The potent clinical responses seen in patients with chronic myeloid leukemia (CML) after administration of donor-specific lymphocytes, as well as the correlation between the presence of antigen specific T cells and prolonged remission in these patients, suggests a role for the immunological control of CML. Here we propose Aldara™, a clinically used formulation of imiquimod, as an agent for augmenting immune responses to CML antigens. Our proposition is based upon 3 tenets: 1) Endogenous dendritic cells (DC) of CML patients, which are known to be derived from the malignant clone, express and present various leukemic antigens; 2) CML-antigen reactive T cell clones exist in the patient but in many situations are ineffectively stimulated to cause significant hematological responses; and 3) Antigen presentation by mature, activated DC, which endogenously express CML-antigens may endow the pre-existing ineffective T cell responses with ability to control CML progression. The practical use of Aldara™ as a localized activator of DC in the context of present day leukemic therapeutics, as well as various properties of this unique immune modulator will be discussed. Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by an indolent chronic phase during which immature myeloid cells increase in the peripheral blood and bone marrow, followed by an accelerated phase, associated with resistance to standard therapies, and terminates in blast crisis where undifferentiated blasts damage vital organs, leading in death. Treatment of CML has undergone several major advancements: a) The development of chemotherapeutic interventions such as busulphan and 6-thioguanine in 1953; b) The introduction of alpha interferon in 1983; c) Bone marrow transplantation in 1986; and d) BCR-ABL-specific tyrosine kinase inhibitors in 1998 . The curThe concept that leukemic cells are immunogenic was introduced in the 1960s when Mathe's group demonstrated a survival advantage in acute lymphocytic leukemia (ALL) patients that were treated with irradiated allogeneic blast cells together with BCG and chemotherapy, in comparison to patients receiving chemotherapy alone . SimilarMolecular evidence for the existence of CML-specific T cell responses was brought forward by Cheever et al who demonstrated that CD4 T cell immunity could be generated toward BCR-ABL expressing cell lines after vaccination of mice with peptides generated from the junctional region of the BCR-ABL fusion protein . Indeed,Utilization of synthetic peptides corresponding to the BCR-ABL junctional areas as immunogens has been performed by numerous groups to activate both CD4 and CD8 T cells from healthy donors ,18,22. IWhile the concept of generating CML-specific T cells is appealing, the question of why such T cells don't naturally control CML arises. As previously mentioned, it appears that there is a functional incapacitation of leukemic-specific T cells in patients. Reports of T cell anergy in CML patients to specific leukemia-associated antigens have included the demonstration of an inability to raise BCR-ABL-targeting T cells from PBMCs derived from CML but not healthy volunteers , as wellFrom these studies it appears that not only is T cell immunity possible to CML cells, but such immunity appears to be relevant to clinical outcome. The fact that antigen-specific suppression of anti-CML responses occurs ,31 but cIn recent years the molecular identification of mechanisms associated with innate recognition of "danger" signals has led to a rebirth of the study of innate immunity. Specifically, the discovery of the toll-like receptors (TLRs) as well as other "sensors of danger" such as the nucleotide-binding oligomerization domain (NOD)-containing proteins has stimulated a plethora of studies assessing the role of innate immune components in areas not traditionally associated with immunology. For example, TLRs have been shown to play a critical role in situations ranging from ischemia-reperfusion injury , to hearNK cells lyse targets that possess low or absent levels of MHC class I molecules . This coThe role of NK cells in CML is very interesting. One reason is that NK cells are found naturally residing in the bone marrow and affect hematopoiesis through secretion of stimulatory factors such as GM-CSF , as wellEx vivo activation of NK cells by IL-2 was performed in bone marrow cultures as a method of "purging" AML cells from the non-malignant marrow for use in autologous bone marrow transplant resulting in clinical remission in a subset of patient . AdditioIn conclusion, while the effector innate immune system definitely is involved in the control of CML, little work has been performed in this area. The essential role of cells like DC and NK in terms of shaping the T cell response is only now beginning to be elucidated. Future studies integrating the activation of both the innate and adaptive immune responses will lead to development of novel therapeutics. What follows is a hypothesis of a novel way of harnessing the innate and adaptive immune responses through the use of a clinically available agent, Aldara™.The active ingredient in Aldara™ is imiquimod, a low molecular weight immune stimulant. Previous to development of Aldara™ as a topical form of imiquimod, oral imiquimod has been assessed clinically for immune stimulatory effects both in cancer and HIV patients in the 1990s. These studies have demonstrated an unacceptable benefit/toxicity profile and as a result administration via the topical route was chosen for development. Specifically, Goldstein et al reported a Phase I study involved 12 patients with HIV infection that were dose escalated by 100 mg/week until toxicity was reached. When toxicity was reached, maintenance dosing of 100 mg/week lower than the toxic dose was performed. Dose-limiting toxicity occurred in 3 patients at 200-mg, 5 at 300-mg, and 3 at 400-mg dose levels. One patient tolerated the 500-mg dose without dose-limiting toxicity. While 7 of the 12 patients completed the 12 week dosing schedule, no consistent effect on viral load was observed. Importantly, serum interferon alpha was observed to rise, as well as neopterin and beta2-microglobulin, which are markers of immune activation . In cancAldara™ is a formulation of 5% imiquimod in an oil-in water vanishing cream base that is approved for use in the United States for treatment of actinic keratosis, superficial basal cell carcinoma and external genital warts . Aldara™In addition to the above date, subsequent data supporting effectiveness of Aldara™ for the above indications were reported. For example, Lee et al examined 146 AK patients which were administered Aldara™ either 2 or 3 times per week for a period of 16 weeks. Recurrence of AK lesions was observed in only 24.7% (19 of 77) of the patients on the 3 times per week regimen and in 42.6% (23 of 54) of the patients on the 2 times per week regimen. With exception to localized irritation, no adverse effects were reported in any of the patients in this 30 center study . The impAlthough the first FDA approval for use of Aldara™ occurred in 1997, the molecular mechanism of action was only identified five years later in 2002 by Hemmi et al, who demonstrated that active ingredient, imiquimod, is a stimulator of the toll-like receptor 7 (TLR7)-MyD88-dependent signaling pathway on immune cells . Other sThe importance of these DC in priming of immune responses subsequent to imiquimod administration was demonstrated in experiments applying a combination of imiquimod and antigen epicutaneously. This type of "trans-immunization" was able to elicit potent cytotoxic T cell responses through the activation of both plasmacytoid, and myeloid-derived DC in a murine model . AlthougIn terms of its clinical use, an immunological mechanism is believed responsible, especially in cases of virally-induced genital warts since imiquimod itself is not active against viruses in the absence of a functional immune system . Indeed,In summary, Aldara™ has been demonstrated to induce various molecular and cellular changes that evoke clinical responses against various viral and preneoplastic/neoplastic conditions. For the remainder of the manuscript we will discuss the therapeutic possibility of medication in regards to CML.CML is a unique disease from the perspective of the tumor immunologist. Since the leukemic clone differentiates into all cells of the myeloid lineage, the majority of DC isolated from CML patients direct progeny of the leukemic stem cell. It is established that in CML patients, DC can not only present the leukemia specific BCR-ABL peptides, but also that such presentation can lead to generation of antigen-specific T-cells capable of inducing lysis of CD34 cells from CML but not control patients in an MHC-I dependent manner. Indeed, CML is different than almost any other cancer in that DC immunotherapy would not require the pulsing of DC with exogenous antigens, since CML DC endogenously express BCR-ABL immunogenic peptides at sufficient concentrations to stimulate a T cell response . This coThe proposition of this paper is that topical administration of Aldara™ would induce the differentiation and expansion of CML DC at the cutaneous sites of administration. This differentiation would then lead to upregulated expression of MHC I and II bearing leukemic peptides, costimulatory molecules, as well as interferon alpha production, in a milieu that would not only rescue the CML-specific T cells from anergy, but also induce their proliferation, expansion, and acquisition of cytotoxic/leukemia inhibitory function. Several questions arise...First, will the DC that are activated by Aldara™ treatment actually express BCR-ABL peptides? Some believe that plasmacytoid DC are only derived from lymphoid lineage and thus will be BCR-ABL negative. To this question we respond by stating the main anti-CML effect of the Aldara™ treatment will not be through the direct activation of the plasmacytoid DC, but through the indirect activation of the myeloid derived DC that occurs as a result of the immune stimulation. For example it was demonstrated that not only does Aldara™ treatment potently induce maturation of myeloid DC through indirect effects , but alsSecond, will the CML-specific T cells not be anergized in vivo, such that even if the presentation of BCR-ABL peptides does occur, the T cells will not be able to mount a productive anti-leukemic response? Although it was previously stated that anergy to CML-specific T cells occurs in CML patients, this anergy seems reversible either by addition of cytokines or antigen-specific vaccination. The recent reports of hematologic and cytogenetic remissions induced by immunization with BCR-ABL peptides support this notion. Interestingly it was observed that higher antigen-specific responses occur in patients that are concurrently receiving interferon alpha -87. ThesThird, what about CML specific antigens that are not expressed endogenously in DC of CML patients? Specifically, antigens such as telomerase , PR1 2929, PASD12 area of skin. Monitoring of therapy would most likely involve ex vivo analysis of cytokine production, as well as in more advanced clinical research settings tetramer analysis for BCR-ABL specific T cells.How will Aldara™ administration be optimally used clinically? In the ideal situation, Aldara™ would be administered as maintenance therapy after induction of molecular remission using bone marrow transplant, IFN-alpha or imatinib. The low cost and tolerability of Aldara™ would make this an interesting immunostimulating adjuvant. Another situation where this therapy may be useful is patients whom are non-responsive to imatinib and cannot tolerate the effects of INF-alpha. Yet another embodiment of this therapy would be application to the skin area where CML-specific vaccines are administered, however, it has been demonstrated that the vaccine stimulating effects do not necessarily require local administration , althougA potential concern for clinical implementation of Aldara™ treatment for CML may be the possibility of activation of immunity that targets not only hematopoietic stem cells of leukemic origin, but also non-leukemic hematopoietic stem cells. Indeed the graft versus leukemia effect observed after donor lymphocyte infusions is correlated with a period of aplasia in which normal hematopoietic stem cells are also targeted . This isWe present a novel hypothesis that administration of the topical TLR-7 agonist imiquimod in the form of Aldara™ may be a useful immunotherapy/adjuvant therapy for CML patients with minimal residual disease. Thus a scientific basis for utilization of this generally innocuous medication is proposed for a lethal disease. Although the current concept may require various modifications before clinical entry, we put forth this idea as a stepping stone for other investigators to expand upon. The lack of significant toxicity and widespread use of this medication makes this hypothesis attractive for testing. Present day therapies for imatinib-resistant, IFN-alpha non-eligible, CML patients are limited to maintenance therapy on hydroxyurea or busulfan. The administration of Aldara™ to this patient subgroup may provide a novel means of life extension without drawbacks of severe myeloablative protocols.


MicroRNAs play critical role in regulating gene expression. MicroRNA profile of particular cell type bears the signature of cell type specific gene expression. Given that viral pathogens replicate by evading host defenses, research is now focused on the miRNA-regulated genes that critically regulate HIV-1 propagation in human host cells. C. elegans lin-4 gene product, a 21 nucleotide non-coding RNA (ncRNA), regulates the expression of lin-14 by partial complementarity to several regions within the 3'-UTR of the target lin-14 mRNA, RNA-mediated gene silencing (RNAi) has taken on new urgency to understand its role in regulating gene expression in mammalian cells. A recent report in Science [Ever since the initial report  that C. Nearly 500 human genes are known to encode ~21 nucleotide miRNAs, which are initially transcribed by RNA Polymerase II as primary (pri-miRNA) that are processed in the nucleus by RNase type III Drosha into precursor (pre-miRNA) and exported to the cytoplasm by exportin 5, to be secondarily processed into miRNA duplexes by the cytoplasmic RNAse type III Dicer. The resulting miRNA duplexes are incorporated into the RNA-Induced Silencing Complex (RISC) where one of the miRNA strands, the 'passenger' is degraded, while the 'guide' miRNA is guided to the target mRNA to either degrade (in case of perfect base complementarity) or to block translation (in case of imperfect sequence complementarity between the miRNA 'seed' sequence and the target mRNA). This general version of miRNA action  that come not only in small forms. In the coming period, one can expect to gain novel insights into the regulation of mammalian gene expression by a better reading of the language of ncRNAs.


A promising application of the huge amounts of genetic data currently available lies in developing a better understanding of complex diseases, such as cancer. Analysis of publicly available databases can help identify potential candidates for genes or mutations specifically related to the cancer phenotype. In spite of their huge potential to affect gene function, no systematic attention has been paid so far to the changes that occur in untranslated regions of mRNA.In this study, we used Expressed Sequence Tag (EST) databases as a source for cancer-related sequence polymorphism discovery at the whole-genome level. Using a novel computational procedure, we focused on the identification of untranslated region (UTR)-localized non-coding Single Nucleotide Polymorphisms (UTR-SNPs) significantly associated with the tumoral state. To explore possible relationships between genetic mutation and phenotypic variation, bioinformatic tools were used to predict the potential impact of cancer-associated UTR-SNPs on mRNA secondary structure and UTR regulatory elements. We provide a comprehensive and unbiased description of cancer-associated UTR-SNPs that may be useful to define genotypic markers or to propose polymorphisms that can act to alter gene expression levels. Our results suggest that a fraction of cancer-associated UTR-SNPs may have functional consequences on mRNA stability and/or expression.SPARC) gene, this report illustrates the utility of a cross-talk between in silico transcriptomics and cancer genetics.We have undertaken a comprehensive effort to identify cancer-associated polymorphisms in untranslated regions of mRNA and to characterize putative functional UTR-SNPs. Alteration of translational control can change the expression of genes in tumor cells, causing an increase or decrease in the concentration of specific proteins. Through the description of testable candidates and the experimental validation of a number of UTR-SNPs discovered on the secreted protein acidic and rich in cysteine ( Genetic variations contribute to the development and maintenance of complex disorders, such as cancer, through alterations in the structure and/or abundance of individual mRNA molecules. The human transcriptome could therefore be considered as a priority target in the fight against cancer. Transcript sequences represent a key source for the search of aberrantly expressed genes and for the identification of genes whose products are deregulated in malignant cells. Among these transcript sequences, Expressed Sequence Tags (ESTs) are partial single-pass sequences of cDNAs made of mRNA from a particular organ, tissue or cell line. Since cDNA libraries are generated from a wide range of cancerous and normal tissues, ESTs can be used both for measuring relative levels of gene expression -6, and fIt is now widely assumed that human genomic DNA contains some level of polymorphism, with single nucleotide polymorphisms (SNPs) being the most common form. Owing to large-scale discovery, SNPs constitute an emerging resource for the study of genetically complex disorders such as cancer ,10. SNPsSPARC) gene, a key factor in cell-matrix interactions and possibly tumour aggressiveness , where 'a' is the number of tumoral variants, 'V' the total number of variants, 'T' the sum of tumoral counts (variant plus reference) and R the total number of reference alleles (Ib being close to 1 in case of strong association).cis acting elements patterns in the regions containing cancer-associated SNPs. UTRScan looks for UTR functional elements by searching through user submitted query sequences for the patterns defined in the UTRsite collection [UTRScan  was usedllection . To testllection .BC072457). The sequences of the primers are listed in Genomic DNA was extracted from bone marrow in patients with acute myeloblastic leukemia (AML) and peripheral blood in healthy donors. The phenol/chloroform method was used for DNA extraction according to standard procedures. Primers to explore SPARC polymorphisms detected by computational procedures were designed based on the DNA sequence from GenBank . Hits are ranked by decreasing p value (see Method section). Sequences for which no annotation is available are referred to as 'NULL'. Bias intensity is given for information. Candidate positive after the multiple testing corrections are underlined. Information concerning the SNPs present on SPARC appears in bold. References appear for validated SNPs from dbSNP (last column). Ambiguity code: R = G↔A; M = C↔A; K = G↔T; Y = C↔T; S = G↔C; W = A↔T. Nucleotide sequence of the reference RNA is shown within a 10-bp interval around each SNP site. Putative cis acting elements located in the regions containing cancer-associated SNPs were identified with UTRScan (see text for details). RNAMute was used to compute distances between variant and reference alleles. Gene symbols were from HGNC (HUGO Gene Nomenclature Committee). UTR-SNPs lying on transcripts for which non-synonymous SNPs were previously identified [Complete list of cancer-associated UTR-SNPs. UTR-SNPs with significantly different allele frequency in normal versus tumoral tissues .Distribution of the different types of simple substitution SNPs . (A) Substitutional patterns observed among UTR-SNPs. Transition rates were 67.2 % in the complete dataset of UTR-SNPs, 72.6 % in the total pool of cancer-associated UTR-SNPs, and 66.3 % in the subset of UTR-SNPs which were positive after the resampling procedure. Of the 358 cancer-associated UTR-SNPs, 260 were transition events while 298 were transversion events. When considering the 92 UTR-SNPs positive after the resampling procedure, 61 were transition events and 31 were transversion events. (B) The proportions for each pair of complementary substitutions are graphed next to each other for ease of comparison. Student t-test not significant (Click here for file


Chlamydia trachomatis, is an important global cause of blindness. A dysregulated extracellular matrix (ECM) proteolysis during the processes of tissue repair following infection and inflammation are thought to play a key role in the development of fibrotic sequelae of infection, which ultimately leads to blindness. Expression and activity of matrix metalloproteinase 9 (MMP-9), a major effector of ECM turnover, is up-regulated in the inflamed conjunctiva of trachoma subjects. Genetic variation within the MMP9 gene affects in vitro MMP9 expression levels, enzymatic activity and susceptibility to various inflammatory and fibrotic conditions.Trachoma, an infectious disease of the conjunctiva caused by MMP9 gene using the high-throughput Sequenom® system. Single marker and haplotype conditional logistic regression (CLR) analysis for disease association was performed.We genotyped 651 case-control pairs from trachoma endemic villages in The Gambia for coding single nucleotide polymorphisms (SNPs) in the MMP9 was found to be associated with lower risk for severe disease sequelae of ocular Chlamydia trachomatis infection. This mutation, which leads to a nonsynonymous amino-acid change within the active site of the enzyme may reduce MMP-9-induced degradation of the structural components of the ECM during inflammatory episodes in trachoma and its associated fibrosis.The Q279R mutation located in exon 6 of This work supports the hypothesis that MMP-9 has a role in the pathogenesis of blinding trachoma. Chlamydia trachomatis, is the commonest infectious cause of blindness. The blinding complications of trachoma are due to progressive scarring of the conjunctiva (trachomatous scarring) eventually leading to in-turning of eyelashes (trichiasis) and corneal opacification. Genital C. trachomatis infection causes similar lesions in the female genital tract contributing to ectopic pregnancy and infertility. Severe and persistent inflammation triggered by repeated conjunctival infections is believed to increase the risk of pathological scarring later in life[Trachoma, a chronic keratoconjunctivitis caused by r in life.C. trachomatis infection upregulates the expression of MMP-9 in the human conjunctival epithelium [Chlamydia muridarum (MoPn) infection found greater MMP-9 transcription and activity during infection in those mouse strains exhibiting increased susceptibility to fibrotic sequelae following infection [The mechanisms of disease pathology are not completely understood. Some evidence suggests that the dysregulated ECM proteolysis seen during the processes of tissue repair following infection and inflammation  may playithelium . MMP-9 aithelium . In addinfection ,6.MMP9 expression is regulated at the transcriptional level in response to pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin 1 beta (IL-1β) [Matrix metalloproteinases (MMPs) are a tightly regulated family of zinc-dependent enzymes that degrade structural proteins of the ECM and basement membranes. Among them, MMP-9 is a major effector of ECM turnover during homeostasis and pathology . MMP9 ex (IL-1β) . Post-tr (IL-1β) .MMP9 gene. Some of them have been reported to affect in vitro MMP9 expression levels, enzymatic activity and susceptibility to various inflammatory and fibrotic conditions [MMP9 affects the risk of scarring sequelae of trachoma.A number of SNPs have been identified in regulatory and coding regions of the IFNγ and IL10 loci in these subjects [One thousand three hundred and fifteen subjects identified by clinical examination using World Health Organization (WHO) criteria were recruited from trachoma endemic villages in The Gambia. They included 651 subjects with scarring trachoma (TS), of whom 307 additionally had trichiasis (TT), and pair-matched by sex, age, ethnic group and village of residence individuals with normal eyelids. The subjects were otherwise healthy. We have previously studied and reported polymorphism at the ® system as previously described [Genomic DNA was isolated from either venous blood in EDTA or buccal brush samples and genotypes were determined by the Sequenomescribed . Primer Haplotypes were inferred from population genotype data and their frequencies were estimated as previously described .A crude analysis (crude Mantel-Haenzsel) was first carried out using chi-square testing to test for differences in allele and haplotype frequencies between cases of TS and TT with their pair-matched controls. In addition, conditional logistic regression (CLR) analysis for disease association was performed. Conditional logistic regression is the analysis of choice for dichotomous paired data (in our case paired cases and controls) where the risk estimates associated with specific attributes (e.g genotype) are required to be adjusted for potential confounders. Reference genotypes were selected to be those that were common in our study population. A CLR test for trend in the odds ratios (OR) was performed to examine dose response effects in the relationship between genotype and disease. All analyses were performed using STATA (v8.0) software.A total of four exonic SNPs of population frequency > 10% were typed at the MMP9 locus  between segregating sites.Table The GCAG haplotype was associated with lower risk of TS and TT Table . The risHaplotypes containing the Q279R G allele, itself associated with decreased risk, were more commonly seen among controls than cases, although the difference in haplotype frequency between cases and controls reach significance only for haplotype GCAG. Conversely, the commonest haplotypes ACCG and ACAG were more frequently seen among cases than controls, and both carry the Q279R A allele. The strength of the single marker association is very similar to, if not greater than that of the haplotype, which supports the suggestion that the Q279R SNP itself may be causal.MMP9 gene to be associated with a lowered risk of trachomatous scarring and trichiasis, which was more marked for trichiasis. This supports the validity of the association because trichiasis is a more advanced form of cicatricial trachoma and also less subject to clinical misclassification than scarring trachoma.The present study found a coding SNP (Q279R in exon 6) within the MMP9 active site. Although the functional impact of this polymorphism on the protein is unknown, this variant is noteworthy; it is located in the coding sequence of one of the highly conserved fibronectin type II-like repeats which confers MMP-9 with high affinity binding to type IV collagen, type I gelatin and elastin [The Q279R mutation leads to the substitution of a positively charged amino-acid (arginine) by an uncharged amino acid (glutamine) at position 279 within the  elastin ,12. The The effect observed in this work was primarily associated with the Q279R GA heterozygous genotype. It is possible that the apparent lack of statistical effect of the GG homozygous genotype may have resulted from the modest sample size within the Q279R GG stratum. However we do not rule out the possibility of this finding being genuine and of biological significance; the increased risk of cicatricial trachoma associated with the Q279R AA homozygous genotype may be linked to the excessive MMP-9-driven proteolytic activation seen in the chronic inflammatory environment of the conjunctiva of subjects with trachoma,4. ConveChlamydia muridarum (MoPn) infection[MMP-9 expression and activity is up-regulated in the inflamed conjunctiva of trachoma subjects and increases with the severity of clinical inflammation ,4. Thereinfection supportsinfection, exhibitMMP9 genetic association analysis, but for practical reasons large numbers of patients with CO are difficult to come by.The blinding lesion in trachoma is corneal opacity (CO), which is thought to result from the lashes rubbing against the eyeball (TT) and damaging the cornea. Epidemiological studies indicate that after successful TT surgery, CO can develop, and theIL10) and interferon gamma (IFNγ) loci [This work supports the hypothesis that MMP-9 has a role in the pathogenesis of blinding trachoma. The risk reductions associated with the Q279R exonic mutation are modest: 26% for scarring and 34% for trichiasis. CLR analysis suggests that these effects are independent of those previously reported in the interleukin 10  declare that they have no competing interests.AN collected, edited, analysed the data and wrote the manuscript. RB directed the study, study design and co-wrote the manuscript. GC study design and contributed to the manuscript. DM, MH and MB directed the study, study design and contributed to the manuscript. DK and KR co-directed the study. OJ and HJ collected clinical data and provide clinical materialThe pre-publication history for this paper can be accessed here:


Investigations of the structure of psychological well-being items are useful for advancing knowledge of what dimensions define psychological well-being in practice. Ryff has proposed a multidimensional model of psychological well-being and her questionnaire items are widely used but their latent structure and factorial validity remains contentious.We applied latent variable models for factor analysis of ordinal/categorical data to a 42-item version of Ryff's psychological well-being scales administered to women aged 52 in a UK birth cohort study . Construct (predictive) validity was examined against a measure of mental health recorded one year later.Inter-factor correlations among four of the first-order psychological well-being constructs were sufficiently high (> 0.80) to warrant a parsimonious representation as a second-order general well-being dimension. Method factors for questions reflecting positive and negative item content, orthogonal to the construct factors and assumed independent of each other, improved model fit by removing nuisance variance. Predictive validity correlations between psychological well-being and a multidimensional measure of psychological distress were dominated by the contribution of environmental mastery, in keeping with earlier findings from cross-sectional studies that have correlated well-being and severity of depression.Our preferred model included a single second-order factor, loaded by four of the six first-order factors, two method factors, and two more distinct first-order factors. Psychological well-being is negatively associated with dimensions of mental health. Further investigation of precision of measurement across the health continuum is required. Recent years have seen a widening interest in research on aspects of well-being -4. ExtenThis new focus has necessitated the theoretical development of new constructs as well as questionnaire items to measure psychological well-being in clinical and population samples. The work of Ryff and colleagues has been at the forefront of this endeavour.Ryff's scales of Psychological Well-being ,9 were dDespite the widespread interest in Ryff's theoretical framework, and application of the Ryff PWB items, the psychometric properties of the proposed sub-scales remain contentious. In particular there has been concern over issues of factorial validity and distinctiveness. Do the items intended to measure each theoretical domain, really do so? Do the items capture information from more than one domain? Are fewer dimensions actually revealed by empirical data collected to test the multidimensional theory?a priori six-factor structure. Authors of existing studies either challenge the value of so many theoretical constructs, whose scores correlate >0.8 or 0.9, or have not confirmed the fit of the proposed model )An annual sub-study of women's health in midlife was undertaken by postal questionnaire between the ages of 47–54. This study included 1,778 (70%) of the original cohort of women; the others had died (6%), previously refused to take part (12%) or lived abroad and were not in contact with the study or could not be traced (13%). The Ryff PWB was sent to the 1,421 women who had completed at least one women's health questionnaire in the previous 2 years. The representativeness of the sample of women who completed the Ryff items at age 52 has not been established in the same terms with respect to population census data. However, we compared the sample of women who completed the PWB and participated in the age 53 follow-up (N = 1108) or age 43 (where 53 data was not available (N = 57)) with those involved in the follow-ups but did not complete the PWB (n = 413). Ryff completers were of higher social class [chi-sq 16.6 df = 1, p < 0.001), more likely to be married  than non-completers and more educated . There was no difference due to employment status. This comparison excluded women (n = 50) who completed the Ryff items but neither the age 53 nor age 43 follow-ups. Comparative socio-demographic data was not available for the excluded group of women.A forty-two item version of the Ryff PWB was included in the women's health questionnaire at age 52 on the recommendation of C.Ryff . The response format for all items comprised six ordered categories labelled from 'disagree strongly' to 'agree strongly'. Twenty PWB items were positively worded and 22 negatively worded. Prior to analysis, negatively worded items were reverse scored so that high values indicated well-being. This made it easier to identify floor and ceiling effects. Full question wording of the 42-items is shown in Table a priori first-order factors which all load (>0.80) on a higher (second) order latent factor capturing psychological distress.One year after the Ryff items were completed, women survey members completed the 28 items of the "scaled" General Health Questionnaire . The GHQplus Version 3.13 [Confirmatory factor analyses were performed treating the six category PWB items as ordinal response variables. Model estimation was performed using robust Weighted Least Squares  ; method factors ; second-order factors (present versus absent).uncorrelated with the construct factors, and with each other. Each method factor was examined separately and then both were modelled simultaneously.We introduced "method" factors in order to isolate nuisance variance due to item wording or content that was unrelated to the constructs being measured ,16,36. Ia priori measurement model, with one based on arbitrary allocation of items to factors . We generated four random item-factor models in order to evaluate the improvement of the a priori model over this scenario. We report the average fit statistics across the four solutions since all four random solutions were similar.The magnitude of some inter-factor correlations reported by previous studies has given rise to the suggestion that the item-factor correspondences for some items are very weak; this can be tested by comparing the fit of the Further structural refinements were identified based on consideration of modification indices and a slightly revised model proposed (see results).In order to examine the association between scores on the psychological well-being constructs, under our preferred model, and another measure of health , we linked the PWB scores for the women to their responses to the GHQ-28 conducted one year later.Our analysis sample includes 1,179 respondents who completed at least 85% of PWB items (36 out of 42 questions); 957 had complete data on all items. Descriptive statistics revealed a general positive skew towards the well-being end of the response scales (Table Each model is described in a single line in Table a priori model against a model with random item-factor associations (A0) and a unidimensional model with all 42 items loading on a single latent factor (A1). Here model A2 is the a priori model, and A3 is extended to incorporate a second-order factor .Our first set of models A0-A3, Table , Table 3a priori model (A2) returned a lower ssaBIC value than unidimensional model (A1). The model with a second-order factor (A3) returned a higher ssaBIC value than the a priori model with only first-order factors.Model fit was poor for all models in terms of all criteria  Table . The wora priori models with both method factors  RMSEA approached 0.08 and TLI approached 0.94, but CFI remained below 0.80. These two models (B4 a priori and B5 a priori plus second-order) were within 110 ssaBIC points but were indistinguishable on all other indices of fit.Our second set of models (B1-B5) repeated A1-A3 with one, or both method factors. Compared to models A0-A3 any model incorporating either or both method factors improved model fit and substantially reduced the ssaBIC, regardless of the number of factors. Even in the model assuming a unidimensional construct of PWB (single first-order factor), but with both method factors, the ssaBIC dropped by a huge amount (>2000 points). In the We report factor loadings from two models A2 and B4) in columns 2 and 3 of Table  in columG2 (I don't want to try new ways of doing things – my life is fine the way it is) and G3  are complex questions, capturing more than one issue, and include both positive and negative item content. Item E1 (I do not fit very well with the people and the community around me) loaded more highly on positive relations (R) than its designated factor ), reflecting the initial part of the question concerned with relationships with others. We therefore chose to model this item on positive relations (R).In a final round of modelling Table  we foundpeople would describe me as a giving person, willing to share my time for others', and 'most people see me as loving and affectionate' and so we allowed correlated residuals between these two items. These small modifications to the a priori model, together with method factors improved fit statistics for TLI and RMSEA (Models PH2 & PH3). The CFI however, still remained below 0.86.Examination of residuals also suggested potential overlap with two questions from positive relations (R) 'We also tested a six-factor model (PH4) where four constructs , personal growth (G), purpose in life (P), self-acceptance (S)) loaded onto a second-order factor, and autonomy (A) and positive relations (R) remained as first-order factors (freely correlated). This model is drawn as a path diagram in Figure a priori first-order PWB factors and the GHQ-28 second-order factor were low (-0.10–0.08) except for environmental mastery (E) (-0.52).The estimated correlation between our second-order PWB factor (model PH3 based on 40 items) and the GHQ-28 second-order factor was -0.45. The correlations among the Figure In this study we provide the first confirmatory test of the factorial validity and structure of Ryff's Psychological Well-being (PWB) scales (42-item version) in the UK. In contrast to previous studies, our sample come from the UK and comprise only women who are surviving members of a national birth cohort study which began in 1946. This sample completed the Ryff items as part of an annual woman's health survey in midlife and also completed a mental health measure one year later.In our psychometric modelling we evaluated the fit of categorical  factor models with single and six construct factors, first and second-order factors, and method factors, as well as providing a reference comparison to a model with random item-factor associations. Like all previous research we were unable to identify a model that fitted the data well, although a number of modelling components appeared to be useful in improving model fit to the data, and therefore determine our conclusions regarding the factorial validity of Ryff's measures, with reference to her theory, and in regard to these 42 items. Our results indicate the following:correlated with the construct factors) to the Ryff PWB model, but we extended this approach to the addition of both positive and negative method factors which were independent of both each other and the measured constructs. Models incorporating a single (either positive or negative) method factor offered an improvement over the same model without this feature, although models incorporating both methods factors had greater impact. Method factors introduce additional latent variables, and model parameters, but ssaBIC comparisons show that these modelling additions improve fit despite penalties for the improvement in the log-likelihood value achieved by the estimation of additional parameters. However we note that these BIC values are taken from traditional linear factor models .1) We found conceptual and empirical value (improved model fit) from the addition of both positive and negative method factors to address methodological artefacts. Springer & Hauser  suggestea priori constructs, we found that in our sample four of the six dimensions of well-being , personal growth (G), purpose in life (P), and self-acceptance (S)), as operationalised by these 42 items, were sufficiently highly correlated to warrant introduction of a general well-being factor, as a second-order general factor, that explained the association among the first-order constructs. We could not justify the inclusion of the remaining two dimensions (autonomy (A) and positive relations (R)) on this second-order construct since they were more independent of these four factors, of the second-order factor, and of each other. This gives some credence to claims that there are fewer than six dimensions under-pinning Ryffs PWB items. However our interpretation is in terms of the hierarchical organisation of the six factors, which seem to span two conceptual levels [2) Regarding dimensionality of the PWB measure, and empirical associations among the l levels . Further3) Finally, we found a strong negative association between a measure of mental health  and the PWB which were measured one year apart. The major contribution to this predictive association came from the environmental mastery items. This replicates a finding reported by Ryff & Keyes  using crWe tested the fit of random-item factor models in our data. Our random item-factor models differed by 1,356 BIC points from the ssaBIC for the theoretical model. This indicates to us that there are still some fragile item factor associations in the six-factor model, otherwise this comparison would yield a much larger reduction in BIC when comparing theoretical to random models.Previous authors have concluded that the empirical data are not consistent with a six-factor model . We do nOur second-order factor model requires the item to factor mapping established for the first-order factors, for its definition, since it is the second-order  factor that is proposed as the explanation for the association among environmental mastery (E), personal growth (G), purpose in life (P), and self-acceptance (S). Examination of item content suggests that this second-order factor may encapsulate a motivational aspect of well-being which incorporates notions of goal orientation and self-direction. Our finding that there are three (rather than six) distinct factors – autonomy, positive relations and motivation/self-direction – is reminiscent of the work of Deci and Ryan ,40 whichMany aspects of our modelling results do suggest that some Ryff items may measure more than one of the six constructs in the theory. This possibility requires further theoretical work that was not undertaken here, but should form an agenda for future research, and for future factorial complexity studies.The factor loadings from our preferred model Figure  indicatewe thank an anonymous referee for distinguishing these two suggestions).Related to this observation, Figure Applied researchers who do not wish to execute complex latent variable models will not be able to distinguish contributions to variance from method versus construct sources and are at a disadvantage in terms of their ability to define and refine both conceptually relevant and psychometrically important variants of multidimensional scale analysis. However, in these instances, a parsimonious account of associations of other variables with Ryff PWB outcomes may be achieved by adding all items loading on the four factors that define our second-order well-being continuum . In samples similar in composition to ours, researchers might wish to consider using our factor loadings as weights, to form sum scores using our loadings in Figure The nature of our data ensures we are undertaking a pure test of the structure of PWB items since our sample are homogeneous with respect to age and gender (women age 52). Our study design therefore minimises the impact of socio-demographic characteristics. Although our sample suggested Ryff completers were more likely to be of higher socio-economic backgrounds than non-completers, comparative studies using the PWB in nationally representative samples e.g. MIDUS and NSFH ,14 do noFuture research could apply further psychometric refinement to the Ryff PWB dimensions, by exploring scoring and effective measurement range using item response theory methodology.Our psychometric analyses of the Ryff 42-item PWB suggests that the addition of two method factors to reflect positive and negative item content improves model fit. A revised model with a single second-order factor, loaded by four of the six first-order factors , two method factors, and two more distinct first-order factors (autonomy and positive relations) provided the most parsimonious solution in this birth cohort sample. Psychological well-being was negatively associated with mental health, but further investigation of precision of measurement across the health continuum is required.SWB subjective well-beingPWB psychological well-beingA autonomyE environmental masteryG personal growthR positive relations with othersP purpose in lifeS self-acceptanceNSHD National Survey of Health and DevelopmentGHQ General Health QuestionnaireRMSEA Root Mean Square Error of ApproximationTLI Tucker Lewis IndexCFI Comparative Fit IndexWLSMV Weighted Least Squares Mean Variance adjustedRWLS Robust weighted least squares ssaBIC Sample size adjusted Bayesian information criteriaML Maximum likelihoodThe author(s) declare that they have no competing interests.RAA undertook the statistical modelling, manuscript preparation and revision. TJC planned and advised on the statistical modelling using latent variables and participated with manuscript preparation and revision. GBP was actively involved with the statistical modelling; FAH advised on the conceptual framework and data interpretation and DK and MEW were responsible for the design and acquisition of the data. FAH, GBP, DK, MEW all advised on manuscript preparation.


Takotsubo cardiomyopathy is an acute cardiac syndrome characterized by transient LV regional wall motion abnormalities , chest pain or dyspnea, ST-segment elevation and minor elevations of cardiac enzyme levelsA 68-year-old woman was admitted to the Emergency Department because of sudden onset chest pain occurred while transferring her daughter, who had earlier suffered a major seizure, to the hospital. The EKG showed sinus tachycardia with ST-segment elevation in leads V2–V3 and ST-segment depression in leads V5–V6, she was, thus, referred for emergency coronary angiography. A pre-procedural transthoracic echocardiogram revealed regional systolic dysfunction of the LV walls with hypokinesis of the mid-apical segments and hyperkinesis of the basal segments. Coronary angiography showed patent epicardial coronary arteries; LV angiography demonstrated the characteristic morphology of apical ballooning with hyperkinesis of the basal segments and hypokinesis of the mid-apical segments. The post-procedural course was uneventful; on day 5 after admission the echocardiogram revealed full recovery of apical and mid-ventricular regional wall-motion abnormalities.Takotsubo cardiomyopathy is a relatively rare, unique entity that has only recently been widely appreciated. Acute stress has been indicated as a common trigger for the transient LV apical ballooning syndrome, especially in postmenopausal women. The present report is a typical example of stress-induced takotsubo cardiomyopathy in a Caucasian Italian postmenopausal woman. Takotsubo cardiomyopathy, also known as transient left ventricular (LV) apical ballooning syndrome, is an acute cardiac syndrome characterized by transient LV regional wall motion abnormalities, chest pain or dyspnea, ST-segment elevation and minor elevations of cardiac enzyme levels . The typA 68-year-old woman was admitted to the Emergency Department because of sudden onset chest pain occurred while transferring her daughter, who had earlier suffered a major seizure, to the hospital. Her cardiovascular risk factors were female sex, tobacco smoking, dyslipidemia and family history of coronary artery disease. Upon admission her blood pressure was 150/80 mmHg and the electrocardiogram (EKG) showed sinus tachycardia with 2–3 mm ST-segment elevation in leads V2–V3 and 1 mm ST-segment depression in leads V5–V6 Figure  and 2B. Takotsubo cardiomyopathy is a relatively rare, unique entity that has only recently been widely appreciated . AlthougThe author(s) declare that they have no competing interests.ML collected the data relative to the Case Report; VZ assisted the patient in the CICU and composed the draft of the manuscript; SM participated in the preparation of the revised manuscript; FC collected the data relative to the Case Report; MM collected the data relative to the Case Report; SL performed the first echocardiogram; PA performed the second echocardiogram; AC performed the coronary angiography; DC helped in revising the manuscript draft; RF is head of the CICU and participated in the coordination and revision of the manuscript; CP is head of the Cath Lab and participated in the coordination and revision of the manuscript; SM is head of the Echo Lab and conceived the Case report and participated in the coordination, data analysis and elaboration and drafting of the manuscript. All authors read and approved the final manuscript.


The use of cytotoxic drug vincristine (VCR) is limited by the occurrence of peripheral neuropathy. A neurotrophic ACTH(4-9) analogue, ORG 2766, is being studied for its protective effect. Possible modulatory effects of ORG 2766 on tumour cell growth and interference with the cytotoxic efficacy of VCR were studied in two human lymphoma cell lines, U937 and U715. The effects of ORG 2766 on cell growth and survival and on VCR-mediated cytotoxicity were investigated using two MTT-based assays to study direct cytotoxic effects and to assess residual growth after pretreatment. Treatment with ORG 2766 alone had no effect on cell growth and survival. Neither did this drug affect VCR cytotoxicity. However, after 96 h pretreatment with ORG 2766 and a culture period of 7 days, a reduction in residual growth and a potentiation of VCR-induced inhibition of growth capacity was observed in U715 cells, and to some extent also in U937 cells. It is concluded that ORG 2766 has no stimulatory effects on tumour growth and does not negatively interfere with VCR-mediated cytotoxicity. Rather it enhances the cytostatic effect of VCR. It is suggested that ORG 2766 can safely be used in clinical trials investigating the ability of ORG 2766 to counteract VCR-induced neurotoxicity.


The risks of second primary cancer were analysed in 2846 patients with Hodgkin's disease treated within the British National Lymphoma Investigation during 1970-87. The relative risk (RR) of leukaemia was significantly greater in women  13.0-59.5) than in men , and showed a significant trend of greater risk with younger age at first treatment (P < 0.001). The relative risk of solid cancers was similar between the sexes, but again significantly greater at young than at older ages of first treatment (P < 0.01). Non-Hodgkin's lymphoma relative risks, although not related to sex or age, were significantly related to histology of the original Hodgkin's disease, and were greatest after lymphocyte predominant Hodgkin's disease . The relative risk of second cancers did not vary significantly according to whether or not splenectomy had been performed. Leukaemia risk was non-significantly greater after splenectomy than with no splenectomy, which accorded with previous evidence of a modest increased risk associated with this operation. If the greater relative risk of solid second cancers after treatment at young than at older ages persists with longer follow-up, the incidence rates of these second primaries in patients treated young for Hodgkin's disease will become very substantial as they age. This emphasises the need to maintain long-term follow-up surveillance of young Hodgkin's disease patients apparently cured of their disease, and to continue to develop new less carcinogenic treatment regimens.


In the aftermath of the SARS epidemic, there has been significant progress in understanding the molecular and cell biology of SARS-CoV. Some of the milestones are the availability of viral genome sequence, identification of the viral receptor, development of an infectious cDNA clone, and the identification of viral antigens that elicit neutralizing antibodies. However, there is still a large gap in our understanding of how SARS-CoV interacts with the host cell and the rapidly changing viral genome adds another variable to this equation. Now the SARS-CoV story has entered a new phase, a search for preventive strategies and a cure for the disease. This review highlights the progress made in identifying molecular aspects of SARS-CoV biology that is relevant in developing disease prevention strategies. Authors conclude that development of successful SARS-CoV vaccines and antivirals depends on the progress we make in these areas in the immediate future. The sequence of epidemiological events that unfolded early in the outbreak gave researchers a glimpse into the first new pathogen of the era of globalization. As the year 2002 drew to a close, multiple reports of an "infectious atypical pneumonia" caught public health officials across the globe by surprise and suggested that a new human pathogen had emerged in the Guangdong Province in China [Following reports of the last case of the in China . By the in China .Electron microscope analysis quickly identified the putative SARS agent as having features associated with coronaviruses. The SARS agent was later unambiguously identified as a new coronavirus member and named SARS-coronavirus (SARS-CoV) -7. CoronThe clinical symptoms of SARS are those of a lower respiratory tract infection and are accompanied by damage to the lungs ,9,10. GaThe virus has been isolated from wild animals  found in the animal markets of Guangdong, China . The actIn the wake of the SARS epidemic, a number of excellent review articles on the clinical and molecular aspects of SARS epidemiology have been published. These reviews have focused primarily on rapid advances made in the identification and characterization of SARS-CoV genomes as well as describing the etiology of the virus and clinical features of the disease -21. Now Coronaviruses are subdivided into three groups based on genetic and serological markers . Groups To date, approximately 61 SARS-CoV genomic sequences have been analyzed representing different phases of the epidemic  and two isolates obtained from palm civets . The SAR-3 to 10-5 per nucleotide [RNA viruses utilize a variety of mechanisms to exchange their genetic repertoire. The viral RNA dependent RNA polymerases (RdRP) have a built in error rate that allows diversification of the genomic sequence as replication proceeds. Estimates put the error rate of an RdRp at 10cleotide . Coronavcleotide . These ecleotide .in vivo may reflect both the effect of viral replication in target cells and host immune responses. The molecular basis for SARS-CoV replication, the signaling pathways affected, and the inflammatory responses provoked by viral infection are not yet clearly understood. Progress in these areas should lead to more effective preventive strategies to counter SARS-CoV infections.Successful viral replication depends upon the ability of the virus to subvert cellular processes to their advantage and counteract cellular defense mechanisms. Such virus-cell interactions represent potential targets for the development of virus-specific antiviral drugs, therapeutics, and prophylactic vaccines. Different viruses, based on their target cell types and entry pathways, differ in their cellular exploitation mechanisms. The mechanism of SARS virus pathogenesis It has been shown that the SARS-CoV N protein selectively activates the Activator Protein-1 (AP-1) signal transduction pathway, which regulates a wide variety of cellular processes including cell proliferation, differentiation, and apoptosis . Such viAs mentioned above, IL-8 induction was shown to be dependent upon AP-1 activation by SARS-CoV S protein and in this process NF-κB was not involved . This maDuring the SARS outbreak that occurred in 2002–2003, the spread of the disease was primarily controlled by strict quarantine protocols and patient-isolation measures as well as by broad-spectrum antibiotics and antiviral regimens with or without administration of corticosteroids ,45. Sinc2+ can bind. This unique structure presents an attractive target for tunnel binding antiviral drugs [Coronavirus genome structure and major gene-product functions have been known for years, but since they cause mild disease, selection of the virus-specific antiviral drugs was not a priority in the past. The SARS-CoV epidemic changed this selective view. Tan et al, 2004, tabulated a screen of available antiviral agents against SARS virus in detail in their recent review . The obval drugs . Finallyal drugs .Vaccines are the best and least expensive prophylactic measures against pathogens that cause epidemics in humans. The fact that high titers of virus neutralizing antibody to SARS-CoV are found in sera of patients recovering from infection and that those infected with the virus show improvement after passive antibody administration suggests a SARS-CoV vaccine is possible and points toward antibody based treatments for the disease ,56-58. HDespite these potential pitfalls in the development of a human vaccine, efforts to develop a vaccine to prevent another SARS outbreak are underway. Several laboratories around the globe are working at an unprecedented pace to develop a SARS vaccine utilizing essentially two different types of SARS-CoV-derived immunogens, 1) inactivated whole virus, and 2) SARS-CoV encoded N and S proteins using recombinant DNA methods. The possibility of producing an engineered live, attenuated SARS-CoV has also been considered.Takasuka et al (2004) have reported that subcutaneous administration of UV-inactivated purified SARS-CoV virion elicits a high level of humoral immunity, resulting in long-term antibody secretion and memory B cells . The antLactococcus lactis bacterium and the N-expressing bacteria were administered to mice by intranasal or oral route [The N protein of SARS-CoV appears to be more conserved than S and M proteins and it has been suggested that this protein may play a role in cell-mediated immunity in SARS-CoV infections and also is an important viral antigen for the early diagnosis. Vaccination of C57BL/6 mice with a SARS-CoV N protein expressed by an E1/partially E3-deleted, replication-defective human adenovirus 5 vector was shown to produce potent SARS-CoV-specific humoral and T cell-mediated immune responses, suggesting the potential of this construct to be used as SARS-CoV vaccine . Along tal route . In thisal route .Although immunization with inactivated viral vaccine provides significant protection in animals against challenge with certain corresponding pathogenic CoVs, in the case of SARS-CoV there remains the threat of introducing live virus into the environment from partially inactivated vaccine, as there are no validated and effective inactivation measures developed yet. To circumvent this obstacle, Chen et al have introduced the S protein into the deletion III region of the live, attenuated modified vaccinia virus Ankara (MVA) vector . This reThe third possibility is a genetically engineered version of live SARS-CoV for traits such as attenuated phenotype, increased immunogenicity, and safe handling (out of BL3+ facility). A full-length SARS-CoV cDNA-containing plasmid has been developed from which synthetic infectious viral RNA can be produced . This sySARS-CoV clearly has pandemic potential. Although progress in SARS-CoV molecular and cell biology research has been remarkable, there remain clear limitations regarding vaccine development due to a lack of complete understanding in the areas of animal models of the disease as well as host immune responses to the evolving molecular diversity of this newly emerged human virus. Caution is warranted when utilizing experimental data originating from one SARS-CoV strain infection in one animal species or cell line in the development of a human vaccine. The rapid development of an effective SARS-CoV vaccine depends upon continuing basic research.A study on the evolving S protein molecular diversity in SARS-CoV isolates and its unexpected profound immuno-functional effects illustrates this point . The S pThe author(s) declare that they have no competing interests.Authors contributed equally to the intellectual content of this review article.The views presented in this article do not necessarily reflect those of the Food and Drug Administration or United States government.


Drosophila pseudoobscura harbors a rich gene arrangement polymorphism on the third chromosome generated by a series of overlapping paracentric inversions. The arrangements suppress recombination in heterokaryotypic individuals, which allows for the selective maintenance of coadapted gene complexes. Previous mapping experiments used to determine the degree to which recombination is suppressed in gene arrangement heterozygotes produced non-recombinant progeny in non-Mendelian ratios. The deviations from Mendelian expectations could be the result of viability differences between wild and mutant chromosomes, meiotic drive because of achiasmate pairing of homologues in heterokaryotypic females during meiosis, or a combination of both mechanisms. The possibility that the frequencies of the chromosomal arrangements in natural populations are affected by mechanisms other than adaptive selection led us to consider these hypotheses. We performed reciprocal crosses involving both heterozygous males and females to determine if the frequency of the non-recombinant progeny deviates significantly from Mendelian expectations and if the frequencies deviate between reciprocal crosses. We failed to observe non-Mendelian ratios in multiple crosses, and the frequency of the non-recombinant classes differed in only one of five pairs of reciprocal crosses despite sufficient power to detect these differences in all crosses. Our results indicate that deviations from Mendelian expectations in recombination experiments involving the D. pseudoobscura inversion system are most likely due to fitness differences of gene arrangement karyotypes in different environments. Drosophila pseudoobscura harbors a rich gene arrangement polymorphism on the third chromosome generated by a series of overlapping inversions The coadaptation model was developed to explain the apparent fitness differences of karyotypes in natural populations and laboratory crosses. Under this model, natural selection maintains favorable combinations of alleles within and between arrangements within populations, and suppressed recombination in arrangement heterozygotes prevents the breakup of coadapted alleles within arrangements D. pseudoobscura third chromosome inversion system confirm suppressed recombination in heterokaryotypic individuals The coadaptation model relies on suppressed recombination in arrangement heterozygotes, but it also assumes equal transmission of homologous chromosomes during meiosis. Previous studies of recombination in the DrosophilaPrevious genetic mapping experiments Using a different set of wild-type arrangements, Levine DrosophilaD. pseudoobscuraThe mechanisms of meiotic segregation differ between male and female D. pseudoobscura third chromosomes carrying phenotypic markers and wild-type chromosomes each carrying one of five naturally occurring third chromosome gene arrangements , Santa Cruz (SC), or Tree Line (TL) arrangement to single males homozygous for chromosomes carrying some of the same mutant markers . The recz = 2.317, p = 0.02). There is no evidence to support the meiotic drive hypothesis because males and females show the same transmission frequency of wild-type non-recombinant chromosomes when heterozygotes carry arrangements that differ by at least one inversion.The frequency of wild-type non-recombinant offspring was significantly greater than 50% for the crosses involving females carrying the wild-type ST and SC arrangements and the crosses involving males carrying the wild-type AR and ST arrangements when summing across replicates . The obsFailure to reject the null hypothesis could occur if the null hypothesis is true or if we lack sufficient power to reject. Significant deviations from Mendelian expectations should be detected using the sum across replicates for any of the arrangements if the actual frequency of transmission of the wild-type non-recombinant chromosome is at least 0.54 (based on our sample sizes). Assuming the wild-type chromosome is transmitted according to Mendelian expectations in heterozygous males, we should be able to detect significant differences in the frequency of wild-type non-recombinants between the sexes when summing across replicates if the wild-type chromosome is transmitted at a frequency of at least 0.54 in heterozygous females carrying the AR, ST, PP, and SC wild-type arrangements and at least 0.55 in TL females (based on our sample sizes). Simulations were carried out to model our cross scheme using different expected frequencies of transmission of the wild-type non-recombinants. For all sets of crosses, we have a power greater than 0.80 to detect significant deviations from Mendelian expectations using the average of replicates when the actual frequency of transmission is at least 0.75 .The aforementioned deviations from Mendelian expectations  may be tThe variance of the frequency of wild-type non-recombinants in the average across replicates was calculated for the simulated data sets in which the observed frequency of transmission of the wild-type non-recombinants (sum across replicates) was used. The observed variance of the frequency of wild-type non-recombinants in the average across replicates is significantly greater than that expected based on the simulations in only two crosses – the crosses involving heterozygous ST males and heterozygous PP females . In all D. pseudoobscura third chromosome inversion system The transmission of gene arrangements was previously observed to depart from the expected one to one Mendelian expectations in experiments examining recombination in the We performed reciprocal crosses to determine whether the deviations from Mendelian expectations are due to fitness effects or non-random disjunction. We observed a significant excess of wild-type non-recombinant chromosomes in only 4 of 10 crosses  if the wild-type chromosome is transmitted at a frequency of 0.50 in males and at least 0.55 in females see . Some ofThe differences between our results and the previously published studies could be a result of differences in rearing temperatures between experiments – our flies were mated and developed at 18°C, while some of the previous experiments were performed at 25°C It is possible that the population density within the vials could have lead to increased deviations from Mendelian expectations in the previous experiments, however Levine and Levine The analysis of variance revealed a significant effect of the gene arrangement carried by the heterozygous parent and the interaction of sex of the heterozygous parent and gene arrangement on the frequency of wild-type non-recombinants . The lacThe amount of variance among replicates is fairly consistent with that expected if all replicates had the same expected frequency of transmission of the wild-type chromosome and if that frequency were determined by the sum across replicates ; we onlyD. pseudoobscura inversion polymorphism. We cannot reject the possibility that environmental effects (such as temperature) or demography (such as population density) may influence allele frequencies. Examining these effects requires further experimentation in which the rearing temperature, size of vial, and number of mated females per vial is varied. Our data support the hypothesis that the D. pseudoobscura third chromosome arrangements are not subject to non-random disjunction because of the inverted regions. This conclusion is consistent with selection maintaining the arrangement polymorphism due to fitness benefits conferred by coadapted gene complexes, which are maintained by suppressed recombination between arrangements.We have no evidence that segregation distortion because of chromosomal inversions is responsible for the maintenance of the orange (or), Blade (Bl), purple (pr), and crossveinless (cv). The or and pr mutations correspond to cinnabar and brown in D. melanogaster, respectively D. pseudoobscura genome project A heterozygous strain for the AR and Cuernavaca (CU) gene arrangements was provided by Wyatt Anderson (University of Georgia). The AR chromosome carries four phenotypic markers: or Bl pr cv marker stock to single males homozygous for a wild-type chromosome carrying either the AR, ST, PP, SC, or TL arrangement  was summed across replicates, and the frequency of wild-type non-recombinants out of all non-recombinant progeny was determined for the “sum across replicates”. The frequency of wild-type non-recombinants was also determined for each replicate with at least thirty non-recombinant progeny (N>30). Both the sample mean of the replicates (“average of replicates”) and the sample variance were calculated for each of the crosses using replicates with N>30. An analysis of variance  was performed using the replicates with N>30 on the frequency of wild-type non-recombinants after an arcsine transformation The number of individuals in each of two non-recombinant classes  was significantly greater than that expected under Mendelian inheritance for each simulated replication of the experiment for all 11 frequencies of transmission. We also calculated the effects of sex of the heterozygous parent, chromosomal arrangement, and the sex by arrangement interaction in our simulated data using an ANOVA as described above. Simulations were also performed using the observed frequency of wild-type non-recombinants from the sum across replicates as the expected frequency of wild-type non-recombinants for each replicate in the simulation. For each of these simulations, we performed an ANOVA (see above) and determined the variance among replicates.Figure S1or, pr, and cv mutations on a ST background, while the second set used a chromosome carrying or, Bl, Sc, and pr on the ST background. Error bars represent 95% confidence intervals, and the dashed line shows the expectation under Mendelian inheritance and no natural selection. The relationships of the different arrangements are shown in Results of previous crossing experiments. The frequency of wild-type non-recombinants is shown for each wild-type arrangement in experiments performed by Dobzhansky and Epling (0.73 MB EPS)Click here for additional data file.Figure S2or, pr, and cv mutations, (B) the marker chromosome carrying the or, Bl, Sc, and pr mutations and (C–G) Levine and Levine's crosses or, Bl, Sc, and pr mutations.Transmission frequencies of mutant alleles in previous crossing experiments. Frequency of transmission for individual mutant alleles in crossing experiments involving wild-type and marker chromosomes carrying the ST arrangement were calculated. Error bars indicate 95% confidence intervals, and the dashed line shows the expectation under Mendelian inheritance. Data taken from Dobzhanksy and Epling's crosses (0.66 MB EPS)Click here for additional data file.


Genotypic antiretroviral resistance testing (GRT) in HIV infection with drug resistant virus is recommended to optimize antiretroviral therapy, in particular in patients with virological failure. We estimated the clinical effect, cost and cost-effectiveness of using GRT as compared to expert opinion in patients with antiretroviral treatment failure.We developed a mathematical model of HIV disease to describe disease progression in HIV-infected patients with treatment failure and compared the incremental impact of GRT versus expert opinion to guide antiretroviral therapy. The analysis was conducted from the health care (discount rate 4%) and societal (discount rate 2%) perspective. Outcome measures included life-expectancy, quality-adjusted life-expectancy, health care costs, productivity costs and cost-effectiveness in US Dollars per quality-adjusted life-year (QALY) gained. Clinical and economic data were extracted from the large Swiss HIV Cohort Study and clinical trials.Patients whose treatment was optimized with GRT versus expert opinion had an increase in discounted life-expectancy and quality-adjusted life-expectancy of three and two weeks, respectively. Health care costs with and without GRT were $US 421,000 and $US 419,000, leading to an incremental cost-effectiveness ratio of $US 35,000 per QALY gained. In the analysis from the societal perspective, GRT versus expert opinion led to an increase in discounted life-expectancy and quality-adjusted life-expectancy of three and four weeks, respectively. Health care costs with and without GRT were $US 551,000 and $US 549,000, respectively. When productivity changes were included in the analysis, GRT was cost-saving.GRT for treatment optimization in HIV-infected patients with treatment failure is a cost-effective use of scarce health care resources and beneficial to the society at large. The advent of potent antiretroviral therapies (ART) a decade ago has led to a substantial decline of morbidity and mortality in HIV infected patients It has been documented in randomized controlled trials that GRT based treatment optimization leads to a higher viral load reduction than standard care alone in treatment-experienced patients Early and more recent cost-effectiveness studies suggested that antiretroviral treatment optimization using GRT is cost-effective We developed a mathematical model of HIV disease to assess the incremental impact of antiretroviral therapy guided by genotypic antiretroviral resistance testing versus expert opinion alone in patients presenting with treatment failure defined as i) a viral log reduction of <1 log HIV RNA copies/ml during the first six months after initiation of antiretroviral therapy (ART), ii) a viral load increase of >1 log HIV RNA copies/ml within two months of ART, or iii) two consecutive viral load assessments >200 copies/ml after reaching undetectable plasma HIV RNA levels (<50 copies/ml) 3, 201–500 cells/mm3, >500 cells/mm3) and viral load , and in patients who experienced an AIDS-indicator disease by CD4 cell count only . Patients are always at risk of dying due to an HIV-related or unrelated cause and were modeled until death. The disease model was populated with data extracted from the SHCS; the starting age of patients is 33 years, the average age of patients enrolled in the SHCS, and 80% of patients are male A state transition model of HIV disease with mutually exclusive health states was developed to describe the course of HIV disease after antiretroviral treatment failure, the starting state of patients entering the model The probability of maintaining the failing antiretroviral regimen or switching to a new regimen with or without information available from GRT was derived from Haupts et al. www.statistik.admin.ch).HIV disease progression was modeled by means of a transition probability matrix derived from the SHCS using the dataset from the period 1996–2004 before GRT became widely available. The probability of transitions to a health state with a viral load above or below 1000 copies/ml, a different CD4 cell stratum, the risk of developing an AIDS-indicator disease and dying was extracted from the SHCS database by pooling observations from patients on highly active antiretroviral therapy over consecutive six-month periods HIV disease may not only affect survival but may also substantially affect the patient's wellbeing st, 2005 .In the analysis from the health care perspective we only included costs due to health care resource consumption . We usedwww.statistik.admin.ch) to estimate productivity changes over a patient's simulated lifetime  or the benefit of Hepatitis B vaccination in newborn babies whose mothers have Hepatitis B (gain in life-expectancy 2 weeks) M. avium complex infection in patients with AIDS ($US 80'000 per QALY gained) However, when only health care costs are considered, the cost-effectiveness ratio of GRT is $US 35'000 per QALY gained, which is similar to the cost-effectiveness ratio of implantable cardioverter-defibrillators ($US 35'000 per QALY gained) In a previous study we have shown that GRT reduces health care costs over a two-year follow-up period due to the reduction of ambulatory and in-patient costs and a reduced consumption of non-HIV medication In the present paper we also considered the most recent methodological developments with respect to the inclusion of productivity costs in economic evaluation to avoid a biased cost-effectiveness ratio Our study has several limitations. We used a mathematical model to approximate the real-world and project long-term costs and outcomes of GRT. However, we used best available evidence from one of the largest cohort studies on HIV disease to reflect a real-world setting Our study has been conducted in Switzerland where health insurance is compulsory by law for all patients. However, our results are equally relevant for other countries such as the USA where health insurance is often funded by employers. The inclusion of GRT in health insurance plans not only leads to a benefit to the patient but also to the employer by increasing job productivity. Public health insurance coverage in the USA, on the other hand, is tied to disability status. Patients with disability status on public health insurance may also benefit from GRT based treatment optimization, as a better health state may increase the likelihood to return to work. Our results suggest that GRT represents high value for money and should be offered to all patients who can benefit from it. Table S1Transition probability matrix for HIV disease progression generated by pooling consecutive six-month observations from patients on HAART enrolled in the SHCS between 1996–2004. Numbers indicate observations, the likelihood is shown in brackets. To calculate the posterior probability with a non-informative prior, the number of observations in each cell with an allowed transition is increased by one.(0.05 MB DOC)Click here for additional data file.


The carboxy termini of proteins are a frequent site of activity for a variety of biologically important functions, ranging from post-translational modification to protein targeting. Several short peptide motifs involved in protein sorting roles and dependent upon their proximity to the C-terminus for proper function have already been characterized. As a limited number of such motifs have been identified, the potential exists for genome-wide statistical analysis and comparative genomics to reveal novel peptide signatures functioning in a C-terminal dependent manner. We have applied a novel methodology to the prediction of C-terminal-anchored peptide motifs involving a simple z-statistic and several techniques for improving the signal-to-noise ratio.We examined the statistical over-representation of position-specific C-terminal tripeptides in 7 eukaryotic proteomes. Sequence randomization models and simple-sequence masking were applied to the successful reduction of background noise. Similarly, as C-terminal homology among members of large protein families may artificially inflate tripeptide counts in an irrelevant and obfuscating manner, gene-family clustering was performed prior to the analysis in order to assess tripeptide over-representation across protein families as opposed to across all proteins. Finally, comparative genomics was used to identify tripeptides significantly occurring in multiple species. This approach has been able to predict, to our knowledge, all C-terminally anchored targeting motifs present in the literature. These include the PTS1 peroxisomal targeting signal (SKL*), the ER-retention signal (K/HDEL*), the ER-retrieval signal for membrane bound proteins (KKxx*), the prenylation signal (CC*) and the CaaX box prenylation motif. In addition to a high statistical over-representation of these known motifs, a collection of significant tripeptides with a high propensity for biological function exists between species, among kingdoms and across eukaryotes. Motifs of note include a serine-acidic peptide (DSD*) as well as several lysine enriched motifs found in nearly all eukaryotic genomes examined.We have successfully generated a high confidence representation of eukaryotic motifs anchored at the C-terminus. A high incidence of true-positives in our results suggests that several previously unidentified tripeptide patterns are strong candidates for representing novel peptide motifs of a widely employed nature in the C-terminal biology of eukaryotes. Our application of comparative genomics, statistical over-representation and the adjustment for protein family homology has generated several hypotheses concerning the C-terminal topology as it pertains to sorting and potential protein interaction signals. This approach to background reduction could be expanded for application to protein motif prediction in the protein interior. A parallel N-terminal analysis is presented as supplementary data. The carboxy tails of proteins are frequent sites of post-translational modification, protein-protein interaction domains and sub-cellular protein sorting motifs. This is presumably due to a high-kinetic cost in burying the termini within the interior of the protein; leaving the head and tail regions of many proteins exposed to the cytoplasm and free to engage in static or dynamic biochemical interactions . Althouga priori mapping of experimentally verified motifs to novel unannotated sequences (scanning) and the ab initio identification of potentially novel motifs without any prior knowledge of motif structure. Over the past decade, rapid advances in high-throughput proteomics and a large body of literature detailing the structure and function of numerous proteins in many species, have focused protein motif prediction on the annotation of novel sequences using motif scanning from an a priori collection of protein domain knowledge in the literature DEL. The DEL SOCT was also absent in the lower animals due to its lack of significance in C. elegans. This may represent the presence of an alternate preferred ER retention consensus motif in worms. The 2 mammalian species (human and mouse) possessed Caax motifs, several PTS1 consensus variants and the HDEL form of the ER retention signal, see Table Using our final filtered z-scores for all species, comparisons were made in SOCT conservation between the plants, the lower animals and the mammals see Fig . In eachTo examine SOCT co-occurrence across all 7 eukaryotic species, the SOCTs were filtered for statistical prevalence (z ≥ 3) in at least 2 genomes and for presence in at least 10 proteins within a species. This later stipulation was introduced to remove rare tripeptides possessing significant z-scores due solely to their genome-wide infrequency and not their terminal abundance. A total of 37 SOCTs emerged at the terminal and second-to-last carboxy positions see Fig . To our In total, 35% of the multi-species tripeptide signals occurring in the last two terminal positions matched well characterized C-terminal anchored peptide motifs in the literature. As well as known C-terminal signals, a variety of uncharacterised and potentially functionally important motifs were identified. These motifs may represent, as of yet, unidentified sorting signals but may also represent components of generic C-terminal biology ranging from structural strategies to protein-protein interaction and post-translational modification motifs. For a complete list of identified SOCTs the reader is referred to the supplemental data [see Additional file The amino and carboxyl termini of proteins are critical components, uniquely positioned to fill a variety of roles in protein biology. Our study has focused on the prediction and identification of novel protein motifs dependent upon C-terminal proximity for proper function. Characterized protein motifs known to function in this manner are largely involved with protein sorting and lipidation . Using iC. elegans). Although these motifs have been demonstrated as conforming to the PTS1 consensus [Of all resulting SOCTs, the peroxisomal targeting signal SKL was most prominent  using C-terminal GFP fusions exhibited differential sub-cellular localization from an EGFP:AAA control during transient assays in A. thaliana and N. benthamiana (data not shown). However, other components of a low-level C-terminal protein grammar, such as structural strategies, protein-protein interaction or post-translational modification may be responsible for the high motif frequencies observed in these particular SOCTs.Given the efficiency with which our analysis was capable of identifying existing C-terminally anchored protein sorting signals, several SOCTs represented across species and within the results of A. thaliana seedlings showed a slowed migration of a GFP:SDSDSD fusion protein [see Additional file H. sapiens SOCT ESE. Also of potential interest are the DSD-6 in RNA polymerase II and ESD-8 in topoisomerase II alpha of H. sapiens. Modification of these residues have also been shown to influence DNA binding and protein stability in their respective proteins [A. thaliana are functionally annotated (Gene Ontology) as nucleic acid binding [see Additional file The terminal tripeptide DSD was highly significant in all species save the proteomes of rice and yeast and similar in significance level to SKL see Fig. . Moreoveproteins ,34. AlthA. thaliana and O. sativa, was the presence of a highly conserved sequence (FSDENPNA-4) proximal to the Caax motif in a group of iso-prenylated plant metalloproteins [see Additional files An interesting, albeit unexpected result within the SOCT intersections of There is a strong presence of Lys among many of the uncharacterised tripeptides at the terminal end of the 7 species "C-terminome". These include: KNK, KNN, KKN, KRK, RRK, KKK, GKK and LKK see Fig . In 2003Overall several intriguing patterns in peptide compositional preferences have been identified. Although the present analysis focuses on the C-terminus, it should be noted that an N-terminal examination was run in parallel and similar biases were observed at the N-terminus [see Additional file ab initio motif discovery. Since the carboxyl group provides a point of reference, C-terminal anchored peptides should appear among peptide frequencies calculated at distinct C-terminal positions [ab initio. Given our success in predicting known motifs, the likelihood of novel yet undefined motifs present in the results seems likely. However, among the previously known motifs identified, the majority are widely prevalent with strong significance values. This suggests that any novel uncharacterised signals present in the data may function more specifically or subtly than other confirmed CTAMs present in the analysis.Several properties of the C-terminal class of anchored motifs make them attractive for ositions . LikewisSince the C-terminus is a frequent site for protein regulation and is often utilized in recombinant protein experiments, it would seem that C-terminal peptide function will continue to increase in relevance as our knowledge of its biological importance progresses. The novel SOCTs identified in our analysis may represent C-terminal peptide motifs functioning in biological roles ranging from protein sorting, post-translational modification or capping and structural strategies. However, based on the prominence of known targeting signals and the lack of novel SOCTs with a distinct pattern, any protein sorting motifs that remain to be characterized are likely to be conserved to a small number of protein families, exhibit species-specific functionality or possess a considerable degree of degeneracy. Overall, our results appear to depict a highly accurate representation of the statistical topography of the "C-terminome" and the methodology could be adapted to protein motif prediction efforts in the protein interior.famMCL and masked for simple sequence stretches using the program seg [Prior to statistical analysis, each predicted proteome was clustered into protein families using the shell script gram seg . Mean anA. thaliana which was obtained from TAIR and O. sativa, which was downloaded from TIGR. O. sativa was downloaded in conjunction with a list of accessions corresponding to transposable elements. This list was then used to filter out transposable elements from the protein summary file with a short shell script.Translated datasets for each species were obtained in fasta format. All datasets were downloaded from NCBI with the exceptions of A. thaliana downloaded from TAIR as ATH1_pep_cm_20040228. [0040228. C. elegans by chromosome translated faa. [ted faa. D. melanogaster by chromosome translated faa. [ted faa. H. sapiens by protein summary. [summary. M. musculus by protein summary. [summary. S. cerevisiae by chromosome translated faa file. [aa file. O. sativa by protein summary. [summary. O. sativa transposable element list by accession for the filtering of above. [f above. fastarand was written in the C programming language [fastarand will create an equal size fasta file on a sequence by sequence basis using one of three randomization models.To generate a collection of randomized fasta sequences, the program language . Given a1) shuffle the amino acids within each protein in the file2) generate each sequence based on the amino acid frequencies in the entire proteome3) resample nmers from the query protein until an equal length protein is reachedThe user is able to specify how many randomized proteomes are to be created with a commandline flag . Model 3 using 3mers was employed in our study.famMCL. famMCL should compile on any POSIX based system and depends upon a functional installation of NCBI standalone blast [MCL clustering algorithm [famMCL and its supporting documentation are available under the GPL [Each species proteome was clustered into gene families using the short shell script ne blast , and thelgorithm . famMCL  the GPL .famMCL performs an all-by-all BLASTP of the provided proteome using the concise output format option of the NCBI standalone BLAST program (-m 8). All bit scores for individual protein comparisons are parsed from the BLAST results to produce an MCL format matrix that is submitted to the MCL clustering algorithm. The resulting MCL output is then parsed to generate lists of protein families by accession and corresponding cluster number. A BLAST similarity cutoff of E ≤ 1e-10 and the default MCL granularity were used. Our strategy for implementation resulted in little to no variation in cluster composition in successive runs over the recommended range of MCL granularity settings and fluctuations in cluster size and composition within famMCL were found to be primarily dependent upon selection of the E-value cutoff. As an all-by-all genome BLAST is computationally intensive, it was performed on an openSSI parallelized cluster of workstations running Debian GNU Linux at the Botany Bioinformatics Cluster in the Department of Cell & Systems Biology, University of Toronto.tripepper was written in the C programming language to determine mean and standard deviation background statistics for respective tripeptides from 100 fastarand randomized proteomes. The PERL script cluster adjuster was then used to calculate position specific tripeptide counts for each simple sequence masked proteome and adjust these counts by subtracting duplicate tallies arising from members of a common gene family as derived from famMCL. These programs collectively constitute the SOCT pipeline software [The program software  and retuk is the number of counts for the tripeptide in the masked proteome, λ is the number of duplicate tripeptide counts due to a common gene family and x and s are the mean and standard deviation respectively for occurrences of tripeptideij across 100 randomized proteomes.where i is a tripeptide permutation between AAA and YYY, j is the position in from the C-terminus (-3 to -100), tripepper was given a fastarand generated directory of randomized proteomes and a corresponding proteome with stretches of simple sequences masked out. Masked proteomes were created by running seg at default settings [seg is used to replace the original proteome read by tripepper. Protein clusters as determined by famMCL were then input to the Perl program cluster adjuster, which uses the tripepper results and the masked proteome to adjust total tripeptides counts by the number of common family occurrences and produce a set of final penalized z-scores.The program settings . Note thgrep, sed, awk) to identify all significant tripeptides (z-score ≥ 3) occurring in at least 2 species and present in at least 10 genes in each proteome. These data were then analysed using the open source plotting program gnuplot [Heatmapper [Comprehensive z-statistics generated for each eukaryotic proteome were processed using common UNIX shell scripting tools Background reduction from TE filtering Background reduction in randomization models (Click here for filefamMCL generated protein families in all speciesClick here for fileAll filtered C-terminal z-statistics for all speciesClick here for fileA. thalianaGO annotation for DSD possessing genes in Click here for fileA. thalianaImmunoblotting against EGFP:SDSDSD transgenic Click here for file


This CD-ROM version of Atlas of Salivary Gland Tumor Cytopathology, Oral & Surgical Pathology is an excellent and concise tool for easy reference during sign out of cytology and surgical cases. It is also invaluable as a teaching tool for residents and fellows.Atlas of Salivary Gland Tumor Cytopathology, Oral & Surgical Pathology CD-ROM (ISBN 0-9736518-0-7), Published by Pathology images Inc Ottawa, Ontario K2B 7L4, Canada The Atlas of Salivary Gland Tumor Cytopathology, Oral & Surgical Pathology as a CD-ROM version is a very welcome edition to the library of any pathologist for quick reference . The patThe authors have organized this CD-ROM into convenient chapters. In the sections on specific tumors, the authors have nicely provided tables, brief cytologic and surgical text and excellent colored photographs with key features highlighted. Some photographs use arrows to illustrate the salient features. The majority of the photographs are of high quality, although an occasional one has been taken at too low power to fully discern the specific features.I would recommend this CD-ROM in the cytology sign out room as a very useful quick reference for differential diagnosis of salivary gland tumors. The CD-ROM is easy to use, very concise in the information provided with high quality colored illustrations.The author(s) declare that they have no competing interests.The author critically reviewed the CD-ROM of Atlas of Salivary Gland Tumor Cytopathology, Oral & Surgical Pathology. Thereafter drafted and revised the manuscript of this review article.