The essential difference between cancer cells and normal cells is that cancer cells evolve. Most cancers arise from a single cell through a sequential evolutionary process of mutation and selection. Cancer cells harbor mutations in a number of critical genes that, at various stages during the evolution of the tumor, provide those cells with a selective advantage. Many of the phenotypes, or physical outcomes, conferred by these mutant genes are subverted from a normal cell's repertoire, including proliferation, invasion, migration, loss of differentiation, and loss of apoptosis (programmed cell death); other phenotypes, such as immortalization, are novel. Tumor evolution is thought to adhere to Darwinian principles, with mutations arising randomly within an individual cell, followed by selection for mutant clones with favorable traits.Support for this idea stems from the observation that end-stage tumors have mutations in a number of genes. But linking mutations in particular genes with defined stages is difficult for most human cancers and especially so for the last and deadliest stage: when cancer disseminates throughout the body during metastasis. It's unclear, for example, whether there are mutations in particular genes or sets of genes that enhance metastasis. That is, do metastatic lesions develop through a continuation of Darwinian evolution, or is metastasis an intrinsic property of the primary tumor, meaning that further genetic evolution is not required? It is also unclear whether there is a “preferred” sequence of mutations, such that selective pressure for particular mutations depends on preexisting mutations.Ras and the loss of the tumor suppressor p53 cooperate to transform cells.Since the earliest days of research on oncogenes—genes that can cause a cell to become cancerous—it has been known that certain oncogenic mutations cooperate to transform normal cells into cancer cells. For example, an “activating” mutation in the oncogene Ras. Using a well characterized mouse model of squamous cell carcinogenesis, which generates a form of skin cancer, the authors examine both the functional and evolutionary relationships between three cancer genes that play major roles in most human cancers: Ras and the tumor suppressors Arf and p53.The paper by Christopher Kemp and his colleagues at the Fred Hutchinson Cancer Research Center sheds light on some of these questions, and many of the issues center around the most notorious oncogene: Ras is the initiating genetic event in this cancer model, while mutation of p53 occurs later, during the benign to malignant transition; and expression of mutant Ras in cells activates p53 via signaling through the protein encoded by Arf.Two seminal early observations set the stage: mutational activation of p53 expression in tumors with Ras mutations is dependent on the presence of Arf. Thus, during the early benign stages of tumor growth, Ras activates Arf, which in turn activates p53, thereby inhibiting tumor progression. This provides strong selective pressure in favor of cells with mutations in either Arf or p53, and these mutations are indeed observed as the tumors progress to malignancy. That Arf and p53 function as tumor suppressors was confirmed by demonstrated accelerated tumor progression in mice lacking either Arf or p53. This answers a longstanding question concerning the nature of the signal that activates p53 during autochthonous tumor development: Mutation of Ras not only initiates tumor development but, through its intracellular signaling through Arf and p53, directly influences the subsequent evolutionary trajectory of the tumors. In this view, secondary evolutionary events are determined by the preexisting genetic lesion, as a result of direct signaling interactions.Kemp et al. confirm that this pathway is active in “autochthonous” tumors—which grow and develop where they are initiated—by showing that Arf or p53 show accelerated metastatic dissemination, a phenomenon rarely seen in mouse squamous cell cancer models. Thus both benign and malignant tumors lacking these tumor suppressors are at high risk for metastasis. As Ras is well known to confer many phenotypes required for the metastatic process, it appears that Ras, together with loss of its inhibitors, Arf and p53, may be sufficient to drive this process. More direct evidence that metastasis does or does not require further genetic evolution awaits.The authors go on to show that tumors lacking


Splice variants of 461 transcription factor loci were analyzed using a new database of splice variants in the mouse transcriptome, MouSDB3, providing quantitative evidence that alternative splicing preferentially adds or deletes domains important to the DNA-binding function of the TFs. Analyzing proteins in the context of all available genome and transcript sequence data has the potential to reveal functional properties not accessible through protein sequence analysis alone. To analyze the impact of alternative splicing on transcription factor (TF) protein structure, we constructed a comprehensive database of splice variants in the mouse transcriptome, called MouSDB3 containing 461 TF loci.Our analysis revealed that 62% of these loci in MouSDB3 have variant exons, compared to 29% of all loci. These variant TF loci contain a total of 324 alternative exons, of which 23% are in-frame. When excluded, 80% of in-frame alternative exons alter the domain architecture of the protein as computed by SMART (simple modular architecture research tool). Sixty-eight % of these exons directly affect the coding regions of domains important for TF function. Seventy-five % of the domains affected are DNA-binding domains. Tissue distribution analyses of variant mouse TFs reveal that they have more alternatively spliced forms in 14 of the 18 tissues analyzed when compared to all the loci in MouSDB3. Further, TF isoforms are homogenous within a given single tissue and are heterogeneous across different tissues, indicating their tissue specificity.Our study provides quantitative evidence that alternative splicing preferentially adds or deletes domains important to the DNA-binding function of the TFs. Analyses described here reveal the presence of tissue-specific alternative splicing throughout the mouse transcriptome. Our findings provide significant biological insights into control of transcription and regulation of tissue-specific gene expression by alternative splicing via creation of tissue-specific TF isoforms. Alternative splicing is a widespread mechanism involved in regulation of gene expression, which enables production of many structurally and functionally different forms of proteins from a single gene, adding to the complexity of the genomes -3. DiffeAn expanding body of expressed sequence data from the human and mouse genomes indicates that alternative splicing is an important mechanism in creating protein diversity, and adds to functional complexity encoded in eukaryotic genomes. Earlier studies indicate that at least 50% of the genes in the human genome are alternatively spliced . ExampleComprehensive analysis of alternative splicing is essential to understand fully the proteomes of organisms . SeveralTranscription is a critical process that specifies the mRNAs and the proteins expressed within a cell. Expression of a given gene is dependent on the interactions of different transcription factors and their cofactors with the regulatory regions of that gene. These transcription factors are in turn regulated by processes that include interaction with other proteins and signaling cascades .Alternative splicing is a mechanism that regulates transcription factor (TF) activity by generating a variety of protein isoforms from a single gene. As noted by Lopez, alternative splicing can affect TF structure in two primary ways : alteratIn this paper, we use an integrated approach to analyze DNA and protein sequence data jointly to determine the potential effect of alternative splicing on protein structure and function. We perform a detailed analysis of tissue-specific distribution of alternatively spliced mouse TFs to gain biologically meaningful insights into regulation of gene expression by alternative splicing.For our joint DNA-protein analysis described here, we developed MouSDB3 , which iOur cluster analysis revealed that out of the 461 TF clusters, 62% are variant, compared to 29% of all genes in MouSDB3  are in-frame. Only 11% of cassette exons are expected to be multiples of three and in codon position 1 randomly. The twofold difference between expected and observed numbers indicates a bias towards in-frame cassette exons. The exons which are a multiple of three and in codon position 2 and 3 comprise 10% and 7%, respectively. When deleted, these exons introduce an amino-acid substitution to the sequence. As exons which are a multiple of three starting at codon position 1 are enriched and do not introduce an amino-acid substitution when deleted, our study focuses on these exons only.As shown in Figure et al. [SMART ,16 and Pet al. , who shoPart two of our analysis assessed the tissue distribution of alternatively spliced transcription factors. We chose 18 tissues from the existing libraries in MouSDB3 on the basis of the fact that they contain both variant and invariant transcripts annotated as TFs. There are a total of 1,413 library names in MouSDB3 imported from expressed sequence records in GenBank and dbEST. Of these, 328 are ambiguous in that they list several different tissues or cell types for a single library, such as 'mixture of brain and testis' or no tissues at all, such as 'embryo or carcinoma'. For the work described here we did not include tissue information from such ambiguous libraries. There are a total of 95 libraries in MouSDB3 for which there are TF transcripts. In addition, to account for library ambiguities within these 95 libraries, we pooled different parts of a tissue into one library. For example, the term 'brain' corresponds to all parts of the brain found in MouSDB3, including cerebellum, thalamus, hippocampus and 16 other libraries. When analyzing the tissue distribution of all genes, only the libraries that contain TF transcripts have been used.Transcript counts within variant loci for 18 pooled libraries indicated that in 14 of the 18 analyzed tissues, the proportion of TFs that are variant is higher than the proportion of all genes that are variant Figure . This fi2 values above 1 on the graph). In salivary gland, skeletal muscle, urinary bladder and testis, the fold-differences are 8.7, 5.6, 3.8 and 3.0-fold respectively. Spinal cord, liver, adipose tissue and eye also exhibit more than twofold differences. These values are independent of the sampling depth of the transcripts from these tissues, as illustrated in Figures x-axis as in Figure For each of the 18 tissues in Figure p-value = 0.04). This is true for both transcription factors and the rest of the genes in the mouse transcriptome.We analyzed the presence of different isoforms of transcription factors within and across these 18 tissues. For this analysis we consider transcripts with coding sequence information only. We ignore variation due to 5' and 3' truncation of transcripts. We consider only cassette exons within coding sequences when assessing the differences between isoforms. Within a cluster we compute homogeneity and heterogeneity within a single tissue by checking for the transcripts from the same library and comparing the cassette exons within their coding sequences. If all transcripts from the same tissue contain the same cassette exons with same splice sites they are termed 'homogeneous within'. If the cassette exon distribution within the coding sequences of these transcripts differ, they are termed 'heterogeneous within'. We compute 'homogeneity across' and 'heterogeneity across' tissues in the same way by taking into account transcripts within the same clusters but from different libraries. As shown in Figure p-value = 0.02). Figures Through integrated analyses of DNA and protein sequences for TF genes, we show that alternative splicing of TFs are more prevalent in the entire mouse transcriptome and in specific tissues when compared to alternatively spliced forms of all the genes. In 78% of the tissues analyzed, higher proportions of TFs exhibit alternative splicing compared to all the genes in the mouse transcriptome. This result, along with the finding that 62% of TF loci are variant, indicates the widespread impact of alternative splicing on regulation of TF function.We also show that alternative splicing changes TF structure by adding or deleting domains. This study reveals that 80% of alternatively spliced TFs have different domain architectures due to introduction of an in-frame cassette exon by alternative splicing. Of the altered domains, 75% have a role in DNA binding. These findings provide quantitative evidence for the role of alternative splicing in controlling the presence of domains in the proteins. They also suggest that alternative splicing might regulate TF activity by changing the architecture of the DNA-binding domains of these proteins.Our analyses revealed that within a single tissue there generally is only one TF isoform, and that across tissues, isoforms differ. This finding indicates tissue specificity of alternatively spliced TFs and suggests that TFs might regulate gene expression in a tissue-specific manner by having different isoforms in different tissues. These findings further indicate the role of alternative splicing in regulation of tissue-specific gene expression. Activation and repression of different sets of genes within different tissues can be regulated through variant TF isoforms created by alternative splicing. These findings will significantly aid further understanding of control of transcription and tissue-specific gene expression. In addition, our study shows that all variant loci in the mouse transcriptome display isoform homogeneity within single tissues and heterogeneity across tissues. This finding demonstrates the presence of tissue-specific alternative splicing across the mouse transcriptome and greatly expands the knowledge on the tissue specificity of alternatively spliced genes.Overall, our study provides quantitative evidence for the effect of alternative splicing on protein structure and sheds light on how alternative splicing might regulate transcription factor function in a tissue-specific manner. This, in turn, reveals the contribution of alternative splicing to regulation of gene expression via tissue-specific TF isoforms. The work described here implies that future high-throughput screens of gene expression analyses should be sensitive to multiple alternatively spliced forms of TFs. Because gene-expression arrays are intended to measure transcription, the next generation of arrays should contain probes specific to all known isoforms of genes represented on the arrays. Given that alternatively spliced exons are highly conserved across species ,22, it wMus musculus entries. All expressed sequences were mapped to a region of the University of California Santa Cruz (UCSC) February 2003 version mm3 of the mouse genome assembly using BLAT [For this analysis, we constructed a database of alternatively spliced mouse transcripts called MouSDB3 , using ting BLAT . BLAT toing BLAT . This waing BLAT  version We identified in-frame cassette exons and extracted from MouSDB3 nucleotide and amino-acid sequences for transcripts containing these exons. The selected amino-acid sequences were then analyzed with SMART ,30 to coFrom MouSDB3, we then extracted library information for the transcripts within clusters and their annotations. We used these data to compute the tissue distribution of variant transcripts as reported in Results. All scripts and README files used to carry out this data-gathering process are available upon request from the Laboratory of Computational Genomics of The Rockefeller University.


Pharmaceutical company representatives (PCRs) influence the prescribing habits and professional behaviour of physicians. However, the skills for interacting with PCRs are not taught in the traditional medical school curriculum. We examined whether an innovative, mandatory workshop for third year medical students had immediate effects on knowledge and attitudes regarding interactions with PCRs.Surveys issued before and after the workshop intervention solicited opinions  from third year students (n = 75) about the degree of bias in PCR information, the influence of PCRs on prescribing habits, the acceptability of specific gifts, and the educational value of PCR information for both practicing physicians and students. Two faculty members and one PCR led the workshop, which highlighted typical physician-PCR interactions, the use of samples and gifts, the validity and legal boundaries of PCR information, and associated ethical issues. Role plays with the PCR demonstrated appropriate and inappropriate strategies for interacting with PCRs.The majority of third year students  had experienced more than three personal conversations with a PCR about a drug product since starting medical school. Five percent (4/75) claimed no previous personal experience with PCRs. Most students  were not aware of available guidelines regarding PCR interactions. Twenty-eight percent of students (21/75) thought that none of the named activities/gifts  should be restricted, while 24.0% (8/75) thought that students should be restricted only from sporting events. The perceived educational value of PCR information to both practicing physicians and students increased after the workshop intervention from 17.7% to 43.2% , and 22.1% to 40.5% (p = .0007), respectively. Student perceptions of the degree of bias of PCR information decreased from 84.1% to 72.9% (p = .065), but the perceived degree of influence on prescribing increased (44.2% to 62.1% (p = .02)).Students have exposure to PCRs early in their medical training. A single workshop intervention may influence student attitudes toward interactions with PCRs. Students were more likely to acknowledge the educational value of PCR interactions and their impact on prescribing after the workshop intervention. Pharmaceutical company representatives (PCRs) influence the prescribing habits and professional behavior of physicians . DespitePhysicians in training may be particularly susceptible to marketing strategies from PCRs. Restricting interactions between physician in training and PCRs is one approach to eliminating adverse effects of contacts with PCRs . HoweverWe sought to examine whether a single workshop intervention had immediate effects on the attitudes of third year medical students regarding interactions with PCRs. The goal of the workshop was to increase the student's knowledge and awareness of ethical issues surrounding the PCR encounter, and to improve the students' interactions with representatives by fostering discussion on the profit motive in pharmaceutical marketing, PCR marketing techniques, and appropriate interactions with the PCR, issues necessary for critical thinking about the potential conflict of interest. Unique to our intervention was the participation of a PCR who role played a typical PCR encounter and who offered a perspective on marketing from the perspective of industry. Our findings have implications for institutions considering strategies for controlling PCR interactions and for medical educators seeking to develop curricula for marketing in medicine.During the ambulatory internal medicine clerkship of the third year medical school curriculum, students were required to attend a ninety minute workshop entitled "Appropriate Encounters with Pharmaceutical Representatives". These workshops took place three times during the calendar year 2001 for three different student groups. To the best of our knowledge, there were no other organized learning experiences in the medical school curriculum about interactions with pharmaceutical representatives, either before or during the study period. We did not seek approval by the institutional review board for ethical research practice at our institution, because at that time the study was conducted, approval of student education projects was considered unnecessary.Two faculty members interested in the subject , and a regional manager of pharmaceutical representatives from a major pharmaceutical company facilitated the ninety minute workshop. The workshop began by soliciting student opinions regarding the characteristics of typical interactions with PCRs. After a list of characteristics was compiled, each characteristic was discussed in more detail and compared with previous personal experiences with PCRs. Salient points of the subsequent discussion included the usefulness of patient assistance programs, the use of samples and gifts in PCR marketing strategies, the validity and legal boundaries of information provided by the PCR, and the ethical and legal aspects of physician-industry relations. The final segment of the workshop involved two student volunteers who role played a typical PCR encounter in the office. After discussion of the first role play, a second role play between one faculty member (CO) and the PCR demonstrated desirable characteristics of the PCR encounter.A pre-intervention survey handed out and collected prior to the beginning of the workshop solicited information about the number of previous personal experiences with PCRs, and whether the student was previously aware of guidelines  for appropriate interactions with PCRs. Using five point Likert scales, the survey solicited student attitudes about the educational value of PCR information for practicing physicians and for medical students, the degree of bias in PCR information, and the degree of influence of PCRs on prescribing habits. One additional question solicited the acceptability to students of specific gifts  from PCRs. A post-intervention survey with the same attitude questions was administered and collected as students left the workshop. The available data comes from three groups – the third, or last student group of academic year 2000–1 and the first two student groups of academic year 2001–2.Students were characterized by gender, age, and number of previous personal contacts with PCRs . For the purposes of understanding the Likert scale responses for student attitudes, we collapsed Likert scores into three categories – scale responses of 1 or 2 to signify disagreement, a scale response of 3 to signify neutral, and a response of 4 or 5 to signify agreement with the attitude question. We compared student attitudes toward the educational value of PCR detailing for medical students with the perceived value for practicing physicians using the Pearson chi square test.The association between previous personal PCR experience and attitudes about the educational value of PCR detailing was explored using analysis of variance. We also compared attitudes before and after the workshop intervention using the Pearson chi square test and a dichotomous variable a response of 4 or 5 (versus 3 or lower) on the Likert scale.A total of 75 students attended one of the three mandatory workshops on "Appropriate Encounters with PCRs". One student did not complete a post-intervention survey. The mean age of students was 26.4 (± 2.4), and males  outnumbered females. Fifty-six percent of students (42/75) had experienced more than three personal conversations about a pharmaceutical product with a PCR since starting medical school. Five percent (4/75) claimed no previous personal experience with PCRs. There was no association between the number of PCR contacts, and either gender, age or time of the academic year.The pre-intervention survey showed that PCR detailing was of educational value to 22.1% (18/75) of students with no perceived difference in educational value to medical students versus that to practicing physicians . While students agreed that the degree of bias from PCR information was substantial , only 44.0% (33/75) of students felt that pharmaceutical representatives were influential with regard to physicians' prescribing habits. No relationship between number of previous personal PCR contacts and educational value to medical students was demonstrated (ANOVA p = .08)Forty-three percent (32\75) of students reported awareness of available guidelines regarding PCR interactions. Fifty percent (16/32) of those students reported familiarity with medical school guidelines, 21.8% (7/32) with federal government guidelines, and 40.6% (13/32) with professional society guidelines. When asked which drug company sponsored activities/gifts targeting medical students should be restricted, 28.0% of students (21\75) thought that none of the named activities/gifts  should be restricted. Twenty-four percent (8/75) of students thought that only sporting events should be restricted.Figure Professional relationships with PCRs begin early in a physician's career. Because physicians typically underestimate the influence of pharmaceutical marketing on prescribing practices, countering this naivete early is warranted. Because students do not yet have prescribing privileges, the effect of PCR detailing on the prescribing decision is less relevant than how PCR contact shapes professional values . SeveralOur data show that students have early exposure to PCRs, perhaps earlier than previously suspected. Only five percent of third year students at this institution had not yet experienced PCR detailing, and many had experienced greater than six such encounters. Although resident physicians understandably have more PCR contacts, on average at least three PCR encounters per month ,16, to oIn this study few students were aware of existing guidelines for PCR interactions. Furthermore, students perceived information from PCRs to have educational value, and a value equivalent to that for practicing physicians. That students felt no more or less susceptible to marketing influence than practicing physicians is a marked contrast to the opinions of many educators that physicians in training need special protection from marketing influences ,2,17.Several interventions have been proposed for educating physicians-in-training about pharmaceutical marketing practices. Shaughnessy et al described a single brief seminar of marketing concepts followed by regular structured evaluation of PCR sales presentations throughout the following year . DespiteSimilar to these interventions, our workshop intervention targeting third year medical students was a one time intervention and brief in duration. Our intervention differed in that the workshop (1) took place during the clinical clerkship year, (2) made use of practicing PCRs and physicians, and (3) encouraged a distanced but amicable relationship with the PCR. In contrast to many educators who oppose PCR contact for trainees, we encouraged respect for the individual PCR and the success of the pharmaceutical company's business model. We were concerned enough about the growing influence of pharmaceutical marketing on trainees to stage this workshop, but we were careful to remember our goals of encouraging critical thinking about the topic rather than simply condemning the PCR contact. As an example, we emphasized the legal limits regarding what the PCR could and could not say to the physician. This may explain, in part, why attitudes toward PCR information improved after the intervention, at the same time that perceived degree of influence on prescribing increased. Teaching students the "rules of the game" in PCR encounters may explain why students thought that PCR information had more educational value after the workshop.The limitations of this study should be recognized. First, our survey took place at a single academic medical center affiliated with a private hospital. Student attitudes might be different at a state supported institution or at institutions where there are restrictions on the activities of PCRs. Second, students may have answered the post-intervention survey in a socially desirable manner. Respect for the PCR and the business model was presented as a balanced perspective toward marketing in medicine. Third, how immediate changes in student perceptions will ultimately translate into durable attitude changes and prescribing practices was not a goal of this study. We are not naïve enough to be certain that a one time intervention on any subject matter related to ethical issues will change student attitudes in a way that is durable. However, our goal as educators should be to make the students think critically, and demonstrating self-reported attitude changes is a necessary first step toward more durable change.Some academic institutions have chosen to ban PCRs from the academic learning environment. McCormick et al showed that restricting access to PCRs during residency training was associated with less informational dependence on the PCR and a decreased frequency of PCR contact after training . UnderlyThe challenge for medical educators is how to incorporate this increasingly important knowledge domain into training programs. What aspects of marketing strategy need to be taught and how? The growing emphasis on social justice and professionalism should encourage the appropriate distance from and respect for marketing pressures in medicine and add support for this curricular element in medical education . Our modMedical students have exposure to PCRs early in their medical education, at least in this setting. Students perceived information from PCRs to have moderate educational value, and a value equivalent to that for practicing physicians. A brief workshop intervention can have a measurable immediate effect on student attitudes.PCR – pharmaceutical company representativeThe author(s) declare that they have no competing interests.JW conceived of the study, participated in its design and coordination, and drafted the manuscript.CO participated in design and coordination of the study, conducted the intervention, participated in data interpretation, and manuscript revision.Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


This problem is particularly apparent in evaluating the developmental role of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the cystic fibrosis (CF) phenotype. A previously described transient in utero knockout (TIUKO) technology was used to address the developmental role of CFTR in the rat lung.Examination of late gestation developmental genes cftr in utero developed pathology that replicated aspects of the human CF phenotype. The TIUKO CF rat developed lung fibrosis, chronic inflammation, reactive airway disease, and the CF Antigen (MRP8/14), a marker for CF in human patients, was expressed.Rat fetuses transiently treated with antisense in utero antisense technology can be used to evaluate genes that exhibit either early lethality or compensating gene phenotypes. In the lung CFTR is part of a developmental cascade for normal secretory cell differentiation. Absence of CFTR results in a constitutive inflammatory process that is involved in some aspects of CF pathophysiology.The transient  These data provided the first insight that CFTR expression during the fetal period could permanently alter the differentiation of lung epithelial cells. The permanent functional changes in the in utero cftr-treated rats included an enhanced resistance to pulmonary bacterial infection three months after birth [The boratory  was origal lungs . At the al lungs . Adminiser birth .At the same time, other laboratories were examining the temporal and tissue-specific expression of CFTR. CFTR lung expression is greatest during the fetal period where it is localized to airway epithelial undifferentiated multipotential cells -9. As thIn addition to its role as a chloride channel in the mature lung, CFTR's expression in undifferentiated epithelial cells suggested another role (or roles) during development. Moreover, this raised the question of how much of CF disease pathology could be attributed specifically to the lack of CFTR expression during differentiation and how much could be attributed to lack of a chloride channel in the mature lung.cftr -/-) mouse following transient in utero expression of cftr [in utero gene therapy did not permanently replace the CFTR-encoded cAMP-dependent chloride channel but rescued the mice from the disease phenotype and reversed biochemical markers specific to the knockout phenotype [cftr -/- mice.These questions prompted further experiments by this laboratory in the CF knockout mouse. Reversal of the lethal phenotype of the CF  technology to inhibit expression of specific genes in the fetal lung and intestine [The al cells . Recombi monkeys ,14-16. Tntestine . In thiscftr included in the [in utero gene transfer into fetal rats at 16 days gestation.An adenovirus was constructed from a ATCC plasmid containing exons 1–6 of d in the  cloned iSprague-Dawley rats were used in these experiments. There were 75 rats from 7 litters in the control group and 114 rats from 11 litters in the TIUKO group. The rats were treated with AdCMVAScftr at 16 days gestation and were evaluated daily from 18–22 days gestation and up to 1 year following birth.cftr constructs nor any sense portion of this gene could be used as a control. An adenovirus with exons 1–6 in the sense direction would not express a truncated product and thus its expression could not be detected as a control. The adenovirus constructs with beta-galactosidase (AdCMVlacZ) and green fluorescent protein (AdCMVgfp) were used in previous experiments in several hundred individual fetuses with no effect on the viability, structure, or function of the lung [The choice of controls for these experiments was a primary consideration. We showed in several publications that over expression of CFTR in normal mice and rats results in altered lung morphology. Thus, neither the lung ,16. ThusThe effects of AdCMVAScftr on CFTR expression in rat tissues was compared to control (AdCMVlacZ-treated) lungs at 24 and 72 hours post antisense therapy by fluorescent immunohistochemistry. As shown in Fig. 8 cells would be affected if one achieved 100% infection efficiency. Transfection efficiency via the amniotic fluid is less than 10% so only between 106–7 cells are affected in the tissue. Thus, real time PCR, northern blots, and western blots lack the sensitivity to detect these changes as shown in our previous publication on the TIUKO c-myc mouse [Reduction of the antisense transgene expression is difficult to measure, because at a maximum only 10yc mouse . Thus, tcftr therapy. Thus, as in our previously published TIUKO c-myc model, the TIUKO cftr therapy decreases expression of the target gene at a critical time in lung development.Statistically significant reduction in CFTR levels was observed at 24 (p = 0.014) and 72 hours (p = 0.005) post-TIUKO Focal areas of fibrosis are seen in the lungs of congenic CF mice as well as humans at autopsy . Rats wein utero antisense cftr treatment.Comparison of the histology of lungs from control and AdCMVAScftr-treated animals during the neonatal period revealed little or no gross structural pathology (data not shown). Development of pulmonary histopathology became apparent in adult rats by 100 days of age following the in utero antisense cftr treated animals as compared to their aged-matched controls. The average internal airway circumference was not statistically different between the treated and control group. These measurements insured that similar sized airways were compared between the two groups.The most notable histologic change was in the airways, which appeared thickened and fibrosis. Morphometric analysis was used to quantitate airway wall dimensions on lung sections from 100 day old rats following staining with hematoxylin and eosin ,21. The Masson's Trichrome was used to differentiate collagen from smooth muscle and elastin surrounding the airways to better visualize and quantitate the extent of airway fibrosis. Collagen following this stain was visualized as a dense bluish-tinged material as shown surrounding the membranous airways in Figure in utero adenoviral-mediated transgene expression decreases rapidly in the 30 days post-transfer [cftr expression. In addition, although these animals were not kept in a germ free environment, repeated bacterial challenge was not required for the induction of either lung inflammation or fibrosis. All animals greater than 60 days of age thus far examined (n = 12) have had significant pulmonary inflammatory infiltrate.Chronic inflammation is another feature of CF lung pathology in humans . At 100 transfer . Thus, tcftr gene. Because of their significance the expression levels of these proteins were confirmed by western blot analysis in both mice and rats following AdCMVAScftr gene therapy.MRP8 and 14 are proteins previously used as clinical markers of cystic fibrosis. These proteins are calcium binding proteins that form a heterodimer, are produced in neutrophils, and are associated with wound healing. Importantly, MRP8 was originally called "CF antigen" because it was found to be elevated in the serum of CF patients. The protein was subsequently found to be a heterodimer of MRP8 and MRP14. It was used to identify CF affected individuals as well as heterozygous carriers prior to the discovery of the cftr therapy in utero . Minimally detected levels of MRP 8 were expressed in control fetuses . Thus, increased CF Antigen expression was correlated with the decreased expression of CFTR following antisense cftr gene therapy.To determine if MRP8/14 expression was directly associated with the inhibition of CFTR expression, and not induced by post-natal events, western blot analysis of expression was followed sequentially over the first 96 hours post antisense in utero antisense cftr on the airway development rats treated at 16 days gestation with AdCMVAScftr or AdCMVlacZ were maintained in filtered cages and analyzed for airway reactivity to acetylcholine at 6–13 months of age. As shown in Fig. Raw) at 3.125 and 12.5 mg/ml concentrations. In contrast, age-matched, antisense cftr treated animals were highly reactive to the low concentrations of acetylcholine. In addition, maximal stimulation at 50 mg/ml in the TIUKO CF rats was over twice that observed in control animals. The differences between control and TIUKO CF rats was highly significant (p < 0.0001)In cystic fibrosis patients, alteration in airway reactivity was previously documented ,25. To ec-myc [c-myc gave rise to severely hypoplastic lungs and stunted villi formation in the intestines, even though fewer than 107 cells were affected by the antisense transgene. This method can be used to dissect the developmental pathway of different epithelial cell types in these organs.The development of the TIUKO procedure permits the examination of mid-gestation developmentally required genes in the absence of both early lethality and compensatory mechanisms that ameliorate the final disease phenotype. Previously, transient expression of the antisense to a known growth factor c-myc  demonstrin utero .One caveat with the TIUKO method, however, is the difficulty in measuring the decrease in expression of the target gene. Because the population of affected multipotential, undifferentiated cells represent a small proportion of the total, rapidly expanding lung population, it is impossible to detect the changes in gene expression via real time PCR, northern blots, or western blots. However, as shown previously two independent methods, antisense and ubiquitin targeted, down regulation of C-MYC  yielded in utero cftr gene therapy using a recombinant adenovirus [Cystic fibrosis is a pleiotropic disease. The seemingly unrelated phenotypic effects of CFTR are largely unexplained by the hypothesis that CF pathology results from the lack of continuous chloride channel expression. Beginning with the reversal of the CF knockout mouse phenotype with transient enovirus , this lain utero gene therapy and reversal of the knockout mouse phenotype [As shown recently , CFTR ishenotype ,13. Thes7) of undifferentiated multipotential cells was performed in using a recombinant adenovirus with a cftr fragment cloned in the 3'-5', antisense, direction. As shown in immunohistochemical examinations of lung tissues  was cloned in 1987 by Dorin et al. [cftr gene was cloned and confirmed to be a chloride channel and also when MRP 8 expression was not found in the preliminary survey of adult and fetal CF lung [cftr gene transfer  and CFTR deficient . Thus, as shown in this paper, the TUIKO CF rat exhibited a CF-related phenotype while a CFTR heterozygous does not show any CF features.Until recently, no distinctive changes in lung structure and function were found in the CFTR knockout mouse. However, recent evaluation by our laboratory of lung function in genotype . Thus, ncftr mutation, or as suggested by Groman et al's [We propose that CFTR is part of a developmental cascade for secretory cells in the lung, intestines, pancreas and other secretory organs Fig. . Disrupt et al's  work, otTransient inhibition of CFTR expression in the lungs results in many features of cystic fibrosis in the mature animal. Increased fibrosis, chronic inflammation, increased airway reactivity, and elevation of CF antigen were observed. These data are consistent with a CFTR requirement for normal lung development.E. coli strain BJ5183, according to the protocol of He et al [lacZ (AdCMVlacZ) and green fluorescent protein  were provided by Dr. J. Kolls . All viruses were CsCl or HPLC purified.A 920 bp human CFTR cDNA that included exons 1–6  was gelHe et al . Recombi8 pfu/ml to the amniotic fluid.Timed pregnant Sprague-Dawley Rats were induced (5%) and sedated (2%) with inhaled Isoflurane. A laparotomy was performed exposing the uterine horns. The individual amniotic sacs of the fetuses were visualized and injected with fine gauge needle containing adenoviral particles in 10% of the amniotic fluid volume. The recombinant adenoviruses in Dulbecco's Minimal Essential Medium delivered final concentrations of 10At the time of sacrifice all animals received a number. This code was used for identification of all histologic and biochemical studies.All tissues were fixed in methanol-free, 4% buffered paraformaldehyde and either mounted in paraffin or OCT for sectioning. Fluorescent immunohistochemistry was performed with goat polyclonal IgG (Santa Cruz) specific for CFTR carboxy (sc-8911) and amino terminal (sc-8909) sequences. Secondary donkey anti-goat ALEXA (Molecular Probes) antibodies were used. All tissues were visualized on a deconvoluting, Lieca, light microscope. Hematoxylin and eosin stain and Masson's Trichrome stain were performed with kits  and tissues examined by standard light microscopy.Morphometry was performed with the identification numbers and treatment groups unidentified by two blinded investigators. Airway thickness was determined following staining with hematoxylin and eosin in 100 day old rats. The area of the wall between the sub epithelial basement membrane and parenchymal epithelium was digitized excluding airway epithelium and cartilage. The corresponding segment of sub epithelial basement membrane was digitized and used as a reference length to normalize airway wall area . QuantitImage analysis based morphometry was performed with the identification numbers and treatment groups unidentified. Morphometric analysis was performed by two blinded investigators. Digitalized images were analyzed for airway thickness using Scion Image ,14 or foPolyacrylamide gel electrophoresis was performed on 18% Tris-HCl gels (Biorad) and transferred to PVDF membranes (Amersham) ,35. PolyflexiVent, SCIREQ Inc. Montreal, PQ, Canada) and ventilated with a tidal volume (Vt) of 10 ml/kg; inspiratory:expiratory ratio (I:E) of 66.67%, respiratory rate of 150 breaths/minute, and maximum pressure of 30 cmH20. Positive end-expiratory pressure (PEEP) was controlled by submerging the expiratory limb from the ventilator into a water trap. Each animal was paralyzed with pancuronium bromide (0.5 mg/kg) and allowed to equilibrate on the ventilator until spontaneous breathing ceased (5 minutes). Zrs measurements at a PEEP level of 3. Data were statistically evaluated using paired t-test.Rats at 12–14 months of age were anesthetized with intra-peritoneal pentobarbital (90 mg/kg), and the trachea was dissected free of surrounding tissue and cannulated with a 20-gauge cannula. The rat was then connected to a small animal ventilator (Zrs), mechanical ventilation was interrupted and the animal was allowed to expire against the set level of PEEP for 1 s. We then applied an 8 second broad-band volume perturbation signal was then applied to the lungs with the flexiVent, after which ventilated was resumed. A PEEP of 3 cmH2O was used. The volume perturbation signal consisted of the superposition of 18 sine waves having frequency spaced roughly evenly over the range 0.25 Hz to 19.625 Hz. Zrs was calculated from the displacement of the ventilator's piston and the pressure in its cylinder as described previously [flexiVent, connecting tubing and the tracheal cannula were performed as described previously [To measure the input impedance of the respiratory system , i is the imaginary unit, α links G and H, and f is frequency. We also calculated a quantity known as hysteresivity (η = G/H), which is believed to increase when regional heterogeneities develop in the lung [where  airways , Iaw is the lung .Raw was calculated for each perturbation.After rats were equilibrated on the respiratory, sequential 30 second challenges with nebulized physiologic saline, 3.125, 12.5 and 50 mg/ml acetylmethylcholine dissolved in physiologic saline were performed. Between each challenge, 18 broad-band volume perturbations were produced by the ventilator at 10 second intervals between each perturbation. CFTR – Cystic fibrosis transmembrane conductance regulator; CF – cystic fibrosisBoth authors were equally responsible for both the laboratory work and design of experiments


Pneumonia remains the leading cause of death worldwide in children. Several vaccines against pneumococcal pneumonia are at various stages of development, but the testing of their efficacy is hampered by the lack of noninvasive tests that are sensitive and specific for the disease. Diagnosis is usually based on chest radiographs, which are not very specific for pneumococcal disease.In their quest for a more specific diagnostic test, Shabir Madhi and colleagues—who are conducting clinical trials on pneumococcal vaccines in children—examined whether serum concentrations of procalcitonin and C-reactive protein could improve the specificity of chest radiographs to diagnose pneumococcal pneumonia and thus be useful in the future evaluation of pneumococcal vaccines. Elevated levels of both proteins are associated with bacterial disease. They might therefore help to differentiate bacterial from nonbacterial causes of pneumonia, and thus allow to “enrich” the analyzed disease cases for those of pneumococcal origin, against which the vaccine is potentially active.This study represents a first step, in which the researchers tested whether adding information about procalcitonin and C-reactive protein levels to data from a completed vaccine trial would affect the outcome regarding vaccine efficacy. When reanalyzing previous trial data under these conditions, the vaccine appeared more efficacious compared with placebo when either elevated procalcitonin or elevated C-reactive protein levels were taken into account. The efficacy estimate was greatest when cases of pneumonia that had elevated levels of both procalcitonin and C-reactive protein were compared against placebo.These data suggest that elevated levels of C-reactive protein and procalcitonin, in conjunction with chest radiography, could improve the specificity of a diagnosis of pneumococcal pneumonia over that of chest radiography alone. This combined diagnostic test could be useful for further evaluation of pneumococcal vaccines. The hope is that among patients identified as having pneumonia by the combined test, a higher proportion would have pneumonia of pneumococcal origin. As a consequence, there would be less “background noise” caused by other forms of pneumonia, and this should make it easier to assess the efficacy of vaccine candidates. However, as the researchers point out, this analysis was not a primary objective of the present trial. This analysis can therefore serve only as a hypothesis-generating study, and as such the hypothesis must be tested in other study settings.The study was sponsored by Wyeth, manufacturers of the pneumococcal vaccine used.


RUNX gene family), but only one in other chordates. Therefore, the common ancestor of vertebrates has been thought to have had a single runt domain gene.The runt domain transcription factors are key regulators of developmental processes in bilaterians, involved both in cell proliferation and differentiation, and their disruption usually leads to disease. Three runt domain genes have been described in each vertebrate genome (the Takifugu rubripes) reveals the existence of a fourth runt domain gene, FrRUNT, in addition to the orthologs of human RUNX1, RUNX2 and RUNX3. The tiny FrRUNT packs six exons and two putative promoters in just 3 kb of genomic sequence. The first exon is located within an intron of FrSUPT3H, the ortholog of human SUPT3H, and the first exon of FrSUPT3H resides within the first intron of FrRUNT. The two gene structures are therefore "interlocked". In the human genome, SUPT3H is instead interlocked with RUNX2. FrRUNT has no detectable ortholog in the genomes of mammals, birds or amphibians. We consider alternative explanations for an apparent contradiction between the phylogenetic data and the comparison of the genomic neighborhoods of human and fugu runt domain genes. We hypothesize that an ancient RUNT locus was lost in the tetrapod lineage, together with FrFSTL6, a member of a novel family of follistatin-like genes.Analysis of the genome draft of the fugu pufferfish (Our results suggest that the runt domain family may have started expanding in chordates much earlier than previously thought, and exemplify the importance of detailed analysis of whole-genome draft sequence to provide new insights into gene evolution. Drosophila segmentation gene runt over a decade ago [Since the initial description of the cade ago , a smallcade ago .RUNX1, RUNX2 and RUNX3. RUNX genes have two promoters  [In humans, three different RD genes were identified  and namectively) -7 separactively) . Extensictively) -11.RUNX genes were identified in mouse. A single RD gene was described in Xenopus, presumed to be orthologous to RUNX1 [RUNX2, and suggested the existence of a single additional RD gene in fugu [RUNX genes [Drosophila [C. elegans [RUNX gene complement. The proposed mechanism of expansion involved large-scale genomic duplications, identifiable today as large paralogous segments [RUNX genes, but their functional relationship to the ancestral RD gene is unknown. The single known RD gene in C. elegans has been shown to be required for the formation of a functional gut; this role has been claimed to be conserved with mouse Runx3 [Orthologs of all three human to RUNX1 . An expe in fugu , while aNX genes . Four RDosophila ,15, whil elegans , sea urc elegans . Based o elegans , and thisegments . The prose Runx3 .Homo sapiens, Mus musculus, Rattus norvegicus, Canis familiaris, Gallus gallus, Takifugu rubripes, Tetraodon nigroviridis, Danio rerio and Xenopus tropicalis, allows us to explore a comprehensive set of vertebrate RD genes and characterize their genomic environments, shedding light on the structure and evolution of this important gene family.The current availability of genome drafts for several vertebrate species, including Our search for RD genes in the fugu draft yielded four distinct genomic scaffolds Fig. , each coRUNX genes . They have been assumed to be their orthologs [FrRUNX1, FrRUNX2 and FrRUNX3  and first exon from the main body of the gene. This intron is usually very long in RUNX genes. It is indeed the longest intron observed in FrRUNT, but it is nevertheless very short, spanning just 1372 bp. There is a local concentration of CpG dinucleotides 200–300 bp upstream of exon 2  has an insertion at the same position . Comparison to the published structure of this domain [FrRUNT is exceptional in that the length of the runt domain (131 residues) varies from the universally conserved 130 amino acids, due to the introduction of an asparagine residue after position 47 in the RD Fig. . This apFrRUNT is "interlocked" with FrSUPT3H . The three RUNX gene pairs are conserved between fugu and Tetraodon . In this analysis, the FrRUNT protein is nearly equidistant from the three RUNX proteins and amphioxus RUNT , we reconstructed a molecular tree  and cephalochordates (including amphioxus). On the other hand, when comparing the nucleotide sequences of the first exon from each human and fugu runt domain gene, we observed that We also studied the relationships between the RUNX1, RUNX2 and RUNX3 orthologs in several vertebrates and found the species trees to be largely as expected Fig. , RUNX2A Drosophila [RUNX genes: they represent an independent family expansion in insects. The four Drosophila RD genes are all linked on chromosome X. Moreover, three of these genes are clustered within a 150 kb region. There is no linkage of RD genes, however, in the human genome: each gene is on a different chromosome. Their genomic environments usually show some conservation: the three human RUNX genes are followed by CLIC genes in the complementary strand  and ENPP4. The presence of remnants of SUPT3H upstream of FrRUNX2 [RUNX2 in zebrafish, lends further support to this hypothesis. Thus, comparative genomic analysis of human, fugu and zebrafish suggests that FrRUNT may be a derivative form of a duplicated RUNX2 gene. However, this contradicts the conclusions from phylogenetic analysis or the protein sequences; we discuss this contradiction below.An exception to the conservation of synteny involves the genes  FrRUNX2 , and of FrRUNT. Immediately downstream to FrRUNT we identified a novel gene (FrFSTL6) from the follistatin family, most closely related to FSTL1. A detailed computational search for additional sequences of this family identified two novel, large human follistatin-like genes, which we called FSTL4 and FSTL5. We then found clear orthologs for both in the fugu genome . The genes in this family share a Kazal-type cysteine-rich domain  have four RD genes; since these genomes are only available as draft assemblies, additional RD genes might be found when the finished genomic sequences are made available.We have taken advantage of the availability of genomic drafts for several vertebrate species, including the finished human genome, to identify the orthologs of all currently known runt-domain (RD) genes, as well as a novel member of this small gene family. Both pufferfish species  runt domain itself has diverged at such an accelerated pace. Furthermore, we found this not to be an artifact of mutational saturation [FSTL6 gene: parsimony considerations could lead one to assume that FSTL6 is a fish-specific duplicate of FSTL1, though contradicting the phylogenetic reconstruction of the evolution of this gene family  became interlocked with SUPT3H. After the teleost/tetrapod divergence, a regional duplication in teleosts created a second copy of RUNX2 and its neighboring genes. In the tetrapod lineage, the ancestral RUNT gene was lost, in conjunction with FSTL6. In the pufferfish lineage, the RUNT gene replaced most of RUNX2A, perhaps by recombination  have a second RD gene, short and divergent enough to escape experimental detection by DNA hybridization [RUNX genes bind to the same DNA motif and modify the expression of target genes through recruitment of transcriptional modulators, which are also shared [RUNX genes are attained by way of tightly regulated spatiotemporal expression patterns. We hypothesize that the ancestral RUNT gene became inessential to amniotes by functional reprogramming of the remaining three RUNX genes. Its loss would therefore represent an example of evolution by reduction in complexity. In pufferfishes, the hypothesized recombination event would have placed the RUNT gene under the regulatory control of the former RUNX2A promoter. The viability of such a sudden regulatory change would in turn suggest a significant level of functional redundancy among the RD genes.Under the proposed scenario, none of the three extant dization . Why waso shared ; functioRUNX2, or a direct descendant of the ancestral chordate RUNT gene. We find the latter hypothesis more reasonable. This novel gene evolved in parallel with the vertebrate RUNX genes, and while it has been preserved in pufferfishes, it appears to have been lost entirely in tetrapods. This suggests that the ancestral vertebrate was more complex than previously suspected.We identified a fourth runt domain gene in the fugu genome, which appears to represent either a pufferfish-specific, fast-evolving derivative of FrFSTL6), we have identified a new family of follistatin-like genes in the human genome. Phylogenetic analysis of the RD protein sequences led to results that contradict those derived from comparative genomics, but we showed that the two could be reconciled into a coherent evolutionary model. These results underscore the importance of obtaining complete genomic sequences of strongly divergent vertebrates, and the value to be derived by performing detailed and integrated analyses of their gene complements.By studying a very limited set of fugu genomic regions, namely the scaffolds related to RD genes, we have identified seven apparently functional fugu genes that are absent from the human genome Fig. , and werTakifugu rubripes "assembly3" genome draft [FrRUNX3. No further evidence was found for an additional RUNX3 gene: we conclude that scaffold 25789 is an assembly artifact. We similarly searched the genome drafts for Tetraodon nigroviridis produced by the Whitehead Institute and the Genoscope [Danio rerio  as queries in a TFASTY  search ime draft  releasedme draft . These denoscope , and Dannstitute . We analnstitute ,34, and Ciona intestinalis [In a second round of analysis, we used the newly identified RD genes as queries for renewed TFASTY searches of the genome drafts of human (July 03), mouse (February 03), Xenopus   and sequenced directly using Applied Biosystems Big dye terminator kit reagents. Whenever necessary, additional pairs of primers were designed for oligonucleotide-directed sequencing to close gaps. Shotgun sequencing data was obtained from the BAC clone OML73850 (b193C08) for part of scaffold 682. OML73850 was fragmented by sonication, end-repaired and electrophoresed to select insert size of 2–5 kb. Insert was ligated into pUC18 vector, transformed and plasmid DNA was made using Eppendorf – 5 Prime PERFECTprep robot and sequenced from both ends. Assembly was carried out using Phrap [Large insert clones spanning the gaps in scaffolds from the version 3 assembly were identified by BLAST searches against the BAC/cosmid database in the v.3.0 JGI website . CosmidsC. intestinalis) RUNT, Oikopleura dioica RUNT (AAS21356), amphioxus (B. floridae) RUNT (AY146617), sea urchin S. purpuratus RUNT (NP_999779) and the four Drosophila melanogaster RD sequences . The tree was rooted using the C. elegans RUN protein (AB027412) as outgroup. We further excluded the first thirteen amino acids of the runt domain, to avoid the topological distortion expected in this region from the highly divergent pufferfish RUNT sequences. For the separate phylogenetic trees of the three RUNX genes, we used the complete protein sequences, with gap opening and extension penalties of 5 and 0.1, respectively. ASaturA analyses were performed using PAM250, Kimura's correction and a cutoff value of 9, with 1000 rounds of bootstrap. The first exons of human and fugu runt domain genes were compared at the nucleotide level. For each exon, we selected the range from 30 nucleotides upstream of the ATG codon, to 15 downstream of the splicing donor site, i.e. 103 nucleotides for each RUNX gene and 94 nucleotides for FrRUNT.The sequences were aligned using ClustalW . PhylogeGG conceived of the study, performed the bioinformatics analyses and prepared the manuscript. AK generated the sequence data required for finishing. LH, the laboratory director, provided guidance and contributed to the preparation of the manuscript. LR supervised the Institute for Systems Biology's contributions to the Pufferfish Finishing Consortium and contributed to the preparation of the manuscript. All authors read and approved the final manuscript.


We describe a system of web applications designed to streamline the interdisciplinary collaboration in outcomes research.The outcomes research process can be described as a set of three interrelated phases: design and selection of data sources, analysis, and output. Each of these phases has inherent challenges that can be addressed by a group of five web applications developed by our group. QuestForm allows for the formulation of relevant and well-structured outcomes research questions; Research Manager facilitates the project management and electronic file exchange among researchers; Analysis Charts facilitate the communication of complex statistical techniques to clinicians with varying previous levels of statistical knowledge; Literature Matrices improve the efficiency of literature reviews. An outcomes research question is used to illustrate the use of the system.The system presents an alternative to streamline the interdisciplinary collaboration of clinicians, statisticians, programmers, and graduate students. In the last decade, the number of relevant data sources available for outcomes research has grown exponentially. In contrast, the number of individual researchers with clinical and statistical expertise required to explore these data sets increase at a much slower pace. As a result, an immense quantity of valuable clinical data are left untouched, never becoming clinical publications that could potentially improve health care.The disproportion between data volume and number of qualified researchers can be explained by the growing complexity involved in outcomes research projects using secondary data analyses. Researchers have to formulate of a clinically relevant and methodologically sound research question, find appropriate data sources, perform statistical analyses, and generate a final manuscript that will be submitted for peer-review. Frequently, individual researchers have the training and time to perform a few of these steps, but the integration of all tasks calls for an interdisciplinary systems approach ,2. This This article describes a suite of web applications developed to facilitate the process of converting outcome databases into clinical manuscripts, to streamline the interdisciplinary collaboration of researchers, and to connect all different steps of the outcomes research process. To illustrate its use, we will describe how a research project has been conducted using this system from its early phase of research question formulation to the completion of the final manuscript.. The following sections will describe each of the Web applications and their application in the answer of a real outcomes research question.The system of Web applications is composed by five different tools: QuestForm, Research Manager, Analysis Charts, and Literature Matrices. These tools were designed to assist researchers in each of the phases encountered in an outcomes research project involving secondary data analysis Figure . All too for an updated list). All databases have been de-identified and do not contain protected health information as specified by the Health Insurance Portability and Accountability Act . The application The application is built using Extensible HyperText Markup Language 1.0 (XHTML) [QuestForm, an acronym for "Question Formulation", is an application designed to assist researchers in the location of clinical databases and formulation of outcome research questions Figure . Clinica (XHTML) .QuestForm starts by presenting researchers with three main strategies to find research databases: use of pre-determined key words that describe the database as a unit Figure , user-deDr. Guller initiated the project searching for an existing database that would allow him to compare surgical outcomes between laparoscopic and open appendectomy procedures in the treatment of acute appendicitis. The outcomes were pre-specified as mortality and infection, although other existing outcomes would also be of interest. Although there are multiple single-institution studies attempting to answer this question ,7, few sAs a first step, Dr. Guller searched across previously formulated Question Diagrams to evaluate whether other studies could have used a similar research design. Since none was found in QuestForm, Dr. Guller searched across more than forty different databases for an existing database that would have the variables to answer his research question. After navigating through multiple data dictionaries, Dr. Guller found that the Nationwide Inpatient Sample (NIS) Release 6, 1997  presenteOnce the database was located, Dr. Guller selected the outcomes of interest , main predictors (laparoscopic versus open procedures), and confounders , inserting each of them into the research question fields in QuestForm. Using built-in search engines for ICD9 codes, Dr. Guller created the definition for each of the above-mentioned variables and defined the inclusion and exclusion criteria. The final research question was then saved as a Question Diagram and immediately submitted to Dr. Pietrobon for feasibility evaluation. Dr. Pietrobon judged that the project was feasible and could be completed using the database indicated by Dr. Guller. At this point, the project was initiated and a detailed project management plan was established using Research Manager.Research Manager is a Web application developed by our group designed to facilitate the project management of clinical research projects. Similar to QuestForm, Research Manager is licensed under the GNU Public License , which aResearch Manager provides multiple features to facilitate project management of clinical research projects. All projects are displayed by category  with a brief description. The internal content of all projects is password protected. All internal tasks within a project are assigned to individual researchers. Project administrators initially assign deadlines that can be modified by task leaders within three days. All participating members of the project receive weekly reports containing details about the activity and the latest electronic file within each task Figure . These fResearch Manager helps identifying such problems and enables their early elimination, thus avoiding delays in the project completion. Finally, since weekly reports are generated to all participating members, Research Manager also provides peer-incentive for project members to complete their tasks in a timely manner.Once the Question Diagram was evaluated by the clinical epidemiologist, this project was transferred to Research Manager. Contact information for each of the researchers involved and deadlines for completion of the main phases of the research process were set, including data extraction and cleaning, data analysis, literature review, and manuscript writing. Weekly reports were generated to update investigators on all tasks of the project, including different versions of the statistical analysis, modifications in the research question, and manuscript sections.With an established project plan, the statistician in charge selected the best methods for analysis. Since the database is a random sample of the United States and requires special survey analysis methods, it was necessary that all involved researchers understood the statistical approach by using Analysis Charts.Analysis Chart is a tool designed to enhance the understanding of statistical methods to a format that is understandable by clinical researchers with different previous levels of statistical knowledge. As such, it is important in the design as well as the analysis phases of a project Figure . The applayers of information. Layer 1 summarizes the general goal of the method. Layer 2 presents previous clinical applications so that the researcher can visualize situations in which the method may be realistically applied. Layer 3 describes the data requirements for the application of the statistical technique. Layer 4 describes the basic statistical underpinnings of the method, initially breaking down equations and then reassembling them. Finally, layer 5 presents a list of available software packages for the implementation of the technique as well as cases studies where all previous layers are applied to real data sets. Each layer ends with a section containing selected references that explain the topic in more detail.Analysis charts are composed by cascading links that display information about quantitative methods in progressive levels of complexity called "layers of information". Each layer explains the statistical method with an increasing level of complexity. In this manner, clinicians interested in simply understanding the method to evaluate whether it can be applied to a research project can simply read the first three layers. In contrast, researchers interested in a direct application of the method to available research data can follow all layers and their respective references. Most commonly, complex techniques are presented using five While deciding on the most appropriate analysis strategy for the Question Diagram, Drs. Pietrobon and Guller consulted the Analysis Chart searching for the most appropriate statistical methods of analysis. Given the nature of the research question and that the NIS database has a sample design, Drs. Guller and Pietrobon opted for an approach involving multiple and logistic regression models while adjusting for sampling weights, strata, and clusters. With a defined analysis protocol, the research question was then transferred to a statistical programmer trained in the translation of Question Diagrams into statistical code. This process was closely evaluated by Drs. Pietrobon and Guller, who scrutinized the statistical code and results from a clinical and statistical perspectives. Once the results were deemed to be accurate, Dr. Pietrobon started the literature review using a Literature Matrix.® or Open Office Calc [Literature matrices consist of a comprehensive but not necessarily exhaustive review of the literature focused on a narrow clinical topic Figure . Each arice Calc .The advantage of the Literature Matrix as implemented in the Web suite is its availability over the web to the whole outcomes research community. This allows Literature Matrices to be constantly updated, with authors receiving their due credit in a list of contributors. Literature Matrices also enable researchers to obtain a complete summary of the literature without going through the cumbersome process of copying and reading a manuscript for the first time. In contrast, researchers' time can be spent more efficiently in reviewing what has already been compiled and attempting to expand the Literature Matrix with other relevant bibliographic references.In order to evaluate the literature, a graduate student performed a thorough literature search. All relevant articles were copied, read, and the data extracted according to the established categories. Once the Literature Matrix had been completed, Drs. Guller compared the current project results with results published in the literature. The structured information in Literature Matrices also allowed Dr. Guller to compare the strengths and weaknesses of the current project in relation to previous publications. Once this phase was completed, Dr. Guller proceeded to the final writing of the manuscript using Output Templates.At the end of the project, Dr. Guller combined the Question Diagram, Analysis Chart, and Literature Matrices to write the final manuscript. While Analysis Charts provided the information concerning the statistical techniques used in this study, Literature Matrices provided the basis for comparison of the study results against previous publications. Although not included in the final manuscript, Dr. Guller also had access to multiple analysis files through Research Manager to orient him in each of the steps taken during the research question formulation, data analysis, and literature review.The use of web applications by individual clinical researchers can be summarized in Figure Since the concept of streamlining the interdisciplinary collaboration preceded the existence of the current suite of web applications, different versions of the central idea have been gradually applied since the second half of 2002. Although our usability, qualitative, and economic studies to evaluate this application are still ongoing, we have noticed a significant improvement in the number and quality of our publications as evidenced by the increasing acceptance rates of our manuscripts for publication in peer-reviewed journals. The Web applications are currently used in research projects involving Duke University and other universities in the United States and abroad, where they have been shown to facilitate inter-institutional collaborations.Improving the efficiency of an interdisciplinary approach to secondary data analyses has multiple potential benefits. These include an increase in the overall clinical significance of the final publication, decrease in the number of failed projects, decrease in the time for completion of individual projects, improvement in the education of future outcomes researchers, decrease in the cost-benefit ratio for individual outcomes research projects, and, perhaps most importantly, an automation of repetitive tasks. This last factor is crucial since eliminating repetitive tasks will allow researchers to concentrate on the design of innovative projects .Surprisingly, few systems and applications have been described to solve the problem of complex interdisciplinary collaboration between clinicians and statisticians. Isolated approaches have usually focused on specific portions of the outcomes research process, without attempting to integrate them into a cohesive system. For example, Marshall  has propAlthough this newly developed system of applications provides a significant improvement in the way secondary data analyses are conducted, it still has limitations. First, because of the lack of a formal evaluation of the effectiveness of this system, we are unable to quantify its real time and cost saving benefits. Second, the system is currently restricted to secondary analyses and does not allow for the planning of prospective data collection. Although one of the main advantages of formulating a research question based on existing data sets is the bounded nature of the process, future applications should attempt to create rules and algorithms that may guide prospective data collection.In summary, we have experienced that this system has significant advantages over the traditional manner of conducting outcomes research based on secondary data analyses. This tool may have important applications, not only resulting in an improvement in the overall efficiency of the outcomes research process, but also affecting the way new outcomes researchers are trained and introduced to a research environment.The Web application is available at XHTML Extensible HyperText Markup Language 1.0JAVA: by Sun MicrosystemsICD9-CM: International Classification of Diseases, Ninth Revision – Clinical ModificationNIS: Nationwide Inpatient SampleGNU: GNU's Not linux General Public LicenseWebMIRS: Web-based Medical Information Retrieval SystemNHANES: National Health and Examination SurveyUR: Uniform ResourceThe author(s) declare that they have no competing interestsRicardo Pietrobon – design, manuscript draftingUlrich Guller – design, manuscript revision for important intellectual contentHenrique Martins – design, manuscript revision for important intellectual content, software programmingAndreia P Menezes – design, manuscript revision for important intellectual contentLaurence D. Higgins – design, manuscript revision for important intellectual contentDanny O. Jacobs – design, manuscript revision for important intellectual contentThe pre-publication history for this paper can be accessed here:


In animal studies of the effects of hormonally active agents, measurement of anogenital distance (AGD) is now routine, and serves as a bioassay of fetal androgen action. Although measurement of AGD in humans has been discussed in the literature, to our knowledge it has been measured formally in only two descriptive studies of females. Because AGD has been an easy-to-measure, sensitive outcome in animals studies, we developed and implemented an anthropometric protocol for measurement of AGD in human males as well as females.We first evaluated the reliability of the AGD measures in 20 subjects. Then measurements were taken on an additional 87 newborns . All subjects were from Morelos, Mexico.The reliability (Pearson r) of the AGD measure was, for females 0.50, and for males, 0.64. The between-subject variation in AGD, however, was much greater than the variation due to measurement error. The AGD measure was about two-fold greater in males  than in females , and there was little overlap in the distributions for males and females.in utero exposure to hormonally active agents.The sexual dimorphism of AGD in humans comprises prima facie evidence that this outcome may respond to  In animal studies of the effects of hormonally active agents, measurement of anogenital distance (AGD) is now routine -16, and in utero androgen status.Although measurement of AGD in humans has been discussed in the literature ,21-23, tA cross-sectional study was conducted among the newborn children of women admitted for delivery to the Dr. Ernesto Meana San Román General Hospital in Jojutla, Morelos, Mexico, in 1999. This hospital provides medical care to low socioeconomic status and uninsured populations. The study included 87 newborn infants, none of whom had congenital defects or had been admitted to the neonatal intensive care unit. All infants were born at term (≥38 weeks gestation), except for one (32 weeks). The infants were of both sexes and were born after spontaneous cephalic delivery or caesarean section. Within 6 hours of birth, a structured questionnaire about family background and obstetric history was administered to the mothers, and anthropometric measurements were taken on the newborns.Anthropometric measurements were taken of weight, length, head circumference, and AGD. AGD was measured as follows: the newborn infant was in the dorsal decubitus position; both hips were flexed and light pressure was exerted on the infant's thighs until the examiner's hand touched the subject's abdomen. Measurements were made with Vernier calipers. Distance was measured from the center of the anus to the posterior convergence of the fourchette (where the vestibule begins) in female infants ; and froBefore any contact with the 87 subjects in the main study, the personnel performing the anthropometry examined 20 other neonates; all of whom were born after ≥38 weeks gestation. In this standardization training, 7 female infants and 13 male infants were measured twice by each observer. A sufficient time interval (30 minutes) was allotted between each measurement so that the second would not be influenced by the observer's memory of the first. These data were used to examine the reliability of measures and sources of variance.The reliability of the anthropometric measures was calculated as the Pearson correlation coefficient between the paired measures. The observations taken by the two observers were not statistically different when compared using a paired t-test (results not shown). Analysis of variance (ANOVA) with a random effect term for subject was used to estimate between-subject, between-observer, and within-observer components of variance, by sex.For the main study, a linear regression analysis was used to evaluate birth weight, birth length, and gestational age as predictors of AGD. Age of the mother, number of pregnancies, and time elapsed between birth and measurement were not important predictors (or confounders) of AGD in univariate or multivariate models and were not considered further in the analysis. To examine influential values and the overall fit of the model, we conducted an analysis of residuals, but found nothing of note.The protocol was approved by human subjects committees at the National Institute of Public Health in Mexico and the National Institute of Environmental Health Sciences in the U.S.Among the 20 subjects in the standardization exercise, the between-subject coefficient of variation was greater for measures of AGD in females than for the other measures Table . The relAmong the 87 subjects in the main study, the birth weight, length, and head circumference were as expected in a population from southern Mexico Table  27]. Th. Th27]. 2 and slightly lower p value, suggesting it may be a marginally better predictor than weight in males. In a model of data for males that included weight, length, and gestational age, the p values for both length and gestation were less than 0.05, although the coefficient for gestation was negative. In a model of AGD based on data for males and females combined (results not shown), after adjustment for weight, the term for sex was clearly important .In the crude models of AGD in females, weight, length, and gestational age all appeared to be predictive Table . The adjThe AGD measures employed in the present study reflect the location of the caudal border of the genital swelling, an embryologic structure that differentiates into the labia majora in females and the scrotum in males. After the indifferent stage of the external genitalia, the critical events determining the sexual dimorphism of AGD in humans begin when, relative to the anus, the genital swelling, urethral folds, and possibly the genital tubercle, move ventrally under the influence of androgens . ElongatDirect comparison of our results with those in the two other studies with measures of anus-to-fourchette (AF) distance in female newborns ,25 is haCompared with established anthropometric measures on newborns, the reliability of the AGD measures were lower. The lower reliability of the AGD measures is likely due to several factors. The AGD measures depend on indistinct landmarks on soft tissues. Structures such as "the center of the anus" or the posterior fourchette are not clearly demarcated. Any slight traction or pressure applied to the perineum or surrounding structures could alter measures. Finally, compared with established anthropometric measures on newborns, the AGD dimensions are smaller, thus measures done with the naked eye on a subject unlikely to hold still are inherently at a disadvantage. Use of two observers, one to restrain the subject and one to do the measurements could result in improved reliability compared to our approach, which employed one observer.Compared with adult humans, the size of the genitals at birth is large relative to the body overall . Yet theA complete assessment of AGD in humans would include more measurements than were done in our study. In neonatal rodents, measurement of AGD is relatively straightforward and is the distance from the genital tubercle to the anus. In older animals or humans of any age, however, questions arise as to which measure is most informative. For example, in human males, rather than a genital tubercle, the presence of the phallus and testicles at birth means that a number of measurements are possible. The measurement in the present study, from the posterior scrotal-perineal junction, represents only one such measurement. Ideally we would have done genital tubercle measurements in males and females, but we did not. Whether sexual dimorphism exists in the distance from the anus to the genital tubule  would be useful to know. While one might expect that penile length may be a good measure of androgenization among males, difficulties obtaining a reliable measure mean that alternative measures, such as AGD, are worth investigating.in utero exposure of females to androgenic agents increased AGD [Effects of endocrinopathies on AGD in humans have been described, but only to a limited degree. A rare form of congenital adrenal hyperplasia that causes incomplete masculine development has been reported to cause decreased AGD in boys . Detailsased AGD .The purported mechanism by which androgens increase AGD in females is by inducing "labioscrotal fusion"  . This mein utero to hormonally active agents remains to be seen.In summary, we have shown that an aspect of genital dimension that reflects migration of the genital swelling is sexually dimorphic in humans. Whether this particular measure, or other measures of AGD in humans, has any utility as markers of exposure AF: anus-fourchetteAGD: anogential distanceANOVA: analysis of varianceCI: confidence intervalNone declared.ES participated in the design of the study, carried out the measurements, and wrote the first draft of the manuscript. PR participated in the study coordination and data management. EY carried out and coordinated the measurements. ML originated the idea that AGD measurements in human males may be useful, revised the manuscript, and analyzed the data. MH conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Schizosaccharomyces pombe cells to loss of the catalytic subunit of telomerase (trt1+) identified two waves of altered gene expression and a continued up-regulation of Core Environmental stress Response (CESR) genes.Gene expression profiling of the response of  Schizosaccharomyces pombe cells lacking the catalytic subunit of telomerase (encoded by trt1+) lose telomeric DNA and enter crisis, but rare survivors arise with either circular or linear chromosomes. Survivors with linear chromosomes have normal growth rates and morphology, but those with circular chromosomes have growth defects and are enlarged. We report the global gene-expression response of S. pombe to loss of trt1+.S. pombe progressing through crisis displayed two waves of altered gene expression. One coincided with crisis and consisted of around 110 genes, 44% of which overlapped with the CESR. The second was synchronized with the emergence of survivors and consisted of a single class of open reading frames (ORFs) with homology both to RecQ helicases and to dh repeats at centromeres targeted for heterochromatin formation via an RNA interference (RNAi) mechanism. Accumulation of transcript from the ORF was found not only in trt1- cells, but also in dcr1- and ago1- RNAi mutants, suggesting that RNAi may control its expression.Survivors with linear chromosomes had expression profiles similar to cells with native telomeres, whereas survivors with circular chromosomes showed continued upregulation of core environmental stress response (CESR) genes. In addition, survivors with circular chromosomes had altered expression of 51 genes compared to survivors with linear chromosomes, providing an expression signature. These results demonstrate a correlation between a state of cellular stress, short telomeres and growth defects in cells with circular chromosomes. A putative new RecQ helicase was expressed as survivors emerged and appears to be transcriptionally regulated by RNAi, suggesting that this mechanism operates at telomeres. Telomeres are the nucleoprotein ends of linear eukaryotic chromosomes. In most organisms, telomeric DNA consists of a simple, repeated sequence with a G-rich strand running 5' to 3' towards the chromosome end, and terminates with a short, single-stranded 3' overhang . The trt1+ dATP using DNA polymerase I Klenow fragment. Probes for pol1+, act1+, the putative helicase and the C, I, L and M chromosome fragments were generated by PCR amplification from a genomic DNA template and were gel purified. Probes were labeled by random-primed transcription of PCR products with the use of [α-32P]dCTP and High Prime Mix (Boehringer Mannheim). Probes specific for the telomeric and telomere-associated sequences were created with the use of gel-purified fragments of pNSU70 [DNA from approximately 2 × 10escribed . After df pNSU70 .8) were collected as described above. Plug preparation, chromosome digestion and electrophoresis were performed exactly as described [2 at 254 nm in a UVStratalinker1800 to nick the DNA, treated with HCl, NaOH and neutralization buffer, and processed as described in 'Southern hybridization'.Cells  was not subjected to first-strand cDNA synthesis, but was otherwise treated identically.RNA was prepared as for microarray analysis and used for RT-PCR . First-strand cDNA synthesis was performed using primers complementary to either the forward or reverse strands. Both primers were present in subsequent cycles of PCR amplification after heat inactivation of reverse transcriptase at 95°C for 15 min. The control reaction lacking reverse transcriptase  by hot-phenol extraction and purification with RNeasy columns (Qiagen) following a published protocol [act1+. A reference pool consisting of RNA from each sample was made, comprising 76% trt1- cells and 24% trt1+ cells. This pool was divided into 10 μg aliquots (henceforth referred to as 'reference RNA') and used as the reference RNA in all hybridization experiments reported here.Whole-cell RNA was isolated from protocol . AliquotA single large batch of YES medium was made at the start of the study and used to culture all cells analyzed by microarrays to prevent batch-to-batch medium variations that might yield artifactual microarray results.S. pombe microarrays used have been described previously [The procedures performed and the eviously . Whole-ctrt1+ biological replicates (strains WT 3 and WT 5) collected on day 1 of the growth curve. Experiments and genes were clustered in GeneSpring using the Pearson correlation around zero (termed the Standard correlation in GeneSpring) with a minimum distance of 0.001 and a separation ratio of 1. Gene annotations were taken from GeneDB at the Wellcome Trust Sanger Institute [Normalized data (Cy5/Cy3 ratios) were evaluated using GeneSpring (Silicon Genetics). All gene-expression values were normalized to the average of two nstitute . Lists onstitute  were taknstitute . BLAST snstitute .The density of genes with changed regulation along the chromosome was determined by using a running window of 20 consecutive genes along each chromosome . For eacdh repeats - that we believe were due to cross-hybridization with the SPAC212.11 transcript . Cross-hybridization becomes apparent with array element sequence identities higher than about 70% [dh repeat microarray probe sequence (83% sequence identity), and both the SPAC212.11 and dh repeat transcripts are expected to be capable of hybridizing to each other's microarray probes. Significant levels of forward and reverse centromeric dh repeat transcripts could not be detected using RT-PCR with RNA from days 1, 7, 9 and 15 of the growth curve  and the reverse transcript of centromeric bout 70% . The SPAe Figure  (indicatTwenty-one microarrays were used in this study, representing two wild-type biological repeats, 15 days of the growth curve, and four strains with circularized chromosomes. The complete raw and normalized data sets are available from ArrayExpress  (Accessi