The library converged over three generations to a high-affinity protein ligand. The ability to genetically encode diverse classes of synthetic transformations enables the in vitro selection and potential evolution of an essentially limitless collection of compound families, opening new avenues to drug discovery, catalyst design, and the development of a materials science “biology.”Biological in vitro selection techniques, such as RNA aptamer methods and mRNA display, have proven to be powerful approaches for engineering molecules with novel functions. These techniques are based on iterative amplification of biopolymer libraries, interposed by selection for a desired functional property. Rare, promising compounds are enriched over multiple generations of a constantly replicating molecular population, and subsequently identified. The restriction of such methods to DNA, RNA, and polypeptides precludes their use for small-molecule discovery. To overcome this limitation, we have directed the synthesis of combinatorial chemistry libraries with DNA “genes,” making possible iterative amplification of a nonbiological molecular species. By differential hybridization during the course of a traditional split-and-pool combinatorial synthesis, the DNA sequence of each gene is read out and translated into a unique small-molecule structure. This “chemical translation” provides practical access to synthetic compound populations 1 million-fold more complex than state-of-the-art combinatorial libraries. We carried out an in vitro selection experiment  on a library of 10 The authors use DNA "genes" to direct the synthesis of combinatorial chemistry libraries, and show in an in vitro selection experiment that specific drugs can be developed Creation of molecular function represents a fundamental challenge. Nature accomplishes the task through evolution, iterating cycles of selection, amplification, and diversification. Multiple generations of selective pressure and reproduction transform a diverse population into one consisting only of molecules fit to survive. Life on this planet thus emerged from a limited chemical palette, comprising proteins, nucleic acids, sugars, lipids, and metabolites. Over the last two decades, technologies that recapitulate this process in the test tube have been developed, and have produced an amazing collection of biopolymers with unprecedented recognition and catalytic properties  libraries represents a first approximation to small-molecule evolution, in that the process roughly mimics the diversification and selection components of evolution. However, amplification and iteration have no functional equivalents in HTS-CC techniques, placing practical limits on library complexity. Amplification and iteration are critical for identifying vanishingly small amounts of material from a complex population. Moreover, these processes make possible the application of bulk selections rather than serial screens to assay libraries, vastly increasing throughput. Accordingly, typical HTS-CC libraries rarely exceed 10 members , whereas members . A state members . If the In order to apply in vitro selection to combinatorial chemistry libraries, each compound must be associated with a gene that specifies its structure. DNA has been utilized previously to record the synthetic history of individual beads in a split-and-pool combinatorial synthesis, but the DNA tags could not direct subsequent resynthesis of the corresponding compound . More reIn vitro selection requires iterated rounds of three steps: conversion of genes to gene products, selection of gene products, and gene amplification . The lasIn general, small-molecule libraries are synthesized by the split-and-pool method which is illustrated in The construct we chose for our DNA support library is shown in Our scheme for DNA-directed split and pool synthesis is shown in We first developed a Sepharose-based resin derivatized with anticodon oligonucleotides complementary to codon sequences . We testWe next addressed chemical synthesis on unprotected DNA. Use of a solid phase in small-molecule synthesis allows for the application of excess reagents, to drive reactions to completion, and simplifies product purification . To realAs an initial chemistry, we chose 9-Fluorenylmethoxycarbonyl [Fmoc]–based peptide synthesis. Synthesis of [Leu]enkephalin on a 340-base ssDNA support, capable of encoding an eight-step synthesis, was analyzed using an electromobility shift assay and the enkephalin-specific 3-E7 antibody . Figure Our chemical translation strategy requires repeated hybridization-directed splitting and coupling of chemical building blocks to DNA. Two different solid phases were utilized for these tasks. To efficiently transfer DNA from anticodon columns to DEAE Sepharose columns, we cyclically pumped 50% dimethylformamide (DMF) over the columns connected in series . ConversTo test and validate our general strategy, we applied in vitro selection to a primarily nonnatural peptide library, with the goal of identifying a high-affinity ligand for the monoclonal antibody 3-E7 . Isolati5-fold by PCR recombination to generate a support library with a complexity of one million, as verified by DNA sequencing  was diversified 10quencing . This liquencing A. The fiIn order to monitor library convergence, DNA from the starting material (round 0) and from after one (round 1) or two (round 2) selection generations was subcloned, and approximately 70 different isolates from each round were sequenced B. In rou1-j2-b3-c4-h5-i6, whereas in the alternate library it was d1-b2-g3-g4-i5-f6. Two rounds of selection enriched the alternate [Leu]enkephalin DNA gene 105-fold (data not shown). The data suggest that little, if any, DNA sequence encoding bias exists in our system. Further, they illustrate the reproducibility of the technology. Together, the results demonstrate conclusively that the DNA display strategy can be used for the in vitro selection of synthetic chemical libraries.To assess generality, we carried out a second [Leu]enkephalin in vitro selection experiment using a peptide library of the same size but constructed with a completely different “genetic code.” Every codon in the alternate library coded for an amino acid different from the one it coded for in the first library. The [Leu]enkephalin codon series in the first library was bPrevious efforts to expand the scope of in vitro selection have utilized nonnatural bases or amino acids incorporated into DNA, RNA, and peptide libraries using polymerases and the ribosome . Such efAn alternative strategy for expanding the chemical diversity of gene products exploits DNA-templated synthesis, where hybridization-induced proximity promotes covalent bond formation . One greDNA display is a general method for the in vitro selection of synthetic combinatorial chemistry libraries. The system is modular, so that chemistry and selection protocols can be easily changed. It can take advantage of existing combinatorial chemistry technology as well as chemical transformations previously carried out in the presence of unprotected DNA . Solid-pIn addition to diverse chemistries, many different library architectures are also possible. The library reported here was synthesized in six encoded steps with ten distinct building blocks per step. However, essentially any combinatorial scheme can be accommodated. The 20-base codon sequences used here were taken from a larger set (>10,000) of 20-base sequences experimentally verified to exhibit orthogonal hybridization properties .(E) is defined as the geometric increase in the fraction of target molecules in a library that results from a single round of synthesis and selection. Fidelity (F) is defined as the fraction of genes recovered from a completed library synthesis that have been correctly translated. The signal-to-noise characteristic of a selection (S/N) is defined as the ratio of the fraction of target molecules selected to the fraction of nontarget molecules selected. In most cases, the fold enrichment reduces to the simple expression at the right of As a first approximation, the highest possible fold enrichment per round of selection can be determined by considering its relationship to translation fidelity and the signal-to-noise ratio of the selection. Fold enrichment 0f denotes target gene fraction in the selection input and p denotes the probability of a nontarget gene being mistranslated to the target gene product. Biological systems have such high fidelity that F can be considered to equal one. However, the fidelity of chemical translation processes is the product of hybridization specificity and chemistry efficiency raised to the power of the number of steps. It is important to consider these parameters when adapting new chemistries and selections to the DNA display format. S/N of 1000 would give a 10-fold enrichment per round at best. If the library included 1012 unique members, at least 12 rounds would be required to achieve convergence.where 12 distinct small molecules per 30 pmol of DNA starting material, a quantity easily manipulated in a microcentrifuge tube.In addition to influencing convergence rates, fidelity also limits achievable library complexity. The maximum effective library complexity corresponds to the product of Avagadro's number, the moles of library, and the fidelity. Based on our observed 90% hybridization efficiency and 95% chemistry efficiency, extension of the library reported here to 13 synthetic steps would produce 1013.Diversification between rounds of selection by recombination makes possible in vitro evolution of libraries with complexities exceeding the physical library size. Thus, a “best” molecule can be pinpointed without exhaustive testing of all potential species. Starting with a working population of compounds that sparsely sample a chemical space, molecules containing parts of an optimal molecular solution often have a selective advantage relative to siblings, and become enriched. Subsequent recombination processes splice together fragments from the numerous partially optimal molecules to form a globally optimal molecule. Thus, the best structure is found, even if the odds were negligible that it existed in the initial working population. The same principle accounts for the striking success of gene shuffling in protein engineering  and of t6-fold more complex than current synthetic libraries) will test the validity of this belief.DNA display enables the use of genetic tools such as complementation analysis and backcrossing to analyze small-molecule populations. The approach can be used to study molecular evolution without potential biases resulting from experiments restricted to RNA, DNA, and peptide polymers. A general scientific problem that will be directly addressed is the relationship between small-molecule library complexity and the quality of molecules discovered. With biopolymers, more complex libraries yield higher-affinity ligands . HoweverDrug discovery would represent one important application for a small-molecule in vitro selection technology. While the cost of drug discovery has increased continuously over the last decade (from less than $15 billion for research and development in 1996 to more than $25 billion in 2002), the number of new molecular entities approved by the FDA has steadily dropped, from 56 in 1996 to 17 in 2002 . A fast,Future extensions of DNA display include the development of massively parallel array-based splitting strategies for the in vitro selection of low-molecular-weight small-molecule libraries (for example a library built in three synthetic steps with 10,000 building blocks per step). Massively parallel syntheses will produce compounds that conform better to Lipinski's “rule of five”  and presThe 3-E7 antibody was purchased from Gramsch Laboratories (Schwabhausen, Germany). PANSORBIN cells were purchased from Calbiochem . BSA was purchased from New England Biolabs (Beverly, Massachusetts, United States). Yeast tRNA was purchased from Ambion (#7119, Austin, Texas, United States). The [Leu]enkephalin peptide and all oligonucleotides were purchased from the Stanford PAN Facility .Solid-phase peptide synthesis was carried out as previously described . 5′ aminThe electromobility shift assay was performed as previously described . No plas2, 1X Promega Taq reaction buffer, and 5 U of Taq DNA polymerase (#M1661, Promega, Madison, Wisconsin, United States). The PCR program was 94 °C for 2.5 min, 58 °C for 1 min, and 72 °C for 15 min. The dsDNA–peptide conjugates were incubated with PANSORBIN cells in 50 μl of Selection Buffer (TBS, 0.1% BSA, and 0.1 μg/μl yeast tRNA) at 4 °C for 1 h to preclear conjugates that nonspecifically bind to the cells. Then, preclear beads were pelleted by centrifugation and removed. Antibody 3-E7 (0.5 μg) was added to the supernatant and allowed to incubate for 1 h at 4 °C. The solution was then mixed with fresh PANSORBIN cells for 1 h at 4 °C. The cells were pelleted and washed at 25 °C three times with 500 μl of Wash Buffer (TBS, 0.1% BSA, 0.1 μg/μl tRNA, and 350 mM NaCl), followed by a single wash with 500 μl of Selection Buffer. The dsDNA–peptide conjugates were eluted by incubation of the cells with 50 μl of 200 μM [Leu]enkephalin in Selection Buffer for 1 h at 25 °C. Selected genes were amplified from 10 μl of elute supernatant with 25-cycle PCR reactions.ssDNA was converted to double-stranded DNA (dsDNA) by one-cycle PCR with a single end primer. The 50-μl PCR reaction contained 20 μM primer, 200 μM of each dNTP, 5 mM MgClHigh performance liquid chromatography analysis of DNA–peptide conjugates, synthesis of anticodon columns, hybridization and transfer of DNA, library assembly, ssDNA generation, and library isolation were performed as previously described .


Salmonella Enteritidis infections, phage type 4 has been the dominant phage type in most countries in Western Europe during the last years. This is reflected in Salmonella infections among Swedish travellers returning from abroad. However, there are differences in phage type distribution between the countries, and this has also changed over time.Among human Salmonella Enteritidis infections in Swedish travellers from 1997 to 2002, and have compared this with national studies conducted in the countries visited.We used data from the Swedish infectious disease register and the national reference laboratory to describe phage type distribution of S. Enteritidis infections among Swedish travellers returning from some countries in southern Europe was observed, and a previously rare phage type (PT 14b) became one of the most commonly diagnosed that year, continuing into 2002 and 2003.Infections among Swedish travellers correlate well with national studies conducted in the countries visited. In 2001 a change in phage type distribution in Surveillance of infections among returning travellers can be helpful in detecting emerging infections and outbreaks in tourist destinations. The information needs to be communicated rapidly to all affected countries in order to expedite the implementation of appropriate investigations and preventive measures. Salmonella Enteritidis is the most common serovar causing food-borne salmonellosis in humans, causing approximately 80% of salmonellosis cases reported in Europe [S. Enteritidis infections was reported in Europe and North America [S. Enteritidis varies between countries; while phage type (PT) 4 is reported to be dominant in most countries in Western Europe, PT 8 is common in North America and also a few European countries [S. Enteritidis contamination of eggs [S. Enteritidis in recent years [n Europe . During  America -4. The mountries -7. Epideountries ,8. Approountries . Based oountries ,10. Thes of eggs  and are nt years ,12.In Sweden, about 85% of the reported salmonella infections are acquired during travel abroad and the levels in domestic animals and food products is low . TherefoS. Enteritidis infections, describe the phage type distribution of S. Enteritidis isolated from Swedish travellers returning from abroad related to country of infection, and discuss possible reasons for the emergence of a new phage type of S. Enteritidis in 2001. A preliminary analysis of a subset of these data was published as a short letter in the Lancet in 2002 [In this study, we investigate trends in travel related  in 2002 .S.Enteritidis notified to SMI from January 1, 1997 through December 31, 2002.In Sweden, salmonellosis is a mandatory notifiable disease. Both clinicians and laboratories are required to report a case to the infectious disease register at the Swedish Institute for Infectious Disease Control (SMI). Based on the patient's travel history, information on probable country of infection is collected on the notification forms. Diagnosis of salmonella infections is made at regional microbiology laboratories, and all isolates are submitted to the national reference laboratory at SMI for serotyping and phage typing. In this study we have included all cases of To investigate trends in travel-related infections, we collected information on air travel from the Swedish Civil Aviation Administration (CAA). The figFor the geographical description of dominant phage types in the different countries the analysis was restricted to infections acquired in countries in Europe during 1997 to 2002, and to countries from which more than ten cases were reported during this period. A phage type was considered dominant if it represented >30% of the isolates and was at least twice as common as the second most common phage type. If none of the phage types fulfilled these criteria, the two most common phage types that together represented >50% of the cases were defined as the dominant types. The pattern for 1997 to 2000 was compared with the pattern for 2001.S.Enteritidis infections notified during 1997–2002, 11,570 cases (87%) were reported as infected abroad, 1,032 (8%) were reported as infected in Sweden, and information of probable country of infection was not available for 669 (5 %). The total number of cases reported each year varied from 1,598 to 2,629 cases, with the highest being reported in 1999 and the lowest in 2002. Imported cases varied between 1,404 and 2,164. The most common countries of infection during the six-year period were Spain, Greece and Turkey, accounting for 34%, 8% and 4% of all cases, respectively.Of 13,271 cases of S. Enteritidis among Swedish travellers were reported each year during 1997 to 2002. These are all popular destinations for leisure travel among Swedes. Figure For six countries – 'Spain, Greece, Turkey, Poland, Thailand and Portugal – >50 cases of infection with Eighty-six percent (10,049) of the isolates from 1997 to 2001 were phage-typed, increasing from 75% in 1997 to 95% in 2001. The most common phage types over the period were PT 4 and PT 1  sent by the Norwegian Public Health Institute gave no response on increases of PT 14b in other countries. Spain reported an outbreak of the same phage in a school in January (unpublished data). But after this event, no further increase was noted. The UK later reported an increased number of the same phage type, both among travellers and among people who had been infected in the UK. However, there the 14b isolates of domestic origin were aerogenic, while isolates associated with travel to Greece were predominantly anaerogenic [The change in phage type distribution observed among Swedish travellers returning from some countries in Southern Europe in 2001 was not observed among inhabitants in the countries visited. In total, the two most common phage types among Swedish travellers were, as in previous years, PT 4 and PT 1. However, the third most common was PT 14b, a phage type hitherto uncommon in Sweden with only 20 to 40 cases reported annually prior to 2001. The majority of the cases were among travellers returning from Greece (90%). More cases were also reported among travellers returning from Spain and Bulgaria than in previous years. During the same time period, an increase in the same phage type had also been registered among Norwegian and Finnish travellers returning from Greece . A requeerogenic . The isoNo explanation for the sudden increase in this phage type among Nordic travellers to different countries in Southern Europe has been found. PT 14b is not a new phage type and outbreaks reported previously have been mainly related to eggs and egg-products (ice-cream, tiramisu ,20) or iSalmonella isolates is not routinely performed in some countries. However, if available, this information will be included in the data reported to Enter-Net. Data from this network has previously been useful in detecting travel-related outbreaks [Our data presented are limited by the small numbers of cases from each country investigated on an annual basis. It is therefore difficult to evaluate trends with any certainty. However, the possibility of using surveillance data of infections among returning travellers to detect emerging pathogens should be further investigated. In addition, data from countries that routinely collect information on travel could be pooled in order to increase the numbers of travel-related infections. In several countries outside Europe, laboratory capacity is limited and it may take a long time to detect the emergence of new pathogens or subtypes. Not all countries in Europe collect information on the probable place of infection and phage typing of utbreaks ,28, and This study demonstrates that surveillance of infections among returning travellers may be helpful in detecting emerging infections and outbreaks in tourist destinations, and provides some useful supplementary data about infectious diseases and trends in other geographical regions. Characterization of isolates from travellers can detect changes in the pathogen and antimicrobial resistance patterns in the destination country. This information may be an important supplement in countries where surveillance systems are deficient or lacking, or where the laboratories have limited capacity to do detailed sub-typing and resistance testing. In addition, infections and outbreaks among tourists may not always affect the local residents and therefore may not be detected by the local public health authorities. If proper investigations, and appropriate prevention and control measures are to be implemented in the countries visited, it is important that the surveillance information compiled from the traveller's home countries is rapidly communicated to the affected countries.None declared.KN performed the data analysis and drafted the manuscript. PJG, YA and BdJ participated in the design and coordination of the study. AO conducted typing and provided advice regarding laboratory issues. JG participated in the design and discussion, and provided advice on data analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Xenopus oocyte maturation.The 26S proteasome is the proteolytic machinery of the ubiquitin-dependent proteolytic system responsible for most of the regulated intracellular protein degradation in eukaryotic cells. Previously, we demonstrated meiotic cell cycle dependent phosphorylation of α4 subunit of the 26S proteasome. In this study, we analyzed the changes in the spotting pattern separated by 2-D gel electrophoresis of α subunits during Xenopus, then prepared antibodies specific for five subunits (α1, α3, α5, α6, and α7). With these antibodies and previously described monoclonal antibodies for subunits α2 and α4, modifications to all α-type subunits of the 26S proteasome during Xenopus meiotic maturation were examined by 2D-PAGE. More than one spot for all subunits except α7 was identified. Immunoblot analysis of 26S proteasomes purified from immature and mature oocytes showed a difference in the blots of α2 and α4, with an additional spot detected in the 26S proteasome from immature oocytes (in G2-phase).We identified cDNA for three α-type subunits (α1, α5 and α6) of Xenopus 26S proteasome are modified in Xenopus immature oocytes and two subunits (α2 and α4) are modified meiotic cell cycle-dependently.Six of α-type subunits of the  Eukaryotic cells, from yeast to human, contain large nonlysosomal proteases called proteasomes . The 26SXenopus and goldfish as α 4 [Xenopus and prepared antibodies for a total of five subunits. Using a set of specific antibodies, we analyzed changes in all α subunits composing the 26S proteasome during the meiotic cell cycle. We demonstrated that 6 of the subunits exist as a heterogeneous population in frog oocytes and identified another subunit in addition to α4 which was modified meiotic cell cycle dependently.Fully grown frog oocytes arrest in the late G2 phase of meiosis. Maturation-inducing hormone (MIH) acts on the oocytes, inducing final maturation and triggering germinal vesicle breakdown (GVBD), and the oocytes arrest again at the second meiotic metaphase until fertilization. The proteasomes are thought to be involved in regulating the maturation and fertilization of oocytes ,7. Previh as α 4 ,9. In thXenopus EST database was conducted using known proteasomal subunit α cDNAs. From the data for each subunit, full-length ORFs were obtained by PCR. The amplified cDNAs were 741, 726 and 786 bp long. The clones encode proteins of 246, 241 and 261 amino acid residues with a predicted molecular mass of 27463, 26402 and 29327 daltons, respectively  [Xenopus 20S proteasome. We named these clones α1_xl, α5_xl and α6_xl (α1, α5 and α6 subunits of Xenopus laevis) according to a systematic nomenclature [Xenopus 20S proteasome. Overall identity between the subunits was 25.1–38.4 %. A consensus sequence for α-type proteasomal subunits was conserved. Interestingly, a conserved sequence for β-type proteasomal subunits was found in the α3 subunit [A BLAST search of the 2–69.1%) ,13 and y2–69.1%) -16  were raised against purified recombinant proteins. The specificity of the antibodies was examined by immunoblotting with the cytosol fraction and the purified 26S proteasome  ioteasome . The phon et al.  (1996) sn et al. . Recent 96 ioteasn et al. ,29. Cycln et al. . Calcium[Xenopus . It can [Xenopus . PossiblXenopus, there is a possibility that the changes in the spots identified in this study may derive from differential expression of alternative subunits from paralogous genes.Recently, alternative subunits of proteasomes have been identified. In Drosophila where alternative α-type, β-type and 19S cap subunits are expressed from separate genes during spermatogenesis  and in Aα1_xl, α5_xl and α6_xl) of X. laevis were identified.(1) cDNAs for three α-type proteasome subunits (X. laevis.(2) Six subunits but not α7_XL are modified in immature oocytes in X. laevis.(2) α2, α4_XLs are modified during the meiotic cell cycle in Xenopus laevis) were purchased from Jo-hoku Seibutsu Kyozai (Shizuoka, Japan) and maintained till used. 26S proteasomes were purified from immature oocytes and ovulated oocytes as described [Frogs (escribed .SDS-PAGE was carried out according to the method of Laemmli  (1970). Xenopus EST database was conducted using known proteasomal subunit α cDNAs. From the data obtained for each subunit, independent sequences were linked and the full-length ORF sequences were eliminated(α1: BG347128 and CB558360, α5: BQ398972 and BJ043946, α6: BJ072624 and BJ091555). The specific primers for amplification of the full-length ORF were α1: 5'-GGAATTCCATATGTCTCGGGGATCTAGCGCG-3' and 5'-CCGCTCGAGGTCACGCTCAGCTAGTGCAAC-3', α5: 5'-GGAATTCCATATGTTCCTAACCCGCTCCGAG-3' and 5'-CCGCTCGAGGATGTCCTTAATAACTTCCTC-3', and α6: 5'-GGAATTCCATATGTTTCGCAATCAGTATG-3' and 5'-CCGCTCGAGGTGCTCCATAGGCTCCTCCTGC-3'), in which EcoRI (5'end) and XhoI (3'end) recognition sequence was added for cloning to the vector pET21a (Novagen). PCR was carried out using KOD DNA polymerase (TOYOBO) or LA taq DNA polymerase (TaKaRa), with Xenopus ovarian cDNA as a template, and the product was cloned to pET21a. The DNA sequencing was performed using a 377A DNA sequencer (Applied Biosystems). The sequences that include the full-length ORF identified here were deposited into GenBank (accession nos. AB164677, AB164678 and AB164679 for α1_xl, α5_xl and α6_xl, respectively). Pairwise comparisons of sequence homology were conducted using the Genetyx-Mac ver.12 computer program (Software Development, Tokyo, Japan).Identification and sequence analysis of cDNAs. A BLAST search of the E. coli BL21 (LysE) and purified by SDS-PAGE as described [The recombinant proteins were produced in escribed . Polycloescribed . Anti sebp, base pair; BLAST, basic local alignment search tool; cDNA, DNA complementary to RNA; EST, expressed sequence tags; kDa, kilodalton; NEPHGE, non-equilibrium pH gradient gel electrophoresis; PCR, polymerase chain reaction; SDS-PAGE SDS-polyacrylamide gel electrophoresis; 2D-PAGE, two-dimensional-PAGE.YW carried out cDNA cloning, expression of recombinant proteins, antibody production and 2D-PAGE analysis. MT and RH participated in cDNA cloning, expression of recombinant proteins. YN and KI participated in coordination of the study. TT carried out the protein purification and also participated in the design of the study and drafted the manuscript.


There is now good evidence that antiplatelet agents  prevent pre-eclampsia, a leading cause of morbidity and mortality for pregnant women and their babies. A Cochrane Review identified moderate, but clinically important, reductions in the relative risks of pre-eclampsia (19%), preterm birth (7%) and perinatal mortality (16%) in women allocated antiplatelets, rather than placebo or no antiplatelet.Uncertainty remains, however, about whether some women (in terms of risk) benefit more than others, what dose of aspirin is best and when in pregnancy treatment should ideally start. Rather than undertake new trials, the best way to answer these questions is to utilise existing individual patient data from women enrolled in each trial.Systematic review with meta-analysis based on individual patient data. This involves the central collection, validation and re-analysis of thoroughly checked data from individual women in all the available randomised trials.The objective is to confirm that antiplatelet agents, given during pregnancy, will reduce the incidence of pre-eclampsia. The review will then determine the size of this effect, and whether antiplatelets delay the onset of pre-eclampsia or its impact on important outcomes for women and their babies. It will also explore whether the effect of antiplatelets differs by womens' risk profile; when commenced during pregnancy; and/or by dose.The PARIS  Collaboration has been formed to undertake the review. This will be the first individual patient data review in the perinatal field. Final results should be available by 2006–7. World-wide, over half a million women die each year of pregnancy related causes with 99% of these occurring in low resource countries ,2. An esHigh blood pressure is common during pregnancy: approximately one in ten women will have their blood pressure recorded as above normal at some point before delivery . For womAlthough the outcome for most women is good, pre-eclampsia and eclampsia (the rare occurrence of seizures superimposed on the syndrome of pre-eclampsia) are major causes of maternal mortality. In low resource countries pre-eclampsia and eclampsia account for 10–15% of maternal deaths  whilst iPre-eclampsia is a multi-factorial condition. Although its aetiology remains unclear, there have been significant advances in the understanding of the pathophysiology of the disorder. The primary lesion is thought to be deficient trophoblast invasion of the maternal spiral arteries in the second trimester, leading to underperfusion of the uteroplacental circulation and placental ischaemia . The resIt is further hypothesised that the effect of antiplatelets may be different if treatment is started before placental implantation is complete. . If thisAlthough it is known that pre-eclampsia is a multi-system disorder, the relationship between the placental pathology and maternal endothelial response is not fully understood. Numerous maternal factors can predispose to the disorder, such as previous pre-eclampsia, diabetes, renal disease, chronic hypertension or other risk factors . The synThe effects of antiplatelet agents were first evaluated in small randomised trials, which reported striking reductions in the risk of hypertension and proteinuria -29. ThesThe updated Cochrane Review identified 51 trials with over 36,500 pregnant women evaluating antiplatelet agents, principally low dose aspirin, for the prevention of pre-eclampsia . Nine ofThe aim is now to extend this review based on aggregate data, to utilise the available data for every individual woman in each trial to help address these remaining questions. The use of individual data for each woman will allow for more powerful and flexible analysis of both subgroups and outcomes [These effects are much smaller than had initially been hoped for but, nevertheless, potentially they have considerable public health importance. The conclusion from this aggregate data review was that for most low-risk women large numbers of women would need to be treated with antiplatelets agents to prevent one episode of either pre-eclampsia or perinatal death. Whether there are specific high risk sub-groups of pregnant women for whom there might be greater benefits, remains unclear, as does the best time to initiate treatment, and at what dose. outcomes ,34. This• Many studies were excluded because publications did not report sufficient information to allow them to be included in the review.• The published aggregate data is variable in the completeness of outcome reporting and in definitions used between trials.• The aggregate data meta-analyses were restricted to analysing outcomes for complete trials. As many trials included women with a wide range of risk profiles at trial entry, trials could only be defined by average values. Such aggregated outcomes such as 'proportion of women developing pre-eclampsia' conceal a range of severity of disorder and so it was not possible to explore fully whether the effectiveness of antiplatelet therapy differed according to risk.• It was also difficult to make precise recommendations about when to start treatment. While most trials could generally be classified as starting treatment at earlier or later in gestational ages, there was a wide within-study variation that could not be explored with aggregate data analyses.• There is a markedly skewed distribution of effect estimates for each trial around the summary effect on pre-eclampsia, with more small positive trials than small negative trials. This suggests the possibilities of publication bias or that the differences in the characteristics of the women enrolled in small and large studies has an important influence on the effects of antiplatelet agents.• Obtaining individual patient data from previously excluded trials will allow assessment of whether these trials are eligible for inclusion, both in terms of available outcomes and methodological quality. This will potentially increase the power and scope of the analyses.• Trial level information obtained by direct discussion with the trialists enables clarification of the definitions and measurements used. Data for measured, but previously unreported, outcomes can also often be obtained and trialists, who are part of a Collaborative group, may be able to provide missing outcome data. Furthermore, we will be able to apply common definitions across trials based on each woman's baseline and clinical data. For example, where possible, we will define pre-eclampsia based on a set of uniform criteria.• Subgroup analyses will be performed for a number of different risk factors using individual patient's specific risk data, rather than using the aggregated risk profile of all women enrolled in a particular trial.• By obtaining gestational age at randomisation for each individual woman, more precise patient-based analyses can be performed exploring whether gestation at treatment commencement alters the effectiveness of antiplatelets.• Formation of a Collaborative group, is likely to lead to a more complete identification of all relevant trials, including those previously unpublished. This may help overcome the potential for publication bias. The patient-level data will also allow us to explore whether there were important differences in the characteristics of women enrolled in small and large trials.The objective of this review is to confirm that antiplatelet agents, given during pregnancy, reduce the incidence of pre-eclampsia. The review will then determine the size of this effect, and whether antiplatelets reduce the severity of pre-eclampsia and/or its impact on important outcomes for women and their babies. It will also explore whether the effect of antiplatelets differs by womens' risk profile, when commenced during pregnancy, and/or by dose.The main questions to be addressed in this review are:• Do antiplatelet agents, primarily low dose aspirin, have clinically important benefits for women at risk of developing pre-eclampsia and their babies?Investigation of this hypothesis will also explore whether the treatment effect differs, in a clinically meaningful way, between women with different risk factors such as those with a history of early onset pre-eclampsia, renal disease, diabetes, chronic hypertension, or autoimmune disease.• Does the planned dose of aspirin affect outcome in terms of preventing or delaying the onset of pre-eclampsia or other adverse outcomes, such as preterm birth or perinatal death?• Do the effects of aspirin differ according to gestation at onset of treatment?The Cochrane Controlled Trials Register and hand searching of relevant journals. Trialists will be asked if they know of any further studies. [See The search strategy to identify potentially eligible studies will include a search of the register of trials developed and maintained by the Cochrane Collaboration Pregnancy and Childbirth Review Group. Details of how this register is maintained are available elsewhere ,36, but ies. See  for the The inclusion and exclusion criteria for the types of study designs, participants, interventions and data completeness to be included in the review are listed below. Each potentially eligible study will be assessed independently by two members of the Secretariat, unblinded to the trial's identity. Any differences of opinion regarding the assessment of the inclusion criteria will be resolved by discussion between the two assessors. If differences cannot be resolved, a third member of the Secretariat will be asked to assess the study. If individual patient data are unavailable from an eligible trial, the trial will remain included in the review and aggregate data will be used.Studies will be included in the review if they were randomised trials. Quasi-random study designs, such as those using alternate allocation, will be excluded. The level of allocation concealment within each trial will be assessed according to the criteria outlined in the Cochrane Handbook , and claParticipants will be pregnant women at risk of developing pre-eclampsia. Women who started treatment postpartum will be excluded, as will those who already have a diagnosis of pre-eclampsia at trial entry .The interventions will be any comparisons of an antiplatelet agent (such as low dose aspirin or dipyridamole), or any combination of antiplatelet agents, compared with placebo or no antiplatelet agent. This is regardless of dose, mode of administration and irrespective of whether the antiplatelet is in combination with another drug. Trials that assessed only physiological outcomes following a short duration of intended therapy will be excluded.The main analyses will include all trials that fulfill the previous inclusion criteria, regardless of completeness of follow-up. Sensitivity analyses will be undertaken to assess the effect of the inclusion of data from trials where only small numbers of enrolled participants have available outcome data. The threshold for an acceptable level of data completeness may vary by outcome. For example, for long-term follow-up of women and children, data may be included if follow-up was less than 80% provided that substantive bias between the groups was unlikely. Other outcomes from each trial may only be included in the analysis if available for 80% or more of women.The individual patient data provided by the Collaborators will be de-identified, re-coded as required and stored in a custom-designed Microsoft ACCESS database. It will not include any patient identifying information such as names or addresses. Electronic data will be located on a secure, password protected network server. Copies of hardcopy data will be stored in locked filing cabinets until converted into electronic format, and will then be securely destroyed. Only authorised personnel will have access to the data. All data will be securely stored and archived according to the policies of the major funder, the Australian National Health and Medical Research Council.The data will be checked with respect to range, internal consistency, consistency with published reports and missing items. Trial details such as randomisation methods, and dose and timing of the interventions will be cross-checked against any published reports, trial protocols and data collection sheets. Integrity of the randomisation process will be examined by reviewing the chronological randomisation sequence and pattern of assignment, as well as the balance of prognostic factors across treatment groups (taking into account stratification factors). Inconsistencies or missing data will be discussed with the individual trialists and attempts will be made to resolve any problems by consensus. Each trial will be analysed individually, and the resulting analyses and trial data will be sent to the trialists for verification.There has been extensive consultation with the PARIS Collaborative Group regarding what data items to collect for each woman in the analyses. The following section contains the list of data items requested from trialists, which has been compiled following this consultation. More detailed definitions for the data items listed below can be found in Tables 1 informed consent2 dates trial opened and closed to accrual3 total number of women randomised4 treatments used in each arm of the trial5 intended duration of treatments6 definitions of key outcomes used in the trial7 method of random allocation8 stratification factors used9 methods of allocation concealment1 unique identifier for the enrolled woman, coded for anonymity2 date of randomisation3 gestational age at randomisation, or best estimate of expected date of delivery or last menstrual period4 woman's date of birth or age5 any previous pregnancy6 blood pressure (systolic, diastolic, whether diagnosed as raised)7 presence of proteinuria8 presence of oedema9 risk factors – multiple pregnancy, autoimmune disease, renal disease, diabetes, chronic hypertension, previous gestational hypertension or pre-eclampsia/eclampsia (including early onset disease), family history of pregnancy-related hypertensive disorders, previous fetal growth restriction, previous perinatal death, abnormal uterine artery Doppler flow and abnormalities on other diagnostic tests1 hypertension during pregnancy (highest blood pressure, diagnosis of severe hypertension)2 proteinuria during pregnancy (including date and/or gestation at onset)3 oedema during pregnancy4 pre-eclampsia5 drug treatment for pre-eclampsia or hypertension6 severe maternal morbidity 7 onset of labour (spontaneous or induced or pre-labour caesarean section)8 mode of delivery 9 antepartum haemorrhage 10 postpartum haemorrhage and/or estimated blood loss at delivery11 maternal mortality1 gestational age at birth and/or date of birth2 birthweight3 gender4 small for gestation age 5 miscarriage or stillbirth: date and/or gestational age at death/loss6 neonatal or infant death: date and/or age at death7 admission to special care baby unit or neonatal intensive care unit8 use of assisted ventilation9 number of days in hospital or date of hospital discharge10 neonatal bleeding (for example, periventricular haemorrhage)11 child growth and development This section contains a summary of the planned analyses. The full, detailed analysis plan will be discussed and agreed upon by the Collaborators before any data have been analysed.2 statistic will also be used to assess consistency of results.Analysis will aim to be of all women ever randomised and will be based on intention to treat. In the main analyses a two stage approach will be taken. Outcomes will be analysed in their original trial and then these separate results will be combined to give an overall measure of effect. A fixed effect model will be used and the assumption of homogeneity of treatment effects will be tested using the chi squared test. The IThe main analyses comparing the effect of antiplatelet agents with placebo or no antiplatelet agents will be undertaken for all outcomes listed below, for the woman and any fetus/baby. The planned sub-group and sensitivity analyses will be restricted to the designated main outcomes listed as follows:• pre-eclampsia• pregnancy loss / neonatal death• pre-term birth• small for gestational age infant• pregnancy with serious adverse outcome• early onset pre-eclampsia• maternal death• severe maternal morbidity• antepartum haemorrhage• placental abruption• induction of labour• Caesarean section delivery• postpartum haemorrhage• gestation at delivery• infant admission to special care or neonatal intensive care unit• infant required assisted ventilation• neonatal bleeding• infant deathThe planned sub-group analyses will be restricted to the designated main outcomes unless there are clear indications for expanding the analyses further.The effect of antiplatelet therapy may vary across the trials in the meta-analysis because they have used different agents in different ways. To explore this further, analyses are planned whereby trials will be grouped according to the agent used and by dose. These analyses will focus on the main outcomes. Trials will be classified into subsets based on the following:Trials will be grouped by the type of antiplatelet agent  given as the active treatment.In trials that used aspirin alone, trials will be grouped by planned aspirin dose (<75 mg, 75–149 mg, ≥ 150 mg).One of the strengths of individual patient data reviews is that it allows us to assess eligibility and outcome using individual women's characteristics. Subgroup analyses will explore whether any particular risk factors act as effect modifiers. That is, are there any particular types of women who benefit more or less from antiplatelet agents? These analyses will take account each individual woman's own characteristics, rather than relying on summary measures of the 'average' risk profile of all participants in an individual trial.Analyses will be undertaken to explore whether there are any particular types of women who benefit more or less from antiplatelet agents based on the following criteria:Women normotensive at trial entry:previous hypertensive disorders of pregnancy: previous early onset (≤33 weeks + 6 days gestation) pre-eclampsia or eclampsia / previous pre-eclampsia / previous gestational hypertension / no previous hypertensive disorders of pregnancy / no previous pregnancy but family history of hypertensive disorders of pregnancy / no previous pregnancy and no family history of hypertensive disorders of pregnancy- diabetes: pre-existing diabetes at enrolment / no pre-existing diabetes at enrolment- renal disease: pre-existing renal disease / no pre-existing renal disease- autoimmune disease: autoimmune disease / no autoimmune disease- multiple pregnancy: multiple pregnancy / singleton pregnancy- maternal age: <20 years / 20–35 years / >35 years. Maternal age may also be analysed as a continuous variable.- diagnostic test results: abnormal uterine artery Doppler scan / other diagnostic test abnormalities / no abnormal diagnostic test results- previous SGA: previous small for gestational age infant / previous infant not small for gestational age / no previous infant- primigravida: first pregnancy with no other risk factors / first pregnancy with one or more risk factors / second or subsequent pregnancy with one or more risk factors / second or subsequent pregnancy with no risk factors- Women with hypertension at trial entry:hypertension: gestational hypertension / chronic hypertension- To determine whether antiplatelet agents are differentially effective if given earlier in pregnancy and to determine the magnitude of any difference, gestational age at randomisation will be primarily analysed as a continuous variable in regression analyses. However, a subgroup analysis with women classified according to the following categories may also be performed: <16 weeks, 16–19 completed weeks, 20–23 completed weeks, 24–27 completed weeks, ≥28 weeks gestation. If numbers are insufficient for any category, categories will be combined.a. To assess whether results are robust to the inclusion or exclusion of particular types of trials or patients, the following sensitivity analyses will be conducted:• exclusion of trials that did not use a placebo for the control group• exclusion of trials of small size• exclusion of poor quality trials b. To assess whether results are robust to different methods of analysis or different definitions of pre-eclampsia ,39, the • comparison of analyses using random effects and fixed effect models• comparison of analyses using individual patient data (IPD) only with analyses using individual patient data and aggregate data where IPD unavailable• comparison of analyses using different definitions of pre-eclampsiaThese sensitivity analyses will be carried out for the main outcomes.Depending on what data are available, the level of heterogeneity encountered and available time a one-stage modeling approach may also be undertaken to further explore important key outcomes as appropriate.Participants in the individual trials have previously given informed consent to participate in their respective trial. The data for this project are to be used for the purpose for which they were originally collected and are available through an agreement between all trialists of the PARIS Collaboration. These trialists remain the custodians of their original individual trial data at all times. Data are provided on the stipulation that all trials have received ethical clearances from their relevant bodies.Membership of the PARIS Collaboration will be representative(s) from each of the trials contributing data to the review with an accompanying project coordination and data management structure as described in this section.The membership and responsibilities of each of these management groups is as follows:1 (co-chair), L Duley2 (co-chair), L Askie1 (project coordinator), M Showell1 (project administrator), B Farrell2, L Stewart3, M Clarke4, J King5, C Roberts.1 The first six members act as the Secretariat.The Steering Groupwill be responsible for project management decisions and will meet approximately 4–6 times per year, usually via teleconference. Membership: D Henderson-Smart1 Centre for Perinatal Health Services Research, University of Sydney, Australia;2 Resource Centre for Randomised Trials, University of Oxford, Oxford, UK;3 Medical Research Council Clinical Trials Unit, London, UK;4 UK Cochrane Centre, Oxford, UK;5 Royal Women's Hospital, Melbourne, Australia.ad hoc meetings. Co-chairs of the Advisory Group: C Redman, C Roberts.The aim of the Advisory Group is to facilitate representative input from the Collaborative Group to the Steering Group. Membership of the Advisory Group will include people who have contributed to the trials included in the project and other international experts. Each trial that recruited over 1000 women will be invited to have a representative on the Advisory Group. This group will not have regular meetings, but may be consulted from time to time by means of email or teleconference, and may have occasional All potentially eligible trialists will be contacted and invited to become members of the Collaborative Group. The corresponding author for each study will be contacted in the first instance. If there is no response, the associated statistician, data manager and/or other authors will be contacted. This process will be updated annually for the duration of the project, to ensure that new trialists are offered the opportunity to join the project and contribute their data.The project will be coordinated from the Centre for Perinatal Health Services Research (CPHSR), University of Sydney, NSW, Australia. The coordination centre will be responsible for the daily management of the project including correspondence, newsletter production, maintaining current trialist contact information and meeting/teleconference organisation.The data management centre, based at the UK Cochrane Centre, will be responsible for the receipt, storage, and analysis of project data as directed by the Collaborative Group via the Steering Group.All members of the Collaboration, including the Steering Group, the Advisory Group, and representatives of each participating trial, will be invited to attend regular Collaborators' meetings. These meetings will be scheduled, where possible, to coincide with the biennial International Society for the Study of Hypertension Pregnancy (ISSHP) congresses. The meetings will be designed to allow maximum input from the participating trialists into the design, conduct, analysis and reporting of the project's results. The final Collaborators' meeting, at which the results will be presented for discussion, is scheduled for 2006 in Oxford, UK. The discussion at this meeting will provide the basis for the paper publication.The National Health and Medical Research Council (NHMRC) of Australia have provided funding for the project through the Centre for Perinatal Health Services Research, University of Sydney. These funds are: a three year project grant (ID: 253636) for the overall project administration base in Sydney, and a Sidney Sax Public Health Postdoctoral Fellowship (ID: 245521), based in Oxford and Sydney. Additional support is being provided by the Resource Centre for Randomised Trials and the UK Cochrane Centre, located in Oxford, UK, and the Medical Research Council Clinical Trials Unit in London, UK.The results of the project's analyses will be presented to, and discussed with, the Collaborative Group before publication. The aim of publication will be presentation of the results, rather than their interpretation. The main manuscript will be prepared by the Secretariat, and then circulated to the Steering and Advisory Groups for comment and revision. The revised draft paper then will be circulated to all members of the Collaborative Group for comment before publication. All publications using these data will be authored in the name of the PARIS Collaboration, as follows: Perinatal Antiplatelet Review of International Studies (PARIS) Collaboration.Despite good evidence that antiplatelet agents  reduce the incidence of pre-eclampsia and its consequences, such as preterm birth and perinatal mortality, uncertainty remains regarding whether some women (in terms of risk) benefit more than others, when in pregnancy treatment should ideally start, and whether treatment effectiveness is dependent on antiplatelet dose.The best way to answer these questions is to utilise existing individual patient data from all women enrolled in trials that have addressed this question. This approach has been described as the 'gold standard' of systematic review methodology as it allows for more powerful and flexible analysis of both subgroups and outcomes.The PARIS Collaboration has been formed to undertake a systematic review of all available trials, with meta-analysis based on individual patient data, to answer these important clinical questions. This will be the first individual patient data review in the perinatal field. Provision of data by the participating Collaborators commenced in 2004, and results will be ready for presentation in 2006. Following consultation and discussion with the Collaborative Group, the main publication is expected in early 2007.The author(s) declare that they have no competing interests.All authors, the named members of the PARIS Collaboration Steering Group, contributed to the development of the protocol, and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:List of eligible trials text document listing potentially eligible trialsClick here for fileSuggested coding sheet table listing variables and suggested codingClick here for fileData provision form form to collect trial level data and data provision proceduresClick here for file


Since many tumor suppressor genes are recessive lethals, we screened our colony for lines that display early mortality and/or gross evidence of tumors. We identified 12 lines with elevated cancer incidence. Fish from these lines develop malignant peripheral nerve sheath tumors, and in some cases also other tumor types, with moderate to very high frequencies. Surprisingly, 11 of the 12 lines were each heterozygous for a mutation in a different ribosomal protein (RP) gene, while one line was heterozygous for a mutation in a zebrafish paralog of the human and mouse tumor suppressor gene, neurofibromatosis type 2. Our findings suggest that many RP genes may act as haploinsufficient tumor suppressors in fish. Many RP genes might also be cancer genes in humans, where their role in tumorigenesis could easily have escaped detection up to now.We have generated several hundred lines of zebrafish  A screen for tumour suppressor genes in zebrafish has identified several genes encoding ribosomal proteins, indicating that these genes might be an unappreciated class of cancer genes The third group of RP mutant lines included five lines that were not tumor prone. These lines, with mutations in L3, L24, LP1, S12, and S15, were indistinguishable from controls in tumor incidence and spectrum . We also analyzed a total of 65 RP heterozygotes with grossly visible tumors (including the fish represented in (NF2a) of two distinct zebrafish genes that are highly homologous to the mammalian neurofibromatosis type 2 gene (NF2). The insertion abrogates expression of this gene in homozygous mutant embryos  at approximately six-week intervals between eight and 14 mo of age. As shown in Dominant mutations that predispose vertebrates to cancer can be activated oncogenes, recessive tumor suppressors, or haploinsufficient tumor suppressors . SeveralIn mice, a tumor cell line has been described in which one copy of an RP gene is deleted and the other copy has suffered a mutation that may contribute to tumorigenesis . To ruleIn yeast, a decrease in the amount of at least some RP genes results in a reduction in the amount of the corresponding ribosomal subunit and a reduction in the number of assembled ribosomes . To deteIn this study, we have found that heterozygous mutations in 11 different ribosomal protein genes predispose zebrafish to cancer, predominantly to zMPNSTs, but also to other rare tumor types. All of these mutations reduce RP gene expression, indicating that these 11 genes are not oncogenes. Moreover, in the tumors we examined, the wild-type allele appeared to be present and did not contain point mutations; thus these genes are not recessive tumor suppressors. Rather, our findings suggest that these 11 genes are haploinsufficient tumor suppressor genes; that is, reducing their activities by about a factor of two increases the likelihood of cancer. These findings raise two important, unanswered questions: first, how do these mutations lead to cancer, and second, do similar mutations cause cancer in humans?S7, S8, S15a, S18, S29, L7, L13, L23a, L35, L36, and L36a, predispose to cancer suggests that a function shared by RPs underlies their role in this phenotype. However, not all RP genes were cancer genes: S12, S15, L3, L24, and LP1 heterozygotes appeared normal. This raises the possibility that the oncogenic RP genes could conceivably share some novel biological function independent of their role in the ribosome and that inhibition of this function leads to tumor formation. Individual RPs have been implicated in a wide variety of biological functions, including cell cycle progression, apoptosis, and DNA damage responses  are associated with a mutation of rpS19 , and thirpS19 . It has rpS19 , but it While these examples from mouse and human are consistent with the idea that mutations in individual RP genes might contribute to tumorigenesis in mammals, they have seemed to be unusual examples, rather than suggesting that RP genes in general might be potential cancer genes. Our study suggests for the first time, we believe, that this is a general property of many RP genes. The possibility that a reduction in ribosome levels might be oncogenic in mammals is further supported by the fact that mutations in DKC1, a pseudouridine synthase that is required for rRNA processing and for properly functioning ribosomes, cause dyskeratosis congenita, a disease characterized by both premature aging and increased tumor susceptibility .rp mutations will prove to increase the incidence of tumors in humans as they do in zebrafish. If so, it may be advantageous to devise diagnostic strategies based on ribosomal protein levels or on a function that these proteins share, for example, in translation, rather than on the analysis of such a large number of individual genes.If RP genes frequently cause human cancers, it is not at all certain that their role would have been detected. Even a deliberate search for their involvement in human cancers would be difficult because there are so many (80) RP genes. This plethora of genes, the fact that it is hard to know which tumor type(s) to examine for RP mutations, and the fact that the mutations might lie in regulatory elements rather than protein-coding regions of the genes would make such a search difficult. Nonetheless, given the high degree of conservation of biological mechanisms among vertebrates, it seems likely that NF2a is a tumor suppressor gene in this system demonstrate the power of large-scale, forward-genetic screens in the zebrafish to identify new disease susceptibility genes.In summary, by examining aging populations of mutant lines of fish with defects in embryonic essential genes, we identified a novel group of cancer genes. The ability to identify cancer genes by screening populations of fish heterozyogous for recessive embryonic mutations and the reassuring finding that The insertional mutagenesis screen was carried out as previously described . Stocks Adult fish were euthanized in ice water and fixed within 30 min in Bouin's solution, embedded in paraffin, and sectioned as previously described .DNA was prepared from tumor tissue or tail tissue isolated from fish prior to fixation for histology. PCR was conducted with one primer complementary to proviral sequence and two primers complementary to sequences on either side of the insertion for the appropriate mutation. Primer sequences were as follows: hi10: 10gen5 (5′-CAGCACAGATTCTTGAAAGCGCC-3′), 10gen3 (5′- GCATATGTAGCATCTCGAAGGTCC-3′), and NU3X (5′- TGATCTCGAGCCAAACCTACAGGTGGGGTC-3′); hi258: 258A5a (5′-GGTACGTCTGTGCTTATGTTTGTGTC-3′), 258A3a (5′-TCTCAAGACTTCATCCATTCATAATTCTGC-3′), and NU3X; hi1974: 1974c1 (5′-CTACACCACAGGTATCTCAAGGG-3′), 1974c1est3 (5′-CCACCACGGACTCTTATTGTGTG-3′), and IPL3 (5′-TGATCTCGAGTTCCTTGGGAGGGTCTCCTC-3′).rpL36a: 10rt5 (5′-CAACCATGGTAAACGTACCGAAG-3′) and 10RTR (5′-CACAAAAGAAGCACTTGGCCCAGC-3′); rpL35: 258RTF2 (5′-GCTGCTTCCAAGCTCTCAAAAATCC-3′) and 258RTR (5′-TGCCTTGACGGCGAACTTGCGAATG-3′); rpS8: 1974RTF1 (5′-TCTCAAGGGATAACTGGCACA-3′) and 1974RTR1 (5′-GAACTCCAGTTCTTTGCCCTC-3′); actin: actinF (5′-CATCAGCATGGCTTCTGCTCTGTATGG-3′) and actinR (5′-GACTTGTCAGTGTACAGAGACACCCT-3′). For visualization of 18S and 28S RNA, two embryo equivalents of RNA were electrophoresed through a nondenaturing agarose gel containing 0.5 μg/ml ethidium bromide. For detection of beta actin RNA, four embryo equivalents of RNA were electrophoresed through a 7.5% formaldehyde/MOPS-buffered agarose gel, blotted to Hybond N+ , and hybridized with a random primed beta actin probe.RNA was prepared from mutant and wild-type embryos using Trizol reagent . For RT-PCR, serial dilutions of first strand cDNA were amplified for 30 (hi1974) or 35 (hi10 and hi258) cycles using the following primers for the genes indicated: Figure S1rpl36 locus, which has its own scale bar.The genomic sequence of part of each of these genes is represented as exonic (boxed) and promoter or intronic (line). White boxes represent 5′ UTR while shaded boxes represent coding exons. Where no white boxes are shown, the location of the 5′ UTR and beginning of the coding region has not been determined relative to the part of the locus shown here. In all cases, at least one coding exon  is downstream of the region of the gene represented here. The position and orientation of the proviruses are shown above each genomic sequence. All drawings are to the scale of the top scale bar, except the (62 KB PDF).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the genes discussed in this paper are L13 (AY561516), L23a (AY561517), L24 (AY099532), L3 (AY561514), L35 (AF506205), L36 (AY561518), L36a (AY099511), L7 (AY561515), LP1 (AY561519), NF2a (AY561520), S12 (AY561510), S15 (AY561511), S15a (AY561512), S18 (AY099517), S29 (AY561513), S7 (AY561508), and S8 (AY561509).The GenBank (


APOE) ε2/ε3/ε4 polymorphism, which also is a significant predictor of variation in the risk of coronary heart disease (CHD) and CHD death. Here, we test the hypothesis that the APOE polymorphism may modulate the cholesterol-raising effect of coffee.The response of serum cholesterol to diet may be affected by the apolipoprotein E  and women (78%) aged 29–65 years, took part in a study with four intervention periods: 1 and 3) a coffee free period of three weeks, 2 and 4) 600 mL coffee/day for four weeks.APOE ε2 positive individuals had significantly lower total cholesterol concentration at baseline (4.68 mmol/L and 5.28 mmol/L, respectively, p = 0.01), but the cholesterol-raising effect of coffee was not influenced significantly by APOE allele carrier status.APOE ε 2 allele is associated with lower serum cholesterol concentration. However, the APOE polymorphism does not seem to influence the cholesterol-raising effect of coffee.The  APOE gene sequence results in the 3 common alleles (ε2, ε3 and ε4), which can produce 6 different genotypes (ε2/ε2, ε2/ε3, ε2/ε4, ε3/ε3, ε3/ε4 and ε4/ε4). The ε2, ε3 and ε4 alleles encode three distinct forms of apoE (E2, E3 and E4) and have approximate frequencies of 8%, 77%, and 15%, respectively, in white populations [APOE ε2 allele display lower levels of plasma cholesterol compared with individuals carrying the APOE ε3 allele, whereas individuals with the APOE ε4 allele show higher levels of plasma cholesterol, especially LDL-cholesterol [APOE ε3/ε4 and ε4/ε4 genotypes absorb cholesterol effectively and have higher non-fasting serum triglyceride values than ε4 negative individuals [APOE gene is shown to be a significant predictor of variation in the risk of coronary heart disease (CHD) and CHD death [APOE ε4 allele. The APOE ε4 allele is also considered a strong risk factor for Alzheimer's disease [Apolipoprotein E (apoE) is a structural component of triglyceride-rich lipoproteins, chylomicrons, very-low-density lipoproteins (VLDL), and high-density-lipoproteins (HDL) . VariatiHD death , but theHD death . Both thHD death  and the HD death  suggest  disease -16.APOE ε2/ε3/e4 polymorphism.The serum cholesterol-raising effect of coffee is due to the diterpenes kahweol and cafestol . EarlierThe study was organised as a prospective, controlled study with four consecutive trial periods. The first and third periods were 3 weeks of total coffee abstention. The second and fourth period consisted of 4 weeks with the subjects consuming 600 mL filter brewed coffee/day.The main outcome or effect variable was total serum cholesterol and the effect was assessed as the difference between the measurements at the beginning and the end of the coffee free periods (coffee abstention) and the difference between measurements at the beginning and at the end of the four weeks of coffee consumption Figure . Trial dParticipants were recruited by advertising in Gothenburg's major newspaper. Inclusion criteria were age-range 30–65 years, free from clinically recognized chronic diseases, such as cardiovascular diseases, cancer, renal disorders, liver disease and diabetes mellitus. The participants were required to be free from anti-epileptic or cholesterol lowering drugs, and had been using coffee on a regular basis for at least five years and were currently non-smokers (at least for the last six months).During the coffee drinking periods the participants were instructed to drink about 600 mL filter brewed coffee/day (4 cups), according to standardised measures. The coffee was provided to guarantee that they were all exposed to the same brand and quality of filter brewed coffee. All participants also got the same kind of standardised coffee filter and measuring spoon.The coffee filters used were of the brand Euro-Shopper, made by Indupa N.V., Zaventem, Belgium. Divergence from the 4 cups was reported. The participants were allowed to drink tea and other caffeine containing beverages during the coffee-free periods.Non-fasting blood samples were drawn at inclusion and at three, seven, ten and fourteen weeks after start of the study. Prior to analysis, prepared serum was stored at -70°C.2) was recorded once during the study. Blood pressure was recorded by manual device and EKG and heart rate were recorded at all five visits.The blood samples were analysed for blood lipids  (Lp(a)) and urate in serum. Serum cholesterol and triglycerides were determined by an enzymatic procedure on a Hitachi 917 analyzer. HDL cholesterol was determined enzymatically after precipitation of VLDL, LDL and chylomicrones by α-cyklodextrinsulphate and dextransulphate. Determination of Lp(a) was done by the method Tint Elize Lp(a) of Biopool International. Serum urate was analysed by Hitachi 917 autoanalyser. Body Mass Index , but not in the ε2-positive group (Table 2-positive and the ε2-negative group according to baseline characteristics as sex, age, body mass index (BMI) and coffee consumption prior to the study (Table Coffee consumption resulted in a significant cholesterol increase in the εThe dietary survey did not reveal any important changes during the four intervention periods . Coffee APOE genotypes differ in the absorption efficiency of cholesterol from the intestine, in the synthesis rates of cholesterol and bile acids, and in the production of LDL apoprotein B [APOE e2/e3/e4 polymorphism [APOE polymorphism [APOE ε4 allele. Here, we investigate for the first time the possible influence of the APOE polymorphism on the cholesterol-raising effect of filtered coffee.Subjects with different rotein B ,26. Thismorphism ,28. One APOE ε4-positive individuals did not differ significantly from ε4-negative individuals with regard to baseline cholesterol concentration or coffee-induced changes in cholesterol concentration. However, we confirm that ε2-positive individuals display significantly lower cholesterol levels at baseline than ε2-negative individuals. A tendency was seen that ε2-positive individuals might be partly protected from the cholesterol increasing effect of coffee consumption. This was, however, only seen in the first trial period and will require further investigations. In conclusion, the APOE ε2/ε3/ε4 polymorphism is not a strong modulator of the cholesterol-increasing effect of coffee. Other genes should be discussed and further investigation is needed to see if there is a genetic factor in the cholesterol-raising effect of coffee.


Drosophila and C. elegans.A high-throughput method for genotyping by mapping InDels. This method has been used to create fragment-length polymorphism maps for  Caenorhabditis elegans and Drosophila melanogaster that facilitate genetic mapping with a minimum of manual input and at comparatively low cost.Small insertions or deletions (InDels) constitute a ubiquituous class of sequence polymorphisms found in eukaryotic genomes. Here, we present an automated high-throughput genotyping method that relies on the detection of fragment-length polymorphisms (FLPs) caused by InDels. The protocol utilizes standard sequencers and genotyping software. We have established genome-wide FLP maps for both  For humans and model organisms, such as worms and flies, the availability of high-density sequence polymorphism maps greatly facilitates the rapid mapping and cloning of genes -3. Key aDrosophila melanogaster [The most direct approach for SNP detection is sequencing of a PCR product spanning the polymorphism, but this is too costly and labor intense for high-throughput genotyping. For this reason, several different strategies and methods have been developed in order to detect SNPs more efficiently. In general, assays can be grouped into strategies, where the nature of the SNP is determined by directly analyzing the primary PCR product and those that require a secondary assay performed on the primary amplification product -6. An imnogaster .Caenorhabditis elegans and Drosophila are used regularly in genotyping projects [C. elegans. However, this approach is labor intensive and requires specialized chemistry and equipment. Using DNA microarrays, large numbers of SNPs can be analyzed in parallel, but the number of individuals that can be analyzed is low because of the high cost per chip [Of the methods that detect the SNP in a secondary assay, restriction fragment length polymorphism (RFLP) analysis are very popular . For thiprojects ,3,11. Hoprojects . Here, aprojects . Swan anprojects  have devper chip ,16.Besides SNPs, short tandem repeats (STRs) or microsatellites represent another class of sequence polymorphisms used for genotyping -21. STRset al. [Drosophila are of the InDel type. Also, two independent studies in C. elegans found that InDels constitute between 25% and 28% of all polymorphisms [Drosophila [C. elegans [Interestingly, a significant proportion of the currently available polymorphisms are caused by small insertions or deletions (InDels). Weber et al.  identifiet al.  who founorphisms ,14. In a elegans ).To take full advantage of this class of small InDel polymorphisms, we have developed a strategy that allows us to detect most, if not all, InDels by analyzing the lengths of primary PCR products on a capillary sequencer at single base-pair resolution. We call these assays fragment length polymorphism (FLP) assays. Importantly, this approach can easily be automated on standard robotic pipetting platforms as it involves a simple PCR reaction setup. Furthermore, allele calling is performed automatically using the Applied Biosystems GeneMapper software commonly used for genotyping STRs .C. elegans (one every 0.9 megabase-pair (Mbp)) and 54 FLP assays at 4 cM resolution for the Drosophila autosomes. This set of FLP assays allows us to rapidly map mutations to small chromosomal subregions with a minimum of manual input. Furthermore, we provide a list of predicted InDels for which additional assays can be readily designed in the chromosomal subregion of interest. Those non-validated FLPs enhance the resolution of the map by a factor of 5.6 and 17.9, respectively.To demonstrate the feasibility of this strategy, we have validated 112 evenly spaced FLP assays at 3 centimorgan (cM) resolution in C. elegans or Drosophila.We show the usefulness of this approach by identifying novel alleles of previously characterized genes. In summary, we have taken advantage of a publicly available dataset to adapt a technology widely used for STR analysis to genetic mapping. Thanks to the complete automation of genotyping, this approach is considerably faster, more reliable and cheaper than previously used mapping strategies in To detect a FLP, the region of interest is amplified in a standard PCR reaction with one fluorescently labeled primer, and the PCR products are directly analyzed on a capillary sequencer. Fragment sizes are determined automatically relative to an internal size standard with AppliedBiosystem's GeneMapper software . The software then allocates fragment sizes to previously calibrated genotypes.Taq polymerase has the tendency to catalyze the addition of adenosine (A) to the 3' end of PCR products. This activity could make it difficult to achieve the single base-pair resolution required to assay all available InDels and may hamper allele-calling . However2R090 and 231 samples homo- and heterozygous for the C. elegans Bristol and Hawaii backgrounds, respectively, for the 1-bp InDel ZH5-16. 2R090 exhibits both stuttering and A addition and hence is especially difficult to resolve . The genotype was correctly and automatically assigned by GeneMapper in all 423 assays. Thus, automated genotyping based on FLPs is sensitive down to single base-pair resolution and is extremely robust. The accuracy of FLP mapping is comparable to other methods such as TaqMan , minisein 2,000 ), and pyin 2,000 ).C. elegans, genetic experiments are performed almost exclusively in the background of the standard wild-type strain N2 (C. elegans variety Bristol) [C. elegans, variety Hawaii) has proved extremely useful [et al. [C. elegans were validated to date . Most failures to detect a FLP are probably due to original sequencing errors. The average distance between neighboring FLP assays is about 0.9 Mbp. This physical distance corresponds to about 3 cM, assuming 300 kb per map unit, and encompasses between 100 and a maximum of 500 genes  . For geny useful . When coy useful . Startin [et al. , 112 FLPe Figure . The cons Figure . The lenDrosophila FLP map, a set of 54 FLP assays (12 to 17 per arm of the two major autosomes) was validated from the list of polymorphisms identified by Berger et al. [C. elegans, the confirmation rate of the predicted Drosophila InDels was 80% (n = 30). Furthermore, another 509 InDels are predicted at 248 sites for which an assay can be established to discriminate between EP and FRT strains . The validated Drosophila FLP assays were evenly spaced on the genetic map with an average distance between neighboring assays of about 4 cM, corresponding to an average resolution of 1.77 Mbp on the physical map encompassing 95,55 Mbp [To establish a 5,55 Mbp ,28. TakiDrosophila FLP assays are in part derived from a set of InDels of size difference 7 bp or more . Howeveleotides , so far C. elegans FLP map, we mapped three previously characterized mutations on chromosome II that exhibit diverse phenotypes. Those were the centrally located let-23(sy1) allele that causes an 80% penetrant vulvaless phenotype [rol-1(e91) in the middle of the left chromosome arm, which causes the animals to roll around their body axis [unc-52(e444) mutation located at the right end of the chromosome, which results in a paralyzed phenotype [1 cross-progeny was selected (F1 self-progeny would exhibit a mutant phenotype). Finally, mutant self-progeny was isolated in the F2 generation and used for genotyping  with one centrally located FLP assay per chromosome  mutation showed linkage to the X chromosome of N2 in addition to chromosome II. This pseudo-linkage could be due to a suppressor of the Rol phenotype present on the CB4856 X chromosome. In a second step, 48 F2 animals for each mutation were analyzed with eight FLP assays along chromosome 2  phenotype, and staining of the adherens junctions with the MH27 antibody showed defects in the formation of the vulval torroid rings , indicating that the zh41 mutation in the lin-11 open reading frame (ORF) is responsible for the vulval phenotype.To demonstrate the usefulness of the henotype , rol-1 and by genotyping existing SNPs in the candidate interval . In manyC. elegans allows us to rapidly map a mutation down to a small region containing, on average, 200 candidate genes by crossing a mutant strain to CB4856 and analyzing 48 F2 animals with 300 to 500 PCR reactions.In summary, FLP mapping in C. elegans, zebrafish (Danio rerio) or Arabidopsis genetics, Drosophila strains are very heterogeneous and of ill-defined origin [Drosophila resembles human genetics in that standard inbred lines do not exist and the genotypes of the parental lines have to be determined first. As genome-wide polymorphism databases for reference strains are available [yw strains for the whole set, four FRT-Minute or FRT-cell-lethal strains at the relevant autosomal arms [2R017, Figure Drosophila strains .In contrast to the well defined genetic backgrounds used for d origin ,9,11. Invailable ,11, a limal arms , as wellVI.29 and EP0755 we recovered three types of recombinant chromosomes: recombinants with a crossover proximal or distal to the mutation, respectively, and double-crossovers . Furthermore, data from this and other FLP mappings in this region allowed us to further refine the genetic map . This kind of experimental data is helpful to space new FLP assays more evenly on the genetic map should the available map turn out to be inexact.In a genetic screen devised to isolate genes that regulate growth and are situated on chromosome 2R, we found a complementation group characterized by a mild overgrowth phenotype . Second, we genotype recombinants with SNPs available in the region of interest and resolve them by RFLP, sequencing or DHPLC ,9. Thirdeletions ,38.  the resC. elegans or Drosophila. For example, 7.3% of the Arabidopsis sequence polymorphisms are InDels [Arabidopsis, C. elegans or Drosophila FLPs.It seems likely that in most organisms the frequency of polymorphisms caused by InDels is in the same range as found in humans, e InDels . Thus, FC. elegans was done according to standard procedures described in [C. elegans alleles used were: LG I: lin-11(zh41), lin-11(n389); LG II: rol-1(e91), let-23(sy1), unc-52(e444). Drosophila strains and the genetic screen have been described previously [Culturing and crossing of ribed in . C. eleglin-11n38; LG II: eviously ,40-42.2, 0.45% NP-40, 0.45% Tween-20, 100 μg/ml freshly added proteinase K) and incubated for 60 min at 65°C followed by heat-inactivation of proteinase K at 95°C for 10 min. Before PCR, 90 μl double-distilled H2O (ddH2O) was added to obtain a total volume of 100 μl per lysate.Adult worms were collected in 10 μl lysis buffer  Debris was allowed to settle for about 5 min, and 50 μl of each supernatant were transferred into a 96-well PCR plate. The reactions were incubated in a thermo-cycler for 30 min at 37°C and finally for 10 min at 95°C to heat-inactivate proteinase K. Before PCR amplification, the crude DNA extracts were diluted 20-fold to reduce the concentration of proteins that might be harmful for the capillary sequencer.DNA from recombinant flies was extracted in bulk by squishing flies through mechanical force in a vibration mill (Retsch MM30) programmed to shake for 20 sec at 20 strokes per second . Single 2, 1x PCR reaction buffer, 0.25 U EuroTaq polymerase (Euroclone). PCR reaction setup was done in 96-well plates using a Tecan Genesis pipetting robot with disposable tips. PCR was carried out in two MJR thermo-cyclers that are integrated into the robot. The current setup allows for the sequential processing of six 96-well plates at a time. Cycling parameters were 2 min 95°C, 20 sec 95°C, 20 sec 61°C (-0.5°C for each cycle), 45 sec 72°C (for 10 cycles) followed by 24 cycles of 20 sec 95°C, 20 sec 56°C, 45 sec 72°C and a 10 min 72°C final extension. Following PCR, reactions were diluted 1:100 in water, and 2 μl diluted PCR products were mixed with 10 μl HiDi formamide containing 0.025 μl LIZ500 size standard (Applied Biosystems). This dilution before analysis on the capillary sequencer is necessary to reduce signal intensity because too strong signals compromise data analysis. In addition, sample dilution reduces the risk of damaging the capillaries with proteins or lipids present in the crude lysates. The dilution was done with standard tips using the Tecan Genesis pipetting station. Carryover of fragments was prohibited by a simple wash step with H2O. Fragments were analyzed on an ABI3730 capillary sequencer using POP7 polymer according to standard procedures. Data were analyzed using AppliedBiosystems GeneMapper software and raw data were treated further with Microsoft Excel.Diluted single-worm lysates (2 μl samples) or single fly extracts were added to 23 μl PCR reaction mix. Final concentrations in the PCR reaction were: 0.4 μM forward/reverse primer, 0.2 mM dNTPs, 2 mM MgClThe following additional data are available with the online version of this article. Additional data file C. elegans mapping results are given in Additional data files C. elegans and Drosophila, respectively. Additional data file Drosophila chromosome 2. Additional non-validated FLPs can be found in Additional data files C. elegans) and Additional data file Drosophila).Further General information on fly geneticsClick here for additional data fileProof-of-principle for chromosomal linkagewith 3 known mutations on chromosome 2. Assays used to assess linkagewere ZH1-01, ZH2-01, ZH3-05a, ZH4-03, ZH5-01 and ZHX-02Click here for additional data filelet-23 to its subchromosomal region (C. elegans)Mapping of Click here for additional data filerol-1 to its subchromosomal region (C. elegans)Mapping of Click here for additional data fileunc-52 to its subchromosomal region (C. elegans)Mapping of Click here for additional data fileC. elegans FLP mapping flow chartClick here for additional data fileDrosophila FLP mapping flow chartClick here for additional data fileElectropherograms demonstrating the accuracy of allele-callingClick here for additional data fileC. elegansTables of primer and sequence data of experimentally verified FLP assays in Click here for additional data fileDrosophilaTables of primer and sequence data of experimentally verified FLP assays in Click here for additional data fileDrosophila chromosome 2A table of the refined genetic distances for FLP assays on the right arm of Click here for additional data fileC. elegans InDels LGI)Additional non-validated FLPs Additional non-validated FLPs Additional non-validated FLPs Additional non-validated FLPs Additional non-validated FLPs Additional non-validated FLPs Additional non-validated FLPs (Click here for additional data file