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 (LSHHSC, New Orleans, LA). 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 (eviously ,37. Correviously using dynZrs in terms of the constant phase model [We interpreted the measurement of se model Raw is a frequency independent Newtonian resistance reflecting that of the conducting airways [Iaw is airway gas inertance, G characterizes tissue damping, H characterizes tissue stiffness (elastance), 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 (of two amino acids, a proline and an isoleucine). 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 (FrFSTL4 and FrFSTL5, respectively). 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 (Zv1/06 assembly, which was produced by the Zebrafish Sequencing Group at the Sanger Institute [We used the human RUNX1, RUNX2 and RUNX3 proteins (SwissProt entries Q01196, Q13950 and Q13761, respectively) 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 (NP_523424, NP_511099, NP_572693 and NP_608398). 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 (, accessed on Aug/04/2004). 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 (mean, 22 mm) than in females (mean, 11 mm), 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 (reviewed in ,2). The trt1+ [Telomeres are also essential for the complete replication of chromosomes, because conventional DNA polymerases do not copy the extreme ends of linear DNA molecules. In the absence of a mechanism to compensate for this 'end-replication problem', progressive telomere shortening leads to replicative senescence, which in yeast is characterized by chromosome instability and low cell viability ,5. Replitrt1+ .Saccharomyces cerevisiae, survivors maintain long, heterogeneous telomeres on linear chromosomes using a RAD52-dependent homologous-recombination pathway [S. pombe cells without telomerase cease dividing after about 120 generations, and can give rise to a subpopulation of survivors [In some cases, cells can endure the loss of telomerase and give rise to a population of survivors. In the budding yeast  pathway . Global  pathway . Human a pathway ,13. S. purvivors . Interesurvivors .S. pombe are believed to form as a result of the genomic instability due to loss of telomeres, which normally prevent end-joining and suppress recombination. Interchromosomal fusions yield unstable dicentric chromosomes, while intrachromosomal fusions produce circular chromosomes. S. pombe, with only three chromosomes, is more likely than other organisms with larger numbers of chromosomes to successfully form exclusively intrachromosomal fusions [S. pombe strains with circular chromosomes also result after concurrent deletion of rad3+ and tel1+, two genes with sequence similarity to human ATM (ataxia telangiectasia mutated) [Circular chromosomes in  fusions ,15. S. pS. pombe survivors with linear chromosomes grow remarkably well and have a morphology similar to wild-type cells, survivors with circular chromosomes display obvious growth defects such as slower growth rates and larger sizes [S. pombe global gene-expression response to loss of trt1+ to investigate changes in expression of genes during senescence, and to compare survivors with circular or linear chromosomes. We report that survivors with circular chromosomes maintain an extended stress response not observed in survivors with linear chromosomes. Furthermore, we present evidence for regulation of a telomeric gene by the RNAi machinery.Although er sizes . Survivotrt1- samples. Before averaging the expression values from the two reference strains, the similarity of their expression profiles was assessed. The dye ratios measured by microarray for each strain were plotted against each other  changed their expression values by more than twofold, and they were only slightly greater; thus, the vast majority of genes do not have altered expression as a result of long-term growth in culture, provided that expression is measured while the cells are in early log phase . These three genes also had expression changes of more than twofold in one or more conditions measured for trt1- cells, but given their variable expression in wild-type cells, these changes were most probably unrelated to the absence of telomerase.To learn whether changes in gene expression would result from subjecting cells to the continuous growth program for 15 days, gene-expression values from strain WT 5 on day 1 of the growth curve were compared with those of the same strain harvested on day 15 Figure . Only thS. pombe cells that were heterozygous for trt1+ and able to maintain full-length telomeres were sporulated, and the resulting trt1+ and trt1- cells propagated through a 15-day growth curve , lack of TAS2 sequence (data not shown), the inability of chromosomes to enter a pulsed-field gel , SPAC2E1P3.04 (a predicted copper amine oxidase) and SPAC2E1P3.05c (unknown function). Hybridizations of genomic DNA to microarrays (data not shown) revealed that genes SPAC2E1P3.04 and SPAC2E1P3.05c were deleted from the genome in all strains except WT 3, WT 5 and C1. Interestingly, these two genes are within about 4 kb of transposable element SPAC167.08 (Tf2-2), suggesting a hotspot for DNA excision. In no case was gene amplification detected by genomic hybridization (data not shown), so the observed increases in expression were most probably due to transcriptional or post-transcriptional regulation, as opposed to changes in gene copy number.The vast majority of expression changes involved upregulation, and only seven genes had downregulated expression of twofold or greater on two or more days of the growth curve. Notably, there were three cases of reduction in expression greater than tenfold: trt1- strains were studied, the identification of genes with consistently altered expression was facilitated by selecting those genes with expression changes of twofold or more in two or more days of the growth curve or, alternatively, in both strains C1 and C5. This criterion was met by 123 genes, of which 54 (44%) overlapped between the growth curve and survivors with circularized chromosomes. In addition, of the 67 genes that had their expression changed twofold or more exclusively in the growth curve, many displayed altered expression just below the cutoff in survivors with circularized chromosomes. Two genes - SPBC1683.06c (a predicted uridine ribohydrolase) and SPBC1198.01  - had expression changes of twofold or more in both strains C1 and C5, but no significant changes during the growth curve. As a measure of confidence, 84 of the 123 genes (approximately 68%) met a more stringent criterion requiring a gene to change its expression in three or more of the 17 conditions. Additional confidence that expression changes scored as significant were not false positives came from the remarkably continuous manner in which gene expression changed throughout the growth curve  also had upregulated expression among a conservative list of CESR genes (P ~ 10-77) [trt1- cells had overlap with the CESR (P ~ 10-81). With respect to specific stress responses [P ~ 10-32), and 11/123 genes in common with the heat stress response (P ~ 10-24). The stress response study found that the DNA damage response and the oxidative stress response have substantial overlap [The telomerase-deletion response had a large overlap with genes that changed expression in response to environmental stresses. Fission yeast stress-response genes can be separated into a CESR, in which genes changed expression in all or most of the stresses studied , and into more specific stress responses . Of the ~ 10-77) , and two~ 10-77)  suggesteesponses , there w overlap . TherefoComparisons of all the gene-expression profiles in this study revealed striking differences between the profiles of survivors with linear chromosomes versus those with circular chromosomes. Survivors with linear chromosomes (days 12-15 of the growth curve) had gene-expression patterns similar to those of cells with native telomeres in the first two days of the growth curve. To illustrate, by day 12 of the growth curve, the gene-expression profiles of survivors became relatively constant and remained so through day 15. The profiles of days 12-15 appear most similar to days 1 and 2 of the growth curve, immediately after cells lost telomerase and were experiencing shortening telomeres Figure . This obP ~ 10-46), and eight genes (15%) overlapped with the oxidative stress response (P ~ 10-14). There were 8/54 genes (15%) that overlapped with the heat stress response (P ~ 10-17). Because of the extensive overlap of the 54 genes with the CESR, we conclude that survivors with circular chromosomes had a sustained stress response.There were 54 genes with clearly altered expression (twofold or more) mainly during crisis in the growth curve that also had altered expression in the survivors with circular chromosomes Table . Of the Of the 54 genes, 51 represent a gene-expression signature that differentiates survivors with circular chromosomes from those with linear chromosomes. As an independent test of whether these 51 genes can serve as a signature for cells with circularized chromosomes, two additional cultures  with circularized chromosomes were grown and analyzed by microarray. Both strains clearly displayed altered expression of the 51 genes whereas survivors with linear chromosomes did not  [clr4 mutants (P < 10-45) [dcr1+, ago1+ and rdp1+ (P ~ 10-22) [trt1+, clr4+, pof3+ and the RNAi machinery.One feature of microarray studies is that genes not previously recognized to be under the control of a common regulator can often be associated by similar expression patterns . On the  pathway ; taz1+ [y [taz1+  and pot1+ [pot1+  encoding+ [est1+ ; homolog [rhp54+ , rad32+  [rad32+  and rhp5 [rhp51+ ; RecQ he+ [rqh1+ ; silenci+ [clr4+ ; and tel+ [pof3+  and rad3+ [rad3+ . Interes< 10-45)  and clr4< 10-45) ; a signi~ 10-22) . These gdh repeats [dh repeats most probably resulted from cross-hybridization with the SPAC212.11 transcript .The second wave of gene-expression changes during the growth curve Figure  consiste repeats . InspectA BLAST search of the SPAC212.11 predicted protein sequence found that the ORF has the most similarity to RecQ DNA helicases of superfamily II Figure  . However, these transcripts do not accumulate in wild-type cells. Reverse-strand centromeric transcripts are synthesized and rapidly processed by the RNAi machinery, while forward-strand synthesis is silenced transcriptionally. RNA-dependent RNA polymerase (Rdp1) associates with centromeric repeat DNA and may use siRNAs corresponding to centromeric transcripts  to primeobserved .dh repeats, but that this silencing may have been disrupted in trt1- cells as a result of genomic instability. Arguing against this hypothesis, however, Southern analysis with probe P5' -PCR analysis indicated that the putative helicase gene was robustly expressed in cells emerging from crisis, but was weakly  expressed in wild-type cells, strains C1 and C5 and survivors with linear chromosomes eroxide  on ME plates [trt1+ by using a standard two-step integration procedure [trt1+ ORF separated by HSV1-tk and KanMX4 [FseI and transformed using the lithium acetate method [h- ade6-M210) and PP69 (h+ ade6-M216). Cells were re-streaked twice on yeast extract low adenine (YEA) + geneticin plates [EcoRI restriction fragment 3' of trt1+ which was present only in integrants. Cells were then plated on YEA + 50 μM 5-fluorodeoxyuridine (5-FUdR) plates to select for those that had excised HSV1-tk, KanMX4 and the XbaI-XhoI fragment (around 5 kb) of trt1+ from their genomes. Random surviving colonies were screened for heterozygous diploids by Southern hybridization to the 3' region of the trt1+ KpnI restriction fragment. The heterozygous state was evidenced by hybridization signals to both full-length trt1+ and a shortened, nonfunctional version. Loss of markers was confirmed by lack of a Southern hybridization signal to HSV1-tk, and by lack of growth on YEA + geneticin plates.The E plates . The parrocedure  with a ld KanMX4 . The plae method  into a dn plates  to selech- ade6-M210 genotype, which were further screened by Southern hybridization for the presence or absence of trt1+ (performed as described in 'Strain construction' below). Colonies were subsequently used as described in 'Growth curve', or alternatively used to create strains C1, C5, H1 and H2.After germination of G4 and growth of spores at 32°C for three days on YEA plates , plates trt1- colonies that were each successively re-streaked on YEA plates 15 times (with growth for 2 to 3 days at 32°C between re-streaks), to permit colonies to form without competition from faster-growing survivors with linear chromosomes. During this time cells were presumed to senesce and give rise to survivors. After the last re-streak, a single colony from each strain was randomly selected and used to prepare freeze stocks.Strains C1, C5, H1 and H2 were created from four separate h- ade6-M210 trt1+) designated WT 3 and WT 5, and a single mutant isolate (h- ade6-M210 trt1-) designated as 'GC Day X', where X represents the day of the growth curve that cells were collected. Single colonies were used to inoculate 5-ml starter cultures in yeast extract full supplements (YES) medium [4 cells/ml, and were grown in an incubator (Innova 4430, New Brunswick Scientific) with continuous shaking at 200 rpm at 32°C. Cell density was monitored by periodic counting, and a portion of the cells was harvested for microarray analysis and Southern hybridization when the density reached 3-5 × 106 cells/ml (early log phase). Cells harvested at this point were referred to as day 1 of the growth curve. The unharvested cells were permitted to continue growing until 24 h from the time of inoculation, at which time cells were counted and used to inoculate a fresh 200-ml YES culture at 2.5 × 104 cells/ml, and the process repeated for 15 days. To harvest cells for microarray analysis, a volume of culture containing approximately 1.6 × 108 cells was gently centrifuged at room temperature (2,000 rpm for 2 min), the supernatant removed, and the cell pellet snap-frozen in liquid N2. For Southern hybridization, approximately 2 × 108 cells were collected by centrifugation, washed twice in H2O, and snap-frozen in liquid N2. A portion of cells for pulsed-field gel analysis was also collected in the same manner as for Southern hybridization at the end of each 24 h period. trt1- cells were collected daily, WT 3 and WT 5 on day 1, and WT 5 on day 15.Three strains were grown: two wild-type isolates  medium  and grow4 cells/ml, and cultures grown with constant shaking at 200 rpm at 32°C. When the cell density reached around 3 × 106 cells/ml (early log phase), cells were collected as described in 'Growth curve' for microarray analysis, Southern hybridization and pulsed-field gel electrophoresis. Strains H1 and H2 are trt1- isolates with circular chromosomes, as evidenced by pulsed-field gel electrophoresis (data not shown)Cells were streaked onto YEA plates from freeze stocks, grown for 3 days at 32°C, and single colonies used to inoculate 5-ml starter cultures in YES medium. After 24 h, cells were counted and 200-ml YES cultures were inoculated at 2.5 × 108 S. pombe cells was prepared as described [EcoRI or HindIII, the DNA was subjected to electrophoresis on a 1% agarose gel in 1 × TBE (90 mM Tris, 90 mM borate, 2 mM EDTA pH 8.3). DNA was denatured by sodium hydroxide treatment and transferred to a nylon membrane (Hybond-N+ membrane, Amersham) by capillary transfer in 10 × SSC (1.5 M NaCl, 0.15 M sodium citrate). DNA was immobilized on the membrane by irradiation with 120 mJ/cm2 at 254 nm in a UVStratalinker1800 (Stratagene). For molecular weight markers, the 1 kb DNA ladder (New England Biolabs) was labeled by filling in 5' overhangs with [α-32P]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 (approximately 1 × 10escribed . DNA wasact1+ sense, -RT) 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 (OneStep RT-PCR kit, Qiagen). 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 (The PCR primers used to generate probes C, I, L, and M have been published previously . The PCR5': 5'-CTTCAAAAACTGCTAGAGATATCGCCGG-3' andP5'-GTACTGGTAGTCCTCTGATGTATGGG-3'3': 5'-ATGCCCCGTACGCTTATCTA-3' and 5'-TTTGCCTTTCTAGCCCATGA-3'Pdh: 5'-CAACACCAATACTGACGATGATG-3' and 5'-GCAATAGAACCAGCGGTTTG-3'Pdh repeats have been published previously [Primers for centromeric eviously .S. pombe cell pellets (~1.6 × 108 cells) 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


The habitual "any other comments" general open question at the end of structured questionnaires has the potential to increase response rates, elaborate responses to closed questions, and allow respondents to identify new issues not captured in the closed questions. However, we believe that many researchers have collected such data and failed to analyse or present it.General open questions at the end of structured questionnaires can present a problem because of their uncomfortable status of being strictly neither qualitative nor quantitative data, the consequent lack of clarity around how to analyse and report them, and the time and expertise needed to do so. We suggest that the value of these questions can be optimised if researchers start with a clear understanding of the type of data they wish to generate from such a question, and employ an appropriate strategy when designing the study. The intention can be to generate depth data or 'stories' from purposively defined groups of respondents for qualitative analysis, or to produce quantifiable data, representative of the population sampled, as a 'safety net' to identify issues which might complement the closed questions.We encourage researchers to consider developing a more strategic use of general open questions at the end of structured questionnaires. This may optimise the quality of the data and the analysis, reduce dilemmas regarding whether and how to analyse such data, and result in a more ethical approach to making best use of the data which respondents kindly provide. The survey is a key method in health services research . The majThere are four types of questions which might require an open rather than closed response , and the amount they actually write. Thus researchers may be able to determine the status of a general open question at the design stage of a study by having a strategy to generate depth and treat the data qualitatively, or by having a strategy to generate shorter responses as a 'safety net' for complementary or new issues. Having such a strategy may help researchers to devise a strategy for analysis and publication.Researchers can determine the status of a general open question at the design stage of a study by having a strategy to generate depth and treat the data qualitatively. For example, at the end of a structured questionnaire about use of Chinese medicine, one researcher invited respondents to tell the 'story' of their use of Chinese medicine, leaving a full one and a half pages of white space, and offering a example of the detail required. The following instructions were given: 'Now tell us your own story, using the space on the next page. We've provided one true patient story to give you an idea of the kinds of details we need. The important subjects are repeated in the list above the space we've provided for you to write in. Also use the back of the page if you wish. Please remember to write clearly.' . This apIn the above example, the 80% response suggests that the stories obtained could be viewed as representative of the population surveyed. However, in qualitative research the validity of the study does not rest on the researcher's ability to demonstrate representativeness with respect to the total population. Rather, it rests on transferability whereby the researcher offers detailed description of the setting in which the research was undertaken . Thus whIf the open question is used to generate qualitative data, then researchers will need to use qualitative analysis techniques and possibly qualitative software as used in the Chinese medicine example discussed previously , and wilGeneral open questions may produce little more than the closed questions on the questionnaire  and rathFrom a quantitative perspective, the strength of a survey approach is representativeness, and thus non-response bias should be a concern. Respondents are less likely to complete a general open question than a closed one on a postal questionnaire : 81% of Those who choose to answer the general open question could be different from respondents overall, either being more articulate or having a greater interest in the survey topic. It is important to consider and report on who has made written comments so that bias can be considered. In a patient satisfaction survey, females were more likely to make comments than males but interestingly there were no significant differences by age group or educational status . In a suFormal analysis must be rigorous so that the findings are useful and convincing. Content analysis may be undertaken ,3, where1. Reads a sub-set of the comments.2. Devises a coding frame to describe the thematic content of the comments.3. Assigns the codes to all the comments. The coding frame can be applied using software designed for this purpose  or manua4. The codes can be entered into a statistical package alongside the data from the closed questions and treated as variables in a quantitative analysis.The coding process is time consuming and requires expertise ,4,6. TheWhen reporting the responses to general open questions it is important that the numbers of respondents making each comment are displayed, with recognition that although a specific number of people mentioned an issue it might be relevant to many more who did not choose to mention it. Although numbers rather than percentages tend to be used when reporting responses to open questions ,12,15,26Publication of responses to open questions can occur within a paper reporting the main findings of the questionnaire or as a separate publication. Where comments elaborate and explain findings from closed questions, it may be most appropriate to publish them in the same paper . Where aAn explicit strategy requires that researchers consider the role of a general open question in the context of their survey, and its status in terms of generating either qualitative or quantifiable data. If the role of the question is to give a voice to participants then the researcher can ensure that comments will be read to identify any concerns and queries expressed by individuals, and that appropriate action is taken with individual comments. If the role is to generate qualitative data then attention can be paid to generating depth of data and the quality issues associated with qualitative research. If the role is to act as a safety net and generate quantifiable data then resources will need to be allocated for reading the comments, and if there appears to be added value, for formally analysing the data with attention to non-response bias and reliability of coding. Having a strategy may reduce any dilemma faced by researchers about whether and how to analyse these questions, may help the researcher to allocate the appropriate time and expertise to this data, and may produce an analysis robust enough for publication in peer reviewed journals. A potential disadvantage of having such a strategy may be that some flexibility is lost and that some important issues are missed. Finally, researchers cannot assume that they know how best to facilitate a respondent to complete a general open question and may need to consider using cognitive aspects of survey methodology to construct the question .• General open questions at the end of structured questionnaires can present a problem to researchers who may face the dilemma of whether or not to analyse them.• They are necessary when piloting questionnaires because they identify further issues for inclusion in the survey, and may be a bonus in the main study because they may increase response rates and may identify issues which complement responses to closed questions.• The value of such questions, and the quality of the data and analysis, may be optimised if researchers make more strategic use of them by being clear about their role, and understanding the type of data they wish to generate when they design their study.• An explicit strategy for generating qualitative data will encourage attention to depth of data and issues important to the analysis of qualitative data such as reflexivity.• An explicit strategy for generating quantifiable 'safety net' data, that is important issues missed by the closed questions, will encourage attention to non-response bias and reliability of coding.The author(s) declare that they have no competing interests.AOC conceived the paper and wrote the first draft. KJT reviewed it critically, and developed the sections on qualitative research. Both authors produced the final draft and read and approved the final manuscript.The pre-publication history for this paper can be accessed here: