the midregional epitopes assayed in MRproANP and MRproADM are less susceptible to exoprotease action, and prohormone fragment assays tend to be more stable than assays for the actual hormone .Many of the new biomarkers introduced have enhanced stability Although there are large numbers of emerging novel biomarkers, our understanding of the roles and biochemistry of these various peptides in the disease process is still fairly limited. It is difficult to draw specific conclusions from the current body of evidence regarding the mechanisms through which a biomarker could affect the prognosis. Many of the studies use death or major adverse cardiovascular events as endpoints because they are easy to measure, but either of these endpoints could be a culmination of a variety of pathophysiological processes. As such, currently available biomarkers have not been able to add much to helping us tailor our treatment (over and above Troponin). Randomised trials based on the use of biomarkers to alter therapy would be very informative. Although there is evidence that combining biomarkers may increase the accuracy of the tests, the best combinations for diagnosis or prognosis need to be defined. Some analogies can be drawn from heart failure studies; NTproBNP has been used as a biomarker for diagnosis of heart failure . It doesThere is some evidence that a multi-marker strategy can improve diagnosis, risk stratification and prognostication of patients . Of the ADM: Adrenomedullin; AMI: Acute Myocardial Infarction; ANP: Atrial natriuretic peptide; AVP: Arginine vasopressin; BNP: B-type natriuretic peptide; CAD: Coronary artery disease; CTproET1: C-terminal pro-endothelin 1; CKMB: Creatine kinase MB isoform; ECG: Electrocardiogram; ECM: Extracellular matrix; ET1: Endothelin-1; ETA: Endothelin receptor type A; ETB: Endothelin receptor type B; GDF-15: Growth differentiation factor 15; GRACE: Global registry of acute coronary events study; H-FABP: Heart type fatty acid binding protein; HsCRP: High sensitivity C-reactive protein; IGF-1: Insulin-like growth factor 1; IL-33: Interleukin 33; LV: Left ventricle; LVEF: Left ventricular ejection fraction; MI: Myocardial infarction; MMP: Matrix metalloproteinase; MPO: Myeloperoxidase; MRproANP: Mid regional pro-atrial natriuretic peptide; MRproADM: Mid regional proadrenomedullin; N-ANP: N-terminal pro-atrial natriuretic peptide; NSTEACS: Non-ST elevation acute coronary syndromes; NTproBNP: N-terminal pro-B type natriuretic peptide; PaPPA: Pregnancy associated Plasma Protein A; ROC AUC: Area under the receiver operating characteristic curve; TIMI: Thrombolysis in Myocardial infarction study; TIMP: Tissue inhibitors of metalloproteinases; Trop: TroponinProf. Leong Ng has submitted patents on behalf of the University of Leicester on some of the biomarkers in this review, and has acted as a consultant to and received grants in aid from BRAHMS AG and Unipath PLC in the past. Dr. Daniel Chan has no competing interests to declare.LN recruited the patients in the reported studies, performed the analyses and drafted the manuscript. DC participated in the analyses and drafting of the manuscript and figures.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/8/34/prepub


Clethrionomys glareolus) kept in the laboratory under barren housing conditions develop high incidences of type 1 diabetes mellitus due to beta cell– specific lysis in association with the appearance of GAD65, IA-2, and insulin autoantibodies. Wild-caught and immediately analyzed voles show no histological signs of diabetes, and the disease may therefore be induced by circumstances related to the housing of the animals in captivity. We tested the possibility that postnatal stress by either maternal separation or water immersion at different intervals would induce diabetes in adult bank voles. We found that low-frequent stress during the first 21 days of life increases, whereas high-frequent stress markedly reduces, the incidence of type 1 diabetes in adulthood. These results differentiate the role of early-experienced stress on subsequent type 1 diabetes development and emphasize that the bank vole may serve as a useful new animal model for the disease.Wild bank voles (


Family history of haematopoietic malignancies appears to be a risk factor for non-Hodgkin's lymphoma (NHL), but whether risk varies by family member's gender is unclear. Among 121 216 women participating in the prospective California Teachers Study, NHL risk varied by type of haematopoietic malignancy and gender of the relative. Evidence from epidemiologic studies shows that the risk of non-Hodgkin's lymphoma (NHL) is increased by approximately twofold for individuals with a first-degree family history  of haematopoietic malignancy  Consortium of NHL case–control studies found an increased risk of NHL for individuals who reported having a first-degree relative with a haematopoietic malignancy   were identified through annual linkages with the population-based California Cancer Registry. Person-time was accrued from the date a participant completed her baseline questionnaire until the first occurrence of either a diagnosis of a B-cell NHL or a censoring event: a move outside of California; a first diagnosis of another haematopoietic malignancy not included in our case definition; death; or 31 December 2005.The self-administered, baseline questionnaire collected detailed information on personal and family history of cancer. Having ‘no family history’ of a haematopoietic malignancy was defined as reporting no first-degree relative with NHL, Hodgkin's lymphoma or leukaemia.We used Cox proportional hazards regression models, stratified by age in years at baseline, to estimate hazard rate ratios (RR) and 95% confidence intervals (CIs) for B-cell NHL, using age in days from baseline until the end of follow-up for the time under observation. We assessed potential confounders including race; history of a full-term pregnancy; baseline residential socioeconomic status, smoking status, height and body mass index (BMI); and alcohol consumption 1 year before baseline. We did not include any of these variables in our final statistical models as none changed the risk estimates by as much as 5%. All statistical analyses were performed using SAS version 9.1 (SAS Institute Inc.).During an average of 9.3 years of follow-up, 478 California Teachers Study participants were diagnosed with B-cell NHL. A history of haematopoietic malignancy in any first-degree relative was associated with a significant increased risk of B-cell NHL  . Risk ofThe association with a parental-based family history of lymphoma or leukaemia was comparable with that of a sibling-based family history (results not shown). Similar to the associations based on gender, women having a lymphoma-affected mother had the elevated NHL risk , as did those having a leukaemia-affected father  (results not shown).Our cohort study results confirm the evidence from population-based case–control studies and registry-based linkage studies of an increased NHL risk among individuals with a family history of haematopoietic malignancy (We found that the association with a first-degree family history of haematopoietic malignancy varied by gender, being stronger for female relatives with a history of lymphoma and for male relatives with a history of leukaemia, as reported earlier from population-based registries (A limitation of our study is that our family history reports were self-reported. Concerns regarding the accuracy of self-reported family history of lymphoma were raised previously in a Swedish study (As we asked about family history of lymphoma separately from that of leukaemia, it is possible that participants with a family history of CLL reported this as a leukaemia, not as a lymphoma, though CLL is part of the NHL case definition (A final limitation is that we did not ascertain cancer diagnoses among participants’ relatives during the follow-up period. Given that cohort members who developed NHL, compared with those who did not, would be more likely to have had additional familial diagnoses of haematopoietic malignancy, our RR estimates may underestimate the true association with first-degree family history.In summary, we confirmed a positive association between family history of haematopoietic malignancy and NHL risk.


Pan paniscus  individuals to assess the detailed mitochondrial DNA (mtDNA) phylogeny of this close relative of Homo sapiens.We have analyzed the complete mitochondrial genomes of 22 dN/dS) among polymorphic positions in bonobos and in 4902 Homo sapiens mitochondrial genomes revealed a remarkable difference in the strength of purifying selection in the mitochondrial genes of the F0F1-ATPase complex. While in bonobos this complex showed a similar low value as complexes I and IV, human haplogroups displayed 2.2 to 7.6 times increased dN/dS ratios when compared to bonobos.We identified three major clades among bonobos that separated approximately 540,000 years ago, as suggested by Bayesian analysis. Incidentally, we discovered that the current reference sequence for bonobo likely is a hybrid of the mitochondrial genomes of two distant individuals. When comparing spectra of polymorphic mtDNA sites in bonobos and humans, we observed two major differences: (i) Of all 31 bonobo mtDNA homoplasies, i.e. nucleotide changes that occurred independently on separate branches of the phylogenetic tree, 13 were not homoplasic in humans. This indicates that at least a part of the unstable sites of the mitochondrial genome is species-specific and difficult to be explained on the basis of a mutational hotspot concept. (ii) A comparison of the ratios of non-synonymous to synonymous changes (Some variants of mitochondrially encoded subunits of the ATPase complex in humans very likely decrease the efficiency of energy conversion leading to production of extra heat. Thus, we hypothesize that the species-specific release of evolutionary constraints for the mitochondrial genes of the proton-translocating ATPase is a consequence of altered heat homeostasis in modern humans. Gorilla gorilla , two from Pan troglodytes  and only a single mitochondrial genome from Pan paniscus [GenBank:NC_001644]. Studies addressing the mitochondrial DNA diversity of non-human hominids were, therefore, restricted to short regions of the mitochondrial genome, most typically parts of the non-coding and highly variable D-loop . Comparison of previously reported short D-loop sequences from 60 bonobo individuals  represent a third distinct linage ('C') of bonobos.A more detailed picture of bonobo diversity is shown by the neighbor-joining tree of all 22 complete mitochondrial genomes using two NC_001644] [NC_001644] is a hybrid sequence of two bonobo individuals. Therefore, we suggest it to be depreciated as reference sequence. Instead, the closest sequence, PP23 [GenBank:GU189661], could be used as the new reference sequence for the Pan paniscus mitochondrial genome.Before the present study, there was only a single complete bonobo mtDNA sequence available in public databases .A set of 28 primer pairs was used to amplify overlapping mtDNA fragments from bonobo DNA samples. To avoid sample mix up, each individual was processed separately. Direct sequencing of the purified PCR products was carried out on an automatic sequence analyzer by a commercial sequencing service . Sequence reads were confirmed manually and complete mtDNA sequences were assembled using the SeqMan module of the Lasergene 8.0 software . Complete mtDNA sequences have been deposited in GenBank .Click here for fileFigure S3. Ratios of non-synonymous to synonymous mutations in mitochondrial protein coding genes. dN/dS ratios of within-group polymorphic sites in the mitochondrial encoded subunits of complexes I, III, IV and V in Pan paniscus and diverse human haplogroups. Numbers of analyzed individual sequences are shown in brackets.Click here for fileTable S1. Non-synonymous mutations in mitochondrial genes of complex V. Scoring of amino acid changes according to Betts and Russell [ Russell . PositivClick here for fileTable S2. Human haplogroup definitions used in the study. Haplogroups defined by the presence (+) or the absence (-) of specific mutations as compared to the revised Cambridge Reference Sequence [Sequence .Click here for file