Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING  method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community.We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious.Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project. Rapid progress being made in genome sequencing projects provides raw material for the potential understanding of gene function, so effective reverse genetic strategies are increasingly in demand . Sequencin silico procedure for Arabidopsis researchers, as searchable databases of flanking sequences from T-DNA and transposon insertions are available on-line [Several general strategies have been used to obtain reduction-of-function or knockout mutations in model organisms, including insertional mutagenesis  and RNA  on-line . RNA supMutator (Mu) and Activator (Ac) transposon families. Mutator transposable elements are frequently present in high copy number and tend to insert in or near genes [Mu insertions in target genes include the Trait Utility System for Corn [Mutator element has been engineered to allow plasmid rescue of the element and flanking genomic DNA into E. coli [Activator transposons are low copy number elements that, like Mu, preferentially insert in or near genes [Ac elements for a reverse genetics database is advantageous, because single to low copy number insertions can be obtained.In maize, the majority of reverse genetic resources have exploited the endogenous ar genes  thus profor Corn , the Maifor Corn , and thefor Corn . Additio E. coli ,11 facilar genes . Large-sReverse genetic strategies based on induced mutations have the potential for general applicability. Two such methods have been described for plants. One is deletional mutagenesis using fast neutron bombardment, which appears to be an effective means of knocking out tandemly repeated genes . AnotherWe have introduced a high-throughput screening method for TILLING based on the use of a mismatch-cleavage endonuclease, followed by fluorescent display of cleaved products on polyacrylamide electrophoretic gels using the LI-COR analyzer system . We systATP demonstrated the practicality of high-throughput TILLING in a production setting. However, the small genome size of Arabidopsis and the ease with which it can be cultured might have made Arabidopsis easier to TILL than a field-grown crop plant with a large genome. To determine whether the procedures that were developed for ATP can be generalized, we chose to TILL maize, a crop plant with a genome that is ~20 times larger than Arabidopsis. We find that essentially the same procedures that provided efficient TILLING of Arabidopsis can be applied to maize, yielding comparable results. Despite the relatively small size of our mutagenized maize TILLING population, we obtained useful mutations. These include a promising allelic series for a chromomethylase gene that had been previously implicated in non-CpG DNA methylation, whose counterpart in Arabidopsis is responsible for epigenetic gene silencing and genome surveillance.TILLING consists of a series of steps, beginning with chemical mutagenesis of reference individuals and culminating in the determination of mutant base pairs by DNA sequencing . High-thFor this study, two separate mutagenized B73 maize populations, designated UI (M2 families from 384 mutagenized lines) and NS , were screened in 4-fold pools (4 UI families or 4 NS plants) in a 96-well format. To compensate for the larger genome size of maize relative to Arabidopsis, we increased the amount of genomic template DNA amplified 20-fold; otherwise, all protocols and default parameters were the same as used for Arabidopsis TILLING . We foun17 or ~1/1010.Of the 21 lesions, 17 appeared to be EMS-induced. These 17 mutations were G/C-to-A/T mutations, as expected for EMS . The preImportantly, each population yielded 8–9 confirmed new mutations, for an overall mutation density of approximately two mutations/megabase. Taking into account the fact that pollen treatment mutagenizes only one of two genomes, whereas seed treatment mutagenizes both , the estimated mutation density for both maize populations is ~3/4 as high as our average for Arabidopsis per mutagenized genome.We have previously demonstrated reliable detection of mutations in 8-fold pools based on analysis of ATP-generated data . For exa® (Cambrex) agarose gels reduced "smearing" of fragments, thus facilitating quantification.One potential source of variability in DNA amount is that degraded DNA is difficult to measure accurately when visualized by agarose gel analysis. We noticed that some of the genomic DNA samples, including NS3471.9, were partially degraded. Inaccuracies in measuring the amount of DNA in a sample will compromise normalization in pools: any variation in the amount of DNA contributed by each plant in the pool will lead to reduced representation of one or more plants. As the amount of DNA in a pool from a particular plant decreases, mutation detection becomes limiting. Recognizing that the quality of genomic DNA could potentially hinder the throughput of mutation detection, we sought an alternative method of sample quantification and normalization. We have found that running samples on 3% MetaphorWhile lower DNA quality and inaccurate normalization could account for missing the base change in NS3471.9, this is not a likely explanation for missing the mutation in UI20291, which was from an apparently undegraded sample. Therefore, we considered that another source of sample-to-sample variation arose from the sampling of leaves from M2 sibling plants in the UI series rather than using M1 plants directly. Individual DNA samples from the UI population were generated by pooling approximately 10 individuals from an M2 family. M1 plants that are heterozygous for a new mutation will yield M2 families segregating 1:2:1 for the new mutation. A sample over a large M2 family should yield DNA that is equal parts wild type and mutant allele, assuming good viability and fertility of the mutant allele. However, a small M2 sample may be biased and contain too large a proportion of wild-type DNA which would then prevent a given target sequence containing a mutation from being detected among 8-fold pooled DNAs, depending on the limit for robust mutation detection.Direct evidence that sampling from M2 families was a problem came from our finding that two of the mutations found in plants sampled as leaves from multiple M2 plants were scored as homozygous by our usual criteria. Pollen mutagenesis can only produce M1 heterozygotes, and we interpret the homogeneity of the mutant as resulting from limited sampling of heterogeneous M2 plants. For example, if only one or two of the planted M2 seed germinated for these families, and the limited sample favored homozygotes, then they would sometimes appear to be purely homozygous in the sequence trace. Similarly, an equal or greater number of mutations will be underrepresented because wild type will be in excess, leading us to miss detecting the mutation in 8-fold, but not in 4-fold pools. This underscores the importance of using DNA from M1 pollen-mutagenized individuals and taking care to avoid collection procedures that could exacerbate degradation during DNA isolation. By assaying DNA concentrations on Metaphor gels and sampling only the M1 generation, we should be able to pool 8-fold without reducting detection efficiency. To test this, we screened a population of 768 M1 W22 maize DNAs (pollen mutagenesis) with similar degradation patterns as the B73 samples described above and normalized using 3% Metaphor agarose gels. We then made 8-fold and 4-fold pools from these 768 samples and screened with four of the 11 primer sets in the original screen. This screening of 6-Mb of total sequence led to the independent detection of the same 3 mutations in both the 4-fold and the 8-pools (data not shown). Therefore, we conclude that even partially degraded DNA from M1 pollen-mutagenized samples, such as might be extracted from material collected in the field, can be used for TILLING.The 17 induced mutations discovered in the screen were distributed as expected, consisting of 10 missense, 7 silent and no truncation mutations, compared with 51% missense, 44% silent and 5% truncation mutations based on ~4000 TILLed Arabidopsis mutations. Considering that about half of missense mutations are expected to be damaging to a typical protein , we expeWe have shown that TILLING is an efficient method for reverse genetics in a crop plant. The density of mutations that we discovered appears to be only slightly lower than what is obtained for Arabidopsis using the same methodology. For Arabidopsis, we currently screen ~2300 M2 plants to obtain a suitable allelic series, which averages ~12 mutations per 1.5-kb segment screened. Based on this work, we estimate that screening ~4000 maize plants will provide a comparable series ([4000] [17 mutations] [1.5-kb/1-kb]÷([11-kb] [750 plants]) = ~12 mutations). Since this study was completed, we have expanded the size of our mutagenized population to ~2500 M1 individuals in the B73 genetic background and ~2000 M1 plants in the W22 genetic background that are ready for screening, and populations of ~10,000 M1 plants are being prepared.For effective pooling, each individual in a pool must be represented at a concentration that is equivalent to the other members in a pool. Failure to accomplish this could result in a mutation represented in the pool below the level of detection. The goal is to maximize throughput by increasing pooling while still detecting all possible mutations. We have shown that in Arabidopsis, heterozygous mutations can be as efficiently discovered as homozygous mutations in 8-fold pools, thus providing a minimum estimate of robust discovery in a production setting of 1 in 16 [Sorting Intolerant From Tolerant [Project Aligned Related Sequences and Evaluate SNPs[Position-Specific Scoring Matrix) Difference score based on alignment blocks (Figure in vivo targets of CMT3-dependent methylation [At least one of the maize genes that we screened yielded an excellent allelic series. We discovered three missense mutations in DMT102, all of which are predicted to damage the protein based on sequence conservation. This analysis used two different programs: SIFT (Tolerant , which uuate SNPs, which phylation . Togethehylation , our TILTILLING has several advantages as a general reverse-genetic tool, especially for organisms for which other options are limited. The high density of mutations resulting from chemical mutagenesis means that, relative to insertional or deletional mutagenesis, far fewer plants are required for screening and much smaller genes can be effectively targeted. EMS is a stable and reliable mutagen, whereas the stability, penetrance and accuracy of RNAi-based silencing is uncertain ,31, and B73 pollen was mutagenized with EMS and applied to the silks of B73 ears . Ears weSamples were prepared from lyophilized leaf tissue essentially as described , except The same procedure used for TILLING Arabidopsis  was adapScreening of mutations using LI-COR gel analyzers was performed as previously described . SequencSHR, KY and EB prepared the samples, CB, CAC, LCE and ARO performed the high-throughput screening and data processing, CW and NS provided the mutagenized plant material, EAG performed the data analysis and BJT, LC and SH participated in the design and execution of the study and wrote the paper.


The unique ability of nucleic acids to replicate has recently been combined with the power of combinatorial chemistry, providing a new approach to the science of drug design At some time almost 4 billion years ago, nature likely was faced with a chemical dilemma. Nucleic acids had emerged as replicable information carriers and primitive catalysts , yet theA small but growing number of researchers have begun to tackle a modern version of this dilemma. While proteins and nucleic acids can be manipulated using powerful molecular biology techniques that enable their directed evolution, the size, fragility, and relatively limited functional group diversity of biological macromolecules make them poorly suited for solving many problems in the chemical sciences. Ideally, researchers would like to apply evolution-based approaches to the discovery of functional synthetic, rather than biological, molecules. A solution analogous to nature's translation of mRNA into protein could, in principle, address this contemporary problem . If a laThe translation of DNA into synthetic molecules is conceptually distinct from the use of DNA simply as a tag during the solidphase synthesis of a molecule that is part of a combinatorial library . The latDNA-templated organic synthesis (DTS) has emerged as one way to translate DNA sequences into a variety of complex synthetic small molecules . In thisPLoS Biology, Harbury and co-workers describe an elegant new approach to translating DNA into synthetic peptides called “DNA display.” Their approach uses DNA hybridization to separate mixtures of DNA sequences into spatially distinct locations. The first paper , and can use reaction conditions such as high temperatures or high pH that may not be compatible with DNA hybridization. These features suggest that DNA display may be able to access structures that cannot be created by DTS. Likewise, because DTS approaches use effective molarity rather than intermolecular reactivity to direct organic synthesis, they enable modes of controlling reactivity (such as using otherwise incompatible reactions in a single solution ) and claevolving libraries of diverse synthetic molecules, rather than simply enriching libraries that already contain at the outset the “most fit” molecule, researchers must develop sophisticated library syntheses that generate remarkable complexity (vast numbers of different compounds) in a relatively modest number of DNA-compatible synthetic steps. True evolution takes place when the theoretical complexity of a population exceeds the number of different molecules that can be created in a single library translation step, and when diversification is required to access compounds in later generations that are more fit than any member of the starting pool.In order for either approach to fully realize its potential of truly 8 compounds). However, because so few copies of a DNA-linked synthetic molecule are required for in vitro selection (8 different structures would represent 10 kg of material, not including the mass of beads or plates associated with the library, while a chemically translated library containing 10,000 copies of 108 different species represents less than 1 µg of total material.To my knowledge, no synthetic library to date contains this degree of complexity (indeed, the total size of the Chemical Abstracts Service database of known chemical substances is presently less than 10election —comparedWhile significant remaining challenges face efforts to develop and apply chemical translation, the promise of marrying evolution and organic synthesis is an irresistible combination for some researchers. The work of Harbury and co-workers described in this issue represents the latest approach to the very ancient problem of translating replicable information into functional structures.


Splenic metastases occur in rare cases with a few case reports of patients in the literature. Generally, splenic metastases mean late dissemination of a disease. Solitary splenic metastases from solid tumors are extremely unusual.We report a case of a patient with ovarian mucinous cystadenocarcinoma who underwent splenectomy for isolated parenchymal metastasis.Ovarian epithelial tumors comprised most of isolated splenic metastases from gynecologic tumor. When isolated splenic recurrence is suspected on image studies and serum tumor markers, intraabdominal gross findings should be examined to exclude peritoneal carcinomatosis. If only spleen was under suspicion of recurrence of ovarian cancer, splenectomy may play a therapeutic role. Splenic metastases from solid tumors occur in late stage of a disease, so those can hardly be an indication for a surgery. Cancers of the ovaries, lung, breast, stomach, skin and colon are known to metastasize to the spleen . Even thWe present a case of mucinous cystadenocarcinoma that recurred in the splenic parenchyma.A 29-year-old woman was admitted our hospital on August 1999, had been diagnosed as mucinous tumor of borderline malignancy a year ago. She was followed up at a local clinic. On the follow-up study, the patient's CEA level was raised to 43.32 U/L and CT scan showed splenomegaly with cystic lesion. Her past medical history was not significant. She already underwent two surgeries. The first surgery, right salpingo-oophorectomy, was performed at the age of 22 after being diagnosed as dermoid cyst. She was healthy thereafter. Seven years later, left ovarian mass was found on the routine check. At the second surgery, left ovarian mass excision, mucinous tumor of borderline malignancy was diagnosed.During follow-up after the second surgery, she was referred to our hospital on the suspicion of carcinomatosis peritonei. On the preoperative evaluation Figure , splenicTo exclude peritoneal carcinomatosis, open laparotomy was perfomed. On opening the abdomen, no abnormal gross findings were found except the splenic lesion, which reported as probable metastatic adenocarcinoma on frozen sections. After splenectomy was carried out, peritoneal washings and multiple biopsies on the omentum, peritoneum, mesentery, and left ovary were performed to rule out possible microscopic peritoneal dissemination. Suspecting the transabdominal metastasis, 100 mg of cisplatin was infused into the peritoneal cavity at the end of the operation; however no intraperitoneal recurrence was confirmed after tissue diagnosis.Final pathologic examination Figure  showed mTwo years after the surgery, 3 × 3-sized mass on the left ovary, which assumed to be recurrence, was detected. She has been followed up at outpatient department receiving symptomatic treatment and chemotherapy.The frequency of splenic metastasis has been reported 2.3 to 7.1 per cent from autopsy series of cancer patients -11. SpleUntil now, in the literature, splenic capsular metastasis has been reported to occur in the case of far advanced stage of a disease or in the case of more than one organ was already involved in terminal stage and especially in the case of peritoneal metastasis. When one or more organ in the thoracic cavity and abdominal cavity was involved, the rate of splenic metastasis was 43% . But isoAmong cases of the isolated splenic metastasis, the ovarian cancers make up the most. After Minazawa et al  firstly CT scanning and measurement of serum tumor markers, especially CA 125, are helpful for detecting the recurrence and the infrequent splenic metastasis.We proposed that splenectomy be a proper therapeutic modality for an isolated splenic metastasis, especially parenchymal metastasis, from an ovarian cancer. When isolated splenic recurrence is suspected on the CT scanning and serum tumor markers, intraabdominal gross findings should be examined meticulously. If only spleen was under suspicion of recurrence, splenectomy would be a proper therapeutic procedure.The author(s) declare that they have no competing interests.YS carried out the literature search and prepared the manuscript. JC Kim, the corresponding author, was the main operator in charge of the case. CK was involved in the operation and the patient active management. Three authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


PU.1 null mice, which cannot raise the standard inflammatory response but which can repair skin wounds rapidly.To define the events in wound healing that are independent of inflammation, gene expression during wound healing was profiled in wild-type mice and  Wound healing is a complex process requiring the collaborative efforts of different tissues and cell lineages, and involving the coordinated interplay of several phases of proliferation, migration, matrix synthesis and contraction. Tissue damage also triggers a robust influx of inflammatory leukocytes to the wound site that play key roles in clearing the wound of invading microbes but also release signals that may be detrimental to repair and lead to fibrosis.PU.1 null sib. The PU.1 null mouse is genetically incapable of raising the standard inflammatory response, because it lacks macrophages and functioning neutrophils, yet repairs skin wounds rapidly and with reduced fibrosis. Conversely, by subtraction, we have determined genes that are either expressed by leukocytes, or upregulated by fibroblasts, endothelial cells, muscle cells and others at the wound site, as a consequence of inflammation. To determine the spatial expression pattern for several genes in each cluster we have also performed in situ hybridization studies.To better define key cellular events pivotal for tissue repair yet independent of inflammation we have used a microarray approach to determine a portfolio of over 1,000 genes expressed across the repair response in a wild-type neonatal mouse versus its PU.1 null sibs distinguishes between tissue repair genes and genes associated with inflammation and its consequences. Our data reveal and classify several pools of genes, giving insight into their likely functions during repair and hinting at potential therapeutic targets.Cluster analysis of genes expressed after wounding wild-type mice versus  Much is known about the sequence of cell and tissue behavior that leads to repair of a mammalian skin wound ,2 but wePU.1 null sibs which are genetically incapable of raising an inflammatory response because they lack key leukocyte lineages.The compelling argument for performing such a study comes from microarray investigations of genes upregulated in fibroblasts in response to serum exposure. Cluster analysis of these results hints at the roles of hundreds of genes by the similarity of their temporal profile with genes whose function as part of the serum response cascade is well characterized ,4. To ovPU.1 is an ETS family transcription factor that is crucial for several lineage decisions in hematopoetic cells; consequently, PU.1 null mice lack a number of hematopoetic cell types [PU.1 null mice can efficiently heal skin wounds [PU.1 null mice results in less indication of fibrosis and an altered cytokine and growth-factor profile compared to wild-type. For example, interleukin 6 (IL6) mRNA, which is robustly expressed at wild-type wound sites, is almost undetectable in PU.1 null wounds, and TGFβ1 mRNA, previously implicated in several fibrosis scenarios, is significantly reduced in PU.1 null wounds, as revealed by RNase protection analyses [ll types . They arll types . Howevern wounds . Indeed,PU.1 null skin, to distinguish those key transcriptional events that are part of the tissue repair process but independent of whether or not there is an accompanying inflammatory response, from those genes that are 'inflammation dependent'. The latter are expressed either by inflammatory cells recruited to the wound, or upregulated by fibroblasts, endothelial cells, muscle cells and others at the wound site, as a consequence of inflammation.In this study we use Affymetrix GeneChip analysis of mRNAs collected at various time points after wounding of wild-type versus in situ hybridization data that also reveals spatial resolution.Using cluster analysis we have grouped more than 1,000 wound-induced genes according to their temporal profiles, with each cluster having a unique temporal profile of expression that correlates with a clear physiological episode during the repair process. For a small sample of genes from each of these clusters, we show PU.1 null animals, which naturally will increase with time after the initial wound insult.For our wound model we chose neonatal mouse back skin which raises a robust inflammatory response to wounding that is not dissimilar to that seen at sites of tissue damage in adult skin, but which heals rapidly, such that incisional lesions are generally fully re-epithelialized by 24 hours. This compression of the repair process reduces the temporal 'noise' and thus the potential loss of gene-expression synchrony between wild-type and PU.1 null sibs, with the only obvious differences apparent in the histology being an absence of inflammatory cells in the PU.1 null wounds . These wound dynamics lead us to believe that the neonatal mouse skin wound model provides a good opportunity to analyze the transcriptional events that regulate the various tissue-repair episodes from initial activation steps through to the 'stopping' signals that occur when the tissue defect has been filled in, both in the presence and absence of an inflammatory response.Resin histology of healing incisional wounds in neonatal mouse skin reveals closure of the wound commencing within 3 hours of the lesion; by 24 hours, the epidermal wound edges have generally met and fused along much, if not all, of the length of the wound. This is true for both wild-type neonates and for s Figure . As prevs Figure . The samPU.1 null mice and their wild-type littermates. Each time-matched pair (PU.1 null and wild-type) were chosen from the same litter to reduce the possibility of differential expression 'noise' due to environmental differences. Wound tissues were harvested at either 30 minutes to identify immediate early genes, 3 hours for early tissue repair effector genes, and 12 or 24 hours to reveal later tissue repair effectors as well as inflammatory genes. Total RNA was then extracted and hybridized to Affymetrix GeneChips, and differentially expressed genes were identified by comparison of expression levels for each time point with unwounded skin samples that served as baseline controls. Genes were selected if transcript levels exceeded a twofold increase over either the unwounded baseline, or between time points, or between the wild-type and PU.1 null wounds. On the basis of these criteria, 1,001 genes were identified as wound-induced .For microarray comparison, a consistent series of horizontal and vertical incisional (criss-cross) wounds were made to the back skin of 2-day-old neonatal Cluster analysis with Spotfire Array Explorer 3.0 software was used to organize the 1,001 wound-induced genes into groups according to the cosine coefficient similarity measurement; this includes within a group all those genes that have a similarly shaped temporal profiles, regardless of the levels of gene expression. Nine clusters were identified in this way, and of these, seven correlated with clear episodes in the repair process. The other two had profiles that, as far as we can tell, do not correspond to any currently understood step in the repair process and so were discarded for further analysis, although they appear in our supplementary data . Of the seven clusters associated with known repair episodes, five contain one or more known genes with good functional associations to that repair episode, and this encourages us to name each cluster according to that physiological episode. This does not provide definitive proof of function for any gene in that cluster, but it gives the best opportunity to predict function, particularly for expressed sequence tags (ESTs) with no further sequence information.PU.1 null skin, where there is none. In both these situations there is full and complete repair, and so we propose that these four clusters represent the basic repertoire of repair genes that are activated during the repair response. Figure Four clusters of genes have profiles that are largely independent of inflammation. Genes in these clusters are expressed with similar profile whether wounds are in wild-type skin, where there is an influx of inflammatory cells, or in PU.1 null wound site at any stage. We have called these two groups of genes, the 'early inflammatory' cluster , JunB, Myc, and I-Kappa-Bα (Nfkbia). We present a heatmap for the 90 genes in this cluster arranged with the most highly expressed at the top of the map  cascade activated during morphogenetic episodes such as dorsal closure in the fly embryo [In situ hybridization shows that the front few rows of wound epidermis express MKP-1, although expression extends less far back from the wound edge than for Krox24  signaling by phosphorylation of extracellular-regulated kinases 1 and 2 (ERK1 and 2), and so may actually function as a brake on the earliest tissue movements activated at the wound site.MKP-1 Figure  is a duay embryo . In situ4 Figure . By analFosl1, Fra1), has previously been associated with epithelial migrations during tumorigenesis but has not been analyzed in a wound-repair model [Fosl1 has a classic activator temporal profile . The exact function of Mig-6 remains elusive but it has been shown to interact with Cdc42, a member of the Rho family of GTPases, via the activation of stress-activated protein kinases (SAPKs) [In situ hybridization reveals clear expression of this gene in wound fibroblasts  . In situs Figure ; togethein situ hybridization data to provide an insight into the spatial localization of some genes in these clusters.Two further clusters of genes have increased expression levels post-wounding in a manner that is also inflammation-independent but where expression occurs at a later time than with the activation genes. The profiles of these two clusters are temporally distinct from one another and so we have called them the early effector and late effector clusters. Between them they contain 184 genes that fit the expected profile of genes that might direct re-epithelialization and granulation tissue assembly events. The temporal profiles of all these genes can be seen by heatmap in Figure Map4k4, in both keratinocytes up to 10-12 cell diameters from the wound edge and a subset of dermal fibroblasts extending a similar distance back from the wound edge , a Fabp/p2/Crbp/Crabp family retinol transporter is expressed in wound epidermal cells approximately 15 cell diameters back from the wound site Figure  and 4l. Keratin 6 (K6), a classic wound-induced gene [K6 encodes a nonconventional keratin which is thought to facilitate the packaging up of other intermediate filaments in activated keratinocytes, so that these cells can migrate forward to re-epithelialize the wound [K6 by the front 10-12 rows of wound-edge keratinocytes were confirmed by in situ hybridization  is expressed in a similar temporal and spatial pattern to K6 , since, in addition to keratinocyte expression, it is expressed at high levels by wound leukocytes. As both the MRP8/MRP14 heterodimer and a homodimer, MRP8 is a potent chemoattractant ,23 and, At the end of the repair process many of the cell behaviors that drive repair - such as migration and proliferation - clearly need to cease as tissues re-establish approximately their pre-wound state. This will be a gradual process and yet we might expect to see such genes depressed during the repair period and becoming upregulated as wound edges meet and closure is finishing. We see a cluster of genes with exactly this profile, suggesting that some of these genes are re-expressed to control the later stages of repair. We have loosely termed this the stop cluster. Because of their known biology, several genes in this cluster make ideal candidates for players in the processes of contact inhibition and epithelial fusion that occurs as cells from the two epidermal wound fronts confront one another.In vitro studies show that both ligand- and receptor-bearing cells become activated upon cell-cell contact [Caenorhabditis elegans, where Eph receptor mutants display defects in the movement of epidermal cells over neuroblasts, and in Eph knockout mice, where various morphogenetic epithelial fusions fail, leading, for example, to cleft palate and hypospadius [The Eph receptors and their ligands, the ephrins, have features that might make them ideal for sensing and responding to stop cues.  contact ,26, and  contact . Furtherospadius ,29. All in situ hybridization studies reveal that this transcriptional regulation is also occurring within leading wound-edge epidermal cells , and during gut cell migratory episodes, which are also dependent on transcriptional activation of the short stop gene [Similarly, the expression levels of the receptor Notch also dip and rise during the repair period, and s Figure . Notch htop gene , the mamSeveral other genes within the stop cluster have characteristics that indicate they may be involved in sensing contact-inhibition cues or be downstream of these signals and operate to adhere epidermal fronts together. They include genes for Plexin 3 (Plxn3), a member of the plexin family of semaphorin receptors , DesmocoAs with the other clusters, suggestive biology is no proof of function, and it is worth noting that several other genes with this temporal profile do not have biology suggestive of a role in these late stages of wound healing. We feel that this cluster, more than any other, can only hint at function, and definitive function testing using knockout or knockdown assays will be necessary to investigate any speculative roles in the repair process.PU.1 null mice reveals most clearly genes that are dependent on the presence of an inflammatory response at the wound site. The heatmaps for early and late inflammatory gene clusters strikingly reveal robust expression in wild-type wounds, but little or no expression in the PU.1 null mice for these genes  is a pan-leukocyte, calcium-dependent, actin-bundling protein that has previously been implicated in macrophage activation and migration, although it is also overexpressed in many types of malignant human tumors . It is fs Figure . In situn Figure , and thes Figure . The wouC3 is expressed at similar levels in unwounded PU.1 null and wild-type skin, but whereas expression is rapidly upregulated by 30 minutes post-wounding and continues until 24 hours in wild-type wounds, upregulation of C3 is delayed and much weaker in the PU.1 null wound , encoding a glycoprotein that can mediate cell-matrix interactions via the engagement of a number of adhesive receptors . Previo bundles . Osteopoe Figure  and in sn Figure .CCr1), a receptor for several chemokines including MIP-1α, CCL5 and Scya7, is expressed as early as 3 hours post-wounding, with expression levels peaking by 12 hours. There is no expression at the PU.1 null wound site  encodes an α-chemokine that functions as a potent chemoattractant for macrophages and T cells, and is upregulated by 12 hours in wild-type wounds but is absent in PU.1 null wounds , suggesting that it is constitutively expressed by cells of the monocyte lineage, rather than being part of the macrophage activation profile. No expression of Cathepsin S is seen in wounded or unwounded skin of the macrophageless PU.1 null mouse  in vitro reveals many of the early and late effector genes that are expressed by keratinocytes at the wound edge [Several previous studies have modeled wound healing od serum . Our resund edge .in vitro, but our study shows that even in the complexity of in vivo healing, many hints as to the genes responsible for these episodes can be gleaned by microarray surveys.Other aspects of the repair process, in particular the stopping phase where migratory and proliferative behaviors cease as wound edges confront one another, have not yet been successfully modeled in vivo, informing us, for example, which chemokines might be key attractive cues by revealing which of the chemokine receptors are expressed by these cells. Of most therapeutic significance are those genes expressed as a consequence of inflammation by host wound fibroblasts, endothelial and muscle cells. These genes are clearly not absolutely essential for repair, or PU.1 skin wound not heal. Rather, they will include genes that contribute to the negative side effects of inflammation at the wound site including retarded re-epithelialization and fibrosis. Dissecting out exactly which genes from the inflammation-associated clusters might sit in such a category will be a major goal of our future studies.Clearly, the most novel aspect of our study is its capacity to highlight those gene responses that are specifically associated with, or a consequence of, the wound-activated inflammatory response. Those genes expressed at the wound site by virtue of their being expressed by the invading leukocytes provide clues as to the migratory machinery of leukocytes PU.1 null wounds in RNase protection assays [Drosophila genes expressed downstream of JNK signaling, which highlighted many such genes but revealed barely any change in expression of the TGFβ family member dpp, for which there is excellent genetic evidence for downstream activation by JNK signaling [How full a survey of the wound healing process is revealed by our microarray study? The Affymetrix GeneChip we used covered approximately half of the mouse genome and so we cannot claim this to be a saturation screen. Moreover, it has not escaped our attention that several well established wound players that we know to be represented on the chips are apparently absent from any of the wound clusters. This is true for several growth factors, most notably TGFβ1, which we have previously shown to be differentially expressed in wild-type versus n assays , and theignaling . For thein situ hybridization studies add spatial resolution to this dataset, revealing whether a gene is expressed by the wound epidermis or connective tissue cells, or by inflammatory cells, but given the vast array of genes expressed at the wound site, how can one dissect each of their roles during the repair process? Using recent developments in Cre driver lines, it will be possible to knockout genes specifically in appropriate cell lineages within the mouse, so that the requirement for a particular chemokine receptor in the recruitment of inflammatory cells can be assessed, or the link between expression of a candidate 'fibrosis' gene by wound fibroblasts and subsequent scarring, can be tested. Complementary studies in which mRNAs for candidate inflammation/fibrosis genes are simply knocked down by local delivery to the wound site of antisense oligodeoxynucleotides (AS ODNs) [Drosophila as a testbed to dissect the genetics of some aspects of the repair process and to determine by mutant analysis precisely which genes are required for which repair episodes.As we have highlighted throughout this paper, revealing a temporal expression profile that coincides with one of the physiological episodes of the repair process in no way proves function for a wound-expressed gene. Although limited to a small sample of genes, our AS ODNs)  will proAS ODNs) -51 have in vivo wound healing process and categorized according to the physiological episodes that best correlate with their temporal expression profile. Such a classified listing provides good clues to the genetic regulation of all the cell behaviors that contribute to healing, and supplies us with a pool of genes whose modulation may prove to be therapeutically beneficial to the repair process.In summary, we present here a portfolio of genes expressed during the PU.1 mice has been described previously [in situ hybridization studies, three incisional wounds were made along the long axis of this patch of skin  according to the manufacturer's instructions with a secondary clean-up stage using an RNeasy kit (Qiagen). RNA was amplified and labeled with biotin as described previously .in vitro transcription using Bioarray High Yield RNA Transcript Labelling kit (Enzo Diagnostics Inc). Fragmented cRNA (10 μg) was hybridized in 100 mM β-mercaptoethanol, 1 M NaCl, 20 mM EDTA, 0.01% Tween20, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated BSA, 50 pM control oligonucleotide and eukaryotic hybridization controls, to Affymetrix MGU74A arrays at 45°C for 16 h. Arrays were washed using Affymetrix protocols in nonstringent buffer (6x SSPE buffer, 0.01% Tween20, 0.005% antifoam) at 25°C and stringent wash buffer (100 mM MES buffer, 0.1 M NaCl, 0.01%Tween20) at 50°C and stained with streptavidin phycoerythrin (10 μg/ml) including an antibody amplification step. Arrays were scanned using an Affymetrix confocal scanner.Double-stranded cDNA was generated from 10 μg total RNA using Superscript Choice kit (Life Technologies) with a T7-poly(T) primer. Approximately 1 μg cDNA was used to generate biotinylated cRNA by PU.1 null. The remaining genes were sorted into nine clusters using Spotfire Array Explorer 3.0 software, of which seven correlated with clear physiological episodes of repair or inflammation and we have loosely named those clusters according to these criteria.The data were analyzed using Microarray Analysis Suite version 4.0 (Affymetrix). The data were scaled to a target intensity of 300. Values representing genes not expressed are unreliable and are given an absent call. All genes were subjected to a filter to identify genes with at least one present call across the full eight time points and that had on one or more occasion a greater than twofold change in gene expression levels between time points or between the wild-type and in situ hybridization studies using the macrophage-specific c-fms probe, using the protocol outlined in [Wound tissues were processed for resin histology with sections cut at 5 μm and stained with Toluidine Blue as previously described . To visulined in  and see In situ hybridization on frozen sections was performed as previously described [Probe details are available in Additional data file 3. escribed , with prin situ hybridization probe source, is a Word document listing the origins of the various RNA probes used in the in situ hybridization studies. Additional data file The following additional data are available with the online version of this paper. Additional data file An Excel file containing an annotated database for the 1,001 differentially expressed genes in the nine temporal gene clusters: activation, early effector, late effector, stop, early inflammatory, late inflammatory; inflammation-maintained, and excluded clusters 1 and 2. For each gene this database provides an Affymetrix ID and GenBank description, together with absolute fluorescence levels and absence/presence calls for each time point, and known functional information gained from GenBank and Swiss-Prot databasesClick here for additional data fileLine graphs displaying the temporal profile of the median expression levels at each time point to give a representation of excluded clusters 1 and 2Click here for additional data filein situ  hybridization studiesA Word document listing the origins of the various RNA probes used in the Click here for additional data fileAn Excel file containing the raw data - absolute fluorescence levels and absence/presence calls - for all genes on the Affymetrix MGU74 arrays at all experimental time pointsClick here for additional data file


Metarhizium anisopliae is being considered as a biocontrol agent for adult African malaria vectors. In the laboratory, work was carried out to assess whether horizontal transmission of the pathogen can take place during copulation, as this would enhance the impact of the fungus on target populations when compared with insecticides.The entomopathogenic fungus Anopheles gambiae sensu stricto were exposed to conidia whilst resting on fungus-impregnated paper. These females were then placed together for one hour with uncontaminated males in proportions of either 1:1 or 1:10 shortly before the onset of mating activity.Virgin female P < 0.05).Males that had acquired fungal infection after mating indicate that passive transfer of the pathogen from infected females does occur, with mean male infection rates between 10.7 ± 3.2% and 33.3 ± 3.8%. The infections caused by horizontal transmission did not result in overall differences in survival between males from test and control groups, but in one of the three experiments the infected males had significantly shorter life spans than uninfected males (An. gambiae s.s. mosquitoes during mating activity is possible under laboratory conditions. Field studies are required next, to assess the extent to which this phenomenon may augment the primary contamination pathway (i.e. direct contact with fungus-impregnated targets) of vector populations in the field.This study shows that autodissemination of fungal inoculum between  Anopheles gambiae (Diptera: Culicidae) continues to rely heavily on application of residual insecticides, either for indoor residual house spraying [Control of the main African malaria vector spraying  or bednespraying . These aspraying , but assspraying ,4, accepspraying ,6 and thspraying -10 underspraying ,12.Metarhizium anisopliae against adult African malaria vectors has recently increased [et al., in preparation). The principal method of contamination of the target insect population with the fungus is through application of conidia on indoor resting targets. However, in order to achieve the highest possible impact on the target population, it is desirable that contamination pathways other than the primary mode of contamination are utilised, for instance horizontal transmission. Horizontal transmission of pathogens within the same host/target species is called autodissemination, and this phenomenon has been suggested for biocontrol of several insect pests [M. anisopliae by honeybees for infection of the pollen beetle Meligethes aeneus [Beauveria bassiana between adult flies of Delia radicum [M. anisopliae and B. bassiana between adult tsetse flies, Glossina morsitans morsitans [M. anisopliae against adult An. gambiae is that host-seeking females, and occasionally also males that rest indoors, will receive primary infections while resting indoors on fungus-impregnated resting targets. Under optimal circumstances, prior to death, this infection may be transmitted to conspecifics upon contact (e.g. during mating). These mosquitoes are, therefore, not infected through direct contact with fungus-impregnated materials, but indirectly, upon physical contact with infected counterparts. It is estimated that approximately half of newly hatched, virgin females take a blood meal before mating [Entomopathogenic fungi are among the biological control agents used against insect pests. Interest in using the hyphomycete ncreased . The funncreased  as well ct pests ,16. Succs aeneus , of Beau radicum  and of Morsitans  confirmsorsitans ,20-22. Te mating ,24. A feAn. gambiae infected with M. anisopliae can transmit the fungus to uncontaminated mosquitoes of the opposite sex through physical contact during the mating process.The objective of this study was to investigate whether adult M. anisopliae can occur during the process of mating:Three bioassays were conducted to assess whether autodissemination of 3) netting cage for one hour during the normal mating period;1) 30 fungus-contaminated virgin females and 30 uncontaminated males were placed in a standard (30 cm2) a single fungus-contaminated virgin female and a single uncontaminated male where placed together in a glass tube for one hour during the normal mating period;3) netting cage for one hour during the normal mating period.3) a single fungus-contaminated virgin female and 10 uncontaminated males were placed in a standard (30 cmA fourth bioassay assessed whether or not:4) males that became infected with the fungus had acquired the infective propagules from contaminated females or rather from contact with the substrate where those females had rested previously.Anopheles gambiae sensu stricto strain used originates from Suakoko, Liberia (courtesy Prof. M. Coluzzi) and has been maintained in the Wageningen laboratories since 1989, under standardized conditions described by Mukabana et al. [The a et al. . ExperimMetarhizium anisopliae var. anisopliae isolate ICIPE30 (courtesy Dr. N.K. Maniania) was originally isolated in 1989 from a stemborer, Busseola fusca Fuller, near Kendu Bay, Western Kenya. Conidia were inoculated on oatmeal agar (OA) and placed in an incubator to grow. Fungal virulence was maintained by passing it through An. gambiae mosquitoes every five cycles after growing on OA. Third instar larvae were infected by applying dry conidia on the water surface. After 48–72 hours, moribund larvae were removed and their thorax opened to remove tissue with blastospores. These were plated on OA and placed in a dark incubator at 27°C. One week after the onset of sporulation of the colonies, conidia were harvested using a 0.05% Triton-X solution and a glass rod. The solvent with conidia was concentrated by removing the supernatant after centrifuging for three minutes at 5000 rpm. Dilutions were made using 0.05% Tween80 to obtain a conidial concentration of 108 conidia ml-1. Vegetable (sunflower) oil was added to obtain an 8% adjuvant oil formulation. Five ml of this suspension was pipetted evenly over a 240 cm2 piece of filter paper resulting in conidial densities of 1.6 × 1010 conidia m-2. The impregnated paper was left to dry at 20°C and 75 ± 5% RH for 48 hours and was then placed on the inside of a plastic cylinder (height 11.3 cm, diameter 3.4 cm) in such manner that the paper neatly lined the upright wall of the tube. The top of the tube was covered with netting material. This setup was used only to infect female mosquitoes. Before any contamination, the viability of the impregnated conidia was checked by placing a 1 cm2 piece of the impregnated paper on a Sabourad Dextrose Agar in an incubator at 27°C in the dark for 16–20 hours. After incubation, the piece of paper was carefully removed and placed under a microscope (X 400) to determine the proportion germinated. For direct contamination of the female mosquitoes with M. anisopliae, around 30 individuals were placed in the cylinder for 24 hours.3 netting cage three hours before the onset of mating. Half an hour before  dusk, which for An. gambiae is the time when mating activity occurs [ad libitum. The experiment was replicated three times, on different days. Control groups were treated similarly, with the difference that the paper lining the contamination tube was void of conidia. Mortality of males and females was checked daily to measure longevity. Dead mosquitoes (both sexes) were removed from the cages and placed on moist filter paper in a Petri dish, which was sealed and examined for fungal growth three days later. An additional similar experiment with only five females and five males was performed to determine whether conidia could be seen on the cuticle of the test males directly after the females were removed. Males were killed by a droplet of chloroform and placed under a microscope (X 400) and examined for attached conidia.Thirty uninfected males were placed in a 30 cmy occurs , thirty y occurs . One houM. anisopliae contaminated female was added 30 minutes before the onset of mating activity. After one hour the couple was separated by gently removing the male, which was placed and kept in a separate glass vial until it died. A wad of cotton wool moist with 6% glucose was placed on top of the vial. Females remained in the vial and were provided with glucose in the same way. Mortality of both sexes was recorded daily. The control group consisted of an equal number of pairs that were handled equally, with the difference that the females were not infected with the fungus. This was repeated with 35 male-female pairs on three different days.A single, uncontaminated 7-day old male was placed in a clean glass vial , to which one 3 netting cage. Half an hour before the onset of mating activity, a single infected female mosquito was added to this cage with the males. After one hour each male was gently removed using a clean 2 cm diameter glass vial and kept alive individually as in bioassay 2. This was done 14 times for the test group and six times for the control group.Ten uncontaminated 7-day old males were placed in a 30 cmTo assess whether the males in the above bioassays had acquired fungal infection from the contaminated females during the process of mating, or from resting on the substrate where fungus-contaminated females had rested previously (glass and netting), two experiments were carried out. In one experiment a total of 46 contaminated females  were gently transferred to a 500 ml glass beaker, sealed of with a glass Petri-dish. The females were killed after one hour by applying a droplet of chloroform in the beaker. They were then removed and placed into a Petri-dish on a piece of moist filter paper. The dish was sealed with parafilm, to be checked three days later for fungal infection. Directly after removal of the contaminated females, a total of 47 uncontaminated males  were placed in the beaker for three days, after which they were killed, removed and checked for fungal infection as described above. During the three days inside the beaker they had continuous access to a cotton wool wad moist with a 6% glucose solution. The second experiment differed only in that a standard netting cage was used instead of a glass beaker, with 64 fungus-contaminated females and 67 uncontaminated males in two separate trials.Mortality data were subjected to Kaplan-Meier pair-wise comparison survival analysis. Mosquito mortality data closely fitted the Gompertz distribution . CompariM. anisopliae.In all four bioassays, female mosquitoes that had been exposed to conidia in the cylinder setup died significantly faster than the control females , with an average of 96.4 ± 2.0% of the cadavers having sporulating All three replicates of bioassay 1 indicated transfer of the fungus from contaminated females to uninfected males, with an average male infection rate of 26.1 ± 5.3% . When the males from the test group were split into those that became infected and those that did not, a decrease in survival of the infected test males compared to the uninfected males was observed, although this effect was not significant . However, the average age at death of 11.3 ± 1.9 days of the fungus-infected males was significantly lower than the 15.6 ± 1.6 days of the uninfected test males (p = 0.001).In the third bioassay, horizontal transmission occurred in nine of the 14 replicates (64.3%). In those nine trials an average of 16.7 ± 3.7% of the 10 males acquired an infection. Calculated over the 14 trials, the infection rate was 10.7 ± 3.2%. As in the other two bioassays, there was no significant difference in survival rates between the test and control groups . However, those males in the test group that were found with sporulating fungus died significantly faster than uninfected males  with the average age at the time of death of 8.4 ± 1.1 days for infected and 17.9 ± 0.6 days for uninfected males, respectively.None of the 114 males in bioassay 4 were found infected with the fungus.M. anisopliae from female to male An. gambiae s.s. was the result of mating. This is strongly supported by the findings from experiment 4 where none of the males that had stayed on the surface area where fungus-contaminated females had rested previously acquired an infection. The average age at death of fungus-infected mosquitoes was quite high when compared to mosquito survival in Scholte et al. [It is generally believed that fungal dissemination within a host population occurs due to activities and movements of the host. The fungus can exploit host behaviour like trophallaxis, tactile communication, grooming  ,30 and me et al. . This iset al., in preparation), however, it was found that a large proportion (44.9%) of the An. gambiae s.l. found indoors were males. This suggests that not only females can deliver fungal inoculum to uninfected males, but that also infected males may infect uninfected females. Further research is needed to determine to what extent this secondary pathway of fungal contamination may contribute to spreading the fungus within mosquito field populations.In order to achieve the highest possible impact of the fungus on the mosquito population, it is desirable that other pathways besides the primary mode of (direct) contamination are utilized. The results of this study show that under laboratory conditions horizontal transmission can occur, which suggests that it may occur in the field. When these experiments were carried out, it was presumed that predominantly females would be infected directly from the indoor resting targets in the field. From a recent field experiment (Scholte M. anisopliae against African anophelines will predominantly depend on direct contamination of adult mosquitoes from conidia-impregnated resting targets such as walls, ceilings and sheets.This study has shown that horizontal transfer of fungal inoculum between mosquitoes is possible during copulation and may contribute to spreading of the fungus within target mosquito populations in the field. However, since conditions under which horizontal transmission is likely to occur are quite specific, field experimentation is required to measure the real impact that autodissemination may have. For now it is concluded that the relatively low infection levels recorded in this study suggest that the impact of biological control with E-JS was directly involved in the experimental work, analysis of the data and drafting of the manuscript. BGJK conceived of the study, obtained funding for it in collaboration with WT and revised the manuscript prior to submission.


Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of arterial aneurysms through increased proteolysis of extracellular matrix proteins. Increased proteolysis due to elevated matrix degrading enzyme activity in the arterial wall may act as a susceptibility factor for the development of coronary aneurysms. The aim of this study was to investigate the association between MMPs and presence of coronary aneurysms.Thirty patients with aneurysmal coronary artery disease and stable angina were enrolled into study (Group 1). Fourteen coronary artery disease patients with stable angina were selected as control group (Group 2). MMP-1, MMP-3 and C-reactive protein (CRP) were measured in peripheral venous blood and matched between the groups.Serum MMP-3 level was higher in patients with aneurismal coronary artery disease compared to the control group (20.23 ± 14.68 vs 11.45 ± 6.55 ng/ml, p = 0.039). Serum MMP-1 (13.63 ± 7.73 vs 12.15 ± 6.27 ng/ml, p = 0.52) and CRP levels (4.78 ± 1.47 vs 4.05 ± 1.53 mg/l, p = 0.13) were not significantly different between the groups.MMPs can cause arterial wall destruction. MMP-3 may play role in the pathogenesis of coronary aneurysm development through increased proteolysis of extracellular matrix proteins. Coronary artery aneurysms are defined as dilated coronary artery segments that are greater than 1.5 times the diameter of adjacent normal segments . The golCoronary aneurysms may occur during the development of coronary atherosclerosis. Previous studies have shown that coronary aneurysms are observed in 1% to 5% of patients with angiographic evidence of coronary artery disease -6. In soPossible factors contributing to aneurysms are matrix-degrading enzymes such as collagenases, gelatinases, and stromelysins ,10. MoreWe investigated the association between MMPs and coronary artery aneurysm by measuring the levels of MMP-1 and MMP-3 (both of which represent markers of proteolytic activity) in patients with coronary artery disease, some of whom had coronary aneurysms (cases) and others who did not (controls).We reviewed the medical records of patients who had undergone coronary angiography between January, 2002 and April, 2003. Among 4,456 cases reviewed, 55 patients (1.23%) diagnosed with aneurysmal coronary artery disease were selected. Sixteen patients with acute coronary syndromes and nine patients with balloon angioplasty history were excluded from the study. The remaining 30 patients with aneurysmal coronary artery disease patients were enrolled into the study. Transverse diameter of an aneurysm and reference vessel were measured using the post-processing software (Schimadzu Corporation, DIGITEX ALPHA Plus System, Kyoto, Japan, 2001). The ratio between dilated coronary artery segment and reference vessel diameter was calculated. The control patients (n = 14) had coronary artery disease, but were free of aneurysmal coronary dilatation. Both groups had positive exercise stress tests and had been diagnosed with stable angina. Blood biochemistry and echocardiography were performed in all patients. No patient had a history of coronary atherectomy or balloon angioplasty. All participants gave informed consent.Autoimmune disease, inflammatory arteritis, chronic or, acute infectious disease, use of steroid or anti-inflammatory drugs within the last three months, renal failure and cancer were accepted as exclusion criteria.Fasting blood samples (8–10 hours fast) were obtained from the antecubital vein at approximately 9:00 a.m. These were centrifuged for 10 min at 3,000 × g at a temperature of about 4°C. Serum was stored at -70°C. Blood samples were analyzed at the Ege University Department of Microbiology, Section of Serology.MMP levels were determined using enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer's instructions . The ELISA kit measured total MMP-1 (pro MMP-1, free MMP-1, MMP1/tissue inhibitor MP-1 complex), total MMP-3 (pro MMP-3, free MMP-3, MMP3/tissue inhibitor MP-1 and MMP3/tissue inhibitor MP-2 complex) at >89% cross reactivity. Samples were incubated in microtitre wells pre-coated with anti-MMP-1 (lyophilized rabbit anti-MMP-1) and anti-MMP-3 (peroxidase labelled Fab antibody to MMP-3) antibodies. The assays use the pro form of a detection enzyme that can be activated (by captured active MMP) into an active detection enzyme. MMP-1 and MMP-3 can be measured in the range of 6.25–100 ng/ml and 3.75–120 ng/ml, respectively. The results received from the optic scanners at 450 nm were converted into ng/ml values from a standard curve. All samples were run in duplicate and were averaged. Within-assay precision values for duplicate determinations were 5.5%, 7.9% and 7.3% at MMP-1 concentrations of 16.89 ± 0.94 ng/ml, 35.53 ± 2.82 ng/ml and 54.08 ± 4.0 ng/ml, respectively. Between-assay precisions for repeated measurements of the same sample were 11.6%, 12.0% and 13.2% at MMP-1 concentrations of 23.19 ± 2.68 ng/ml, 55.27 ± 6.65 and 98.04 ± 12.93, respectively. The within-assay precisions for duplicate determinations were 4.8%, 2.4% and 2.1% at MMP-3 concentrations of 13.7 ± 0.66 ng/ml, 33.7 ± 0.83 ng/ml and 83.2 ± 1.76 ng/ml, respectively. Between-assay precisions for repeated measurement of the same sample were 13.3%, 11.7% and 8.8% at MMP-3 concentrations of 11.2 ± 1.49 ng/ml, 27.6 ± 3.24 and 75.4 ± 6.63, respectively.Serums were obtained by centrifugation of vacutainer-clotted tubes at 3,000 rpm for 10 minutes. High sensitivity C-reactive protein (hs-CRP) samples were stored at -30°C and analyzed by latex particle-enhanced immunoturbidimetric assay. The total median inter-assay and intra-assay coefficients of variation for the assays were <6% for CRP. All results were recorded in the patients' files.All values are reported as mean ± SD. Chi Square test was used in the comparison of categorical variables while student unpaired-t test or Mann-Whitney Rank Sum tests were used, where appropriate, in the univariate analysis. Statistical analyses were performed with SPSS statistical software. A value of p < 0.05 was considered to be statistically significant.There were no significant differences in baseline characteristics between cases and controls. High-density lipoprotein, low-density lipoprotein, total cholesterol and triglyceride levels were not statistically different between the groups. Clinical characteristics of and medication use by the groups are shown in Table Mean serum MMP-1 (13.63 ± 7.73 vs 12.15 ± 6.27 ng/ml, p = 0.52) and CRP levels (4.78 ± 1.47 vs 4.05 ± 1.53 mg/l, p = 0.13) were not significantly different between cases and controls. Mean serum MMP-3 values were significantly higher in the cases than in controls (20.23 ± 14.68 and 11.45 ± 6.55 ng/ml respectively, p = 0.039). MMP-1, MMP-3 and hs-CRP levels are shown in Figure Essential factors contributing to the formation of coronary aneurysms include vessel media degradation and ulceration due to increased proteolytic activity. Connective tissue integrity, another factor contributing to aneurysm development, depends on the balance between degradation and repair of the extracellular matrix. Activation or inhibition of degrading enzymes affects extracellular matrix modeling ,10, whic2(PGE2)-cAMP- dependent pathway. G-proteins have been implicated in this pathway [Matrix-degrading enzyme activity is a tightly controlled process that involves transcription, activation of latent pro-enzymes and inhibition of proteolytic activity -13. A ke pathway . Transcr pathway -19. Seve pathway ,20,21.MMP activity is also regulated by tissue-specific inhibitors. There are four known tissue inhibitors of metalloproteinases (TIMP-1, -2, -3 and -4). The TIMPs are secreted by a variety of cell lines, including smooth muscle cells and macrophages. Their activity is increased by growth factors and either increased or decreased by different interleukins . IncreasMMP-1  and MMP-3 (stromelysin-1) are members of a family of proteinases that degrade one or more components of the extracellular matrix. In our study, it appears that elevated MMP-3 activity may represent a risk factor for coronary aneurysm formation. This finding is concordant with previously published studies. The mechanisms underlying this association are unclear. MMP-3 gene disruption may be responsible. Lamblin et al.  have repOthers have reported that MMP-3 is expressed in atherosclerotic plaque cells, but not by cells in normal arteries -37. In aMMP levels are elevated in patients with acute myocardial infarction, unstable angina and coronary angioplasty ,41,42. ACRP reflects systemic inflammatory activity. In this study, we did not observe increased CRP levels in those patients with coronary aneurysms. One explanation for similar CRP expression between cases and controls might be that all study subjects had been diagnosed with stable angina pectoris.Varying degrees of inflammation are reported among individuals with abdominal aortic aneurysms. This variation may relate to possible confounding due to clinical manifestations (asymptomatic or symptomatic) and aneurysm progression rates (cm/year). Other investigators have failed to observe increased CRP levels among asymptomatic patients with abdominal aortic aneurysm .2/cAMP-dependent pathway [Because elevated MMP-3 levels likely contribute to the development of coronary aneurysms, this matrix-degrading enzyme may represent an important therapeutic target. Luan et al.  reported pathway . Theoret pathway .In reporting our findings, we acknowledge that measurement of TIMP levels between cases and controls would have provided useful information about the possibility of proteolytic imbalance. Similarly, measurement of locally produced inflammatory cytokines, hormones and growth factors would be interesting to know about, since these regulate matrix-degrading enzyme expression -19. ThisWe conclude that MMP-3 overexpression due to a proteolytic imbalance may lead to coronary aneurysm development through degradation of matrix components, especially lamina elastica. New medical therapeutic options targeted specifically against MMP-3 may prove useful in the prevention of aneurysm formation.


Prospective users of preventive therapies often must evaluate complex information about therapeutic risks and benefits. The purpose of this study was to evaluate the effect of relative and absolute risk information on patient decision-making in scenarios typical of health information for patients.Factorial experiments within a telephone survey of the Michigan adult, non-institutionalized, English-speaking population. Average interview lasted 23 minutes. Subjects and sample design: 952 randomly selected adults within a random-digit dial sample of Michigan households. Completion rate was 54.3%.When presented hypothetical information regarding additional risks of breast cancer from a medication to prevent a bone disease, respondents reduced their willingness to recommend a female friend take the medication compared to the baseline rate (66.8% = yes). The decrease was significantly greater with relative risk information. Additional benefit information regarding preventing heart disease from the medication increased willingness to recommend the medication to a female friend relative to the baseline scenario, but did not differ between absolute and relative risk formats. When information about both increased risk of breast cancer and reduced risk of heart disease were provided, typical respondents appeared to make rational decisions consistent with Expected Utility Theory, but the information presentation format affected choices. Those 11% – 33% making decisions contrary to the medical indications were more likely to be Hispanic, older, more educated, smokers, and to have children in the home.In scenarios typical of health risk information, relative risk information led respondents to make non-normative decisions that were "corrected" when the frame used absolute risk information. This population sample made generally rational decisions when presented with absolute risk information, even in the context of a telephone interview requiring remembering rates given. The lack of effect of gender and race suggests that a standard strategy of presenting absolute risk information may improve patient decision-making. In contemporary U.S. society, prospective users of preventive therapies often must evaluate complex information about therapeutic risks and benefits. While health information is increasingly available, it is frequently incomplete and sometimes inaccurate ,2. For pPrior research on presentation formats that are most effective, comprehensible, and likely to lead to effective decision-making suggests that a critical element is the format of risk information . Mazur  suggestsA key format difference is absolute versus relative gain or loss ,10. AbsoPrior clinical studies have found that presenting information in absolute versus relative risk formats can affect the interpretation of information about medications and personal risks of developing diseases. For example, in a sample of family practice patients, Hux and Naylor  found thData were collected as a part of a cross-sectional telephone survey of the adult, non-institutionalized resident population of Michigan. The telephone survey was part of a continuing series of quarterly surveys in Michigan known as the State of the State Survey (SOSS) conducted by Michigan State University's Institute for Public Policy and Social Research (IPPSR). 952 interviews were completed within the designated interviewing period. The margin of sampling error for the sample as a whole is ± 3.2%. The average interview lasted approximately 22.8 minutes . A more detailed description of the SOSS methodology, sample design, procedures, and content, is provided in Hembroff and Silver .The sampling design for the State of the State Surveys is a disproportionate stratified random sample of phone numbers across the state's regions, over-sampling the less populated regions, and the city of Detroit. Trained interviewers called the random-digit dial (RDD) sample of telephone numbers to identify those that were actually working household phone numbers. Within identified households, interviewers interviewed a single adult selected using the "next birthday" technique  in whichThe data being reported have been weighted to adjust appropriately for the unequal probabilities of respondent selection based on the number of phone lines to the household and the number of adults within the household. Cases were also weighted to adjust for differential response rates among categories of race, gender, age and to adjust for regional disproportionate sampling. The final weighted data file very closely matches the demographic profile of Michigan's adult population.Interviews were conducted using the computer assisted telephone interviewing facility (CATI) of IPPSR's Office for Survey Research (OSR). Interviewing took place over a six week time period during June and July of 1997. The overall completion rate among eligible households was 54.3%. The calculated response rate is based on assigning final outcome dispositions codes according to Standard Definitions, the American Association of Public Opinion Research  guidelinThe interview contained a split-ballot experiment to examine the effects of alternative ways of communicating risks. All respondents were presented with a baseline scenario. In this case, we asked respondents to ..."Suppose you had a friend who was told that she was very likely to get a bone disease that would gradually make her crippled. Suppose the doctor said that there was a medication she could take on a daily basis that would greatly reduce her chances of getting the bone disease."Respondents were asked if they would recommend that their friend take or not take the medication. Respondents were then randomly assigned to be presented one of four follow-up questions. Each follow-up question provided additional information about the hypothetical medication in terms of its benefits or risks. Half of the respondents were told that the medication would increase the patient's risk of breast cancer while the other half were told that the medication would decrease the patient's risk of heart disease. For each half, however, the information was presented in one of two randomly assigned alternative formats. Regarding a decreased risk of heart disease, respondents were told either that:"by taking the medication she would also reduce her risk of heart disease by more than half," or that"by taking the medicine she would also reduce her risk of heart disease from 1 in 200 to 1 in 500."Regarding the increased risk of breast cancer, respondents were told either that"by taking the medication she would also double her risk of breast cancer," or that"by taking the medication she would also increase her risk of breast cancer from 1 in 10,000 to 2 in 10,000."Based on the additional information received, respondents were asked what they would recommend that their friend do. Subsequently, each of these four groups was randomly divided again and given the additional information about increased risk of breast cancer or decreased risk of heart disease that they had not yet received. Then they were asked one more time to indicate what they would recommend to the friend about taking the medication. Thus, respondents were asked to indicate their treatment recommendations three separate times, under three of nine sets of conditions represented by the table below.An initial treatment recommendation was made based on the information given in condition 1, and then again based on the combinations of information represented by conditions 2, 3, 4, or 7. A third and final recommendation was made based on the combination of information represented in conditions 5, 6, 8, or 9. Comparing the results for conditions 2 through 9 to the results from the baseline in condition 1 allows an assessment of the incremental effect of each additional piece of information on the treatment recommendation.The dependent variable is the decision to recommend the medication or not. We hypothesized responses to vary by risk format and number of competing risks. For example, the number of "don't know" responses would be greater in conditions where complete information about competing risks was given. The benefits-only format would produce a majority of positive recommendations. However, we could not predict how large those proportions might be. Direction of change was expected to be positive for added benefit information, negative for more risk information, and reflecting trade-offs when risks and benefits were present. That is, we expected respondents to make a "rational" choice based on the perceived greatest expected utility.To assess whether additional risk information about breast cancer or heart disease affected decisions in conditions 2 through 9, we compared the distributions of change responses for each condition. A "non-change response" occurs when the respondent gives the same answer to the follow-up question as to the baseline question. All other patterns of answers, such as a "don't know" or a "don't take the medication response to the follow-up after giving a "take the medication" response to the baseline, or vice versa, constitute change responses. Although some error variance would be expected, analyzing the distributions of change responses across baseline decisions for the individual treatment conditions determines whether or not the experimental manipulations had any systematic effect.If the added information supports rational decision-making, the percentage of "don't knows" and recommendations against taking the medication would go down if the net effect of the additional information is further risk reduction. Conversely, if the net effect of the additional information is to increase the risk of health problems, the percentage of change responses would be greater among those who initially said "don't know" or to take the medication. The greater the magnitude in the new absolute risk, the greater should be the percentage of respondents who should change their decisions. To test for the effect of risk information format, we aggregated the respondents' decisions. We dummy-coded whether the risk information had been presented in absolute risk terms or not. We also dummy-coded whether the information was given for breast cancer (= 1) or heart disease (= 0). To test the effect of format using logistic regression, we also aggregated recommendations. A dummy variable was created to indicate whether or not the risk information for breast cancer was presented in absolute risk terms (= 1), and similarly for heart disease risk.To develop a profile of those who could not make a recommendation, we dummy-coded recommendations to the baseline scenario into those recommending either to take or to not take the medication (DONTKNOW = 0) and those who said they did not know what to recommend (= 1). To develop a profile of those who made seemingly non-rational recommendations, we dummy-coded recommendations to the baseline scenario into those recommending to take the medication (WRONG = 0) and those who recommended not to take the medication or said they did not know what to recommend (WRONG = 1).All analyses were performed using SPSS for Windows 7.5.A total of 952 individuals were interviewed. In the weighted datafile, 54% were female; 12% were African Americans and 3% were Other non-whites; 26% were under age 30, 40% were 30–49, 18% were 50–64, and 16% were 65 or older; and 43% had a high school education or less, 31% had some college, and 26% had a college degree or more education. The reported median household income was between $40,000 and $50,000. In terms of health indicators, 15% rated their health as only "fair" or "poor;" 14% reported engaging in no leisure time exercise; 25% were current smokers; 38% were overweight or obese; and 1% were heavy drinkers. This profile shows that the sample obtained was representative of the general population of Michigan. All demographic and clinical characteristics were within the survey's margin of sampling error for the prevalence rates for these reported by the Centers for Disease Control and Prevention (CDCP) for Michigan in 1997 except for leisure time exercise.not take the medication.In the baseline condition, two-thirds of the respondents (66.8%) said they would recommend their friend take the medication, one in nine (11.2%) said they did not know what to recommend, and 22.0% recommended that their friend Table As shown in Table Similarly, in conditions 5, 6, 8, and 9, the percentage of respondents who changed their recommendation from baseline differed. That is, the information in conditions 5, 6, 8 and 9 were similarly effective at creating systematic changes in respondents' recommendations. Furthermore, those who changed a response from "don't know" at baseline, made changes consistent with the health outcome logically effected. This was true across all eight non-baseline conditions.Table Table Table Where the increased absolute risk of breast cancer due to the medication was given along with information about the decreased relative risk of heart disease, decisions to recommend the medication were nearly as great as in the baseline condition. However, the evidence clearly suggests respondents paid attention to both additional pieces of information. Where the very low absolute risk of breast cancer was presented, the increased risk appeared to be more acceptable in the face of the benefit in reducing the risk of heart disease. Where respondents were given information in absolute risk terms for both breast cancer and heart disease, twice as many chose to recommend the medication as did where both risks were given only in relative terms. This was only two-thirds as many as did so in the baseline condition. Compared to the baseline condition, respondents seemed to use both additional pieces of information. This is consistent with Expected Utility theory predictions, but was clearest under absolute risk conditions.Table Table The table (again) shows that the interaction term is significant, indicating that the effect of the risk information format for each disease differs depending on what is known about the other disease. In this case, the table indicates that communicating risk information about both heart disease and breast cancer in absolute risk terms significantly changes positive recommendations compared to scenarios in which respondents received risk information in other combinations of formats.A third of the cross-sectional sample chose something other than to recommend taking the medication in the baseline condition. While the primary purpose of this experiment was to explore the effect of information format on treatment decisions, additional analyses were performed to better understand individuals who seem to make decisions contrary to the evidence given, or who indicated an inability/unwillingess to make a decision. The two dummy variables constructed for this purpose, DONTKNOW and WRONG were analyzed against a broad set of demographic and clinical variables, including gender, race, ethnicity, living as a couple or not, age (in categories), rural/urban dwelling, education (in 4 categories), current smoker, current heavy drinker, perceived health as only fair or poor, physical activity, overweight, employed, and avoids medical care or uses alternative sources of care. (Heavy drinker and overweight definitions were taken from the Centers for Disease Control and Prevention (CDCP) Behavioral Risk Factor Surveillance System.Table Table In general, the risk presentation format appeared to influence the resulting decisions. Without knowing the absolute risks, respondents appeared to assume the risk of breast cancer was much greater than the risk of heart disease, or that the perceived likely consequences of breast cancer were significantly less acceptable than those of heart disease. The pattern of recommendations was made to avoid the greater perceived risk. When the competing risks were stated in absolute risk terms, respondents changed their recommendations appreciably. The results of the experiment strongly suggest that people's prospective preventive treatment decisions can be greatly affected by the manner in which potential risks and benefits are communicated to them. When given information regarding the absolute risks associated with a proposed treatment, individuals make remarkably "rational" decisions, clearly indicating the weighing of risks and benefits.It appears that presenting risks in absolute rather than relative risk formats may be more effective in helping respondents make decisions that are consistent with maximization of expected utility. It appears, in the scenarios devised here, this may be due to the previously documented finding that people exaggerate the risk of cancer -17. TheyOur findings seem to indicate that, in the absence of explicit information regarding the absolute risks of particular outcomes, patients interpret information about relative benefits or risks based on whatever preconceptions they may have about their chances of getting the disease in question. That is, they make reasonable use of the relative risk information if they do not know the base risk rates. However, to the extent that these preconceptions may be quite inaccurate, relative risk information may lead them to make decisions that seem irrational and inconsistent with their actual preference. Without information about the absolute risks, health information may become misleading and individuals may reach conclusions that go quite counter to what they actually think about the appropriate balance of benefits and risks of treatments.Framing the scenarios as recommendations to a friend made the decision-making relevant to both males and females. The logistic regression analyses found no significant differences in decisions based on race, gender, or other demographic characteristics in this relatively large and representative sample. This suggests that these results may represent a general population response pattern. It is clear from the results that members of the public will generally use explicit absolute risk information in quite rational ways when it is available. Consequently, providers of health information, both public and clinical, would be well-advised wherever possible to provide patients with the necessary risk/benefit information in terms of the absolute risks associated with treatment options. Making the best information readily available and comprehensible may reduce reliance on errant preconceptions and opinions. This research suggests that having absolute risk information routinely available to patients and physicians for use in making clinical decisions may be an important part of improving health care decisions.This study is unique in surveying the general public rather than clinical populations on the effects of risk formats on decision-making. However, it was limited to presenting risks only in terms of the chances of an outcome occurring. It did not address the magnitude of the outcome's impact on the individual's life or the acceptability to the individual of the relevant alternatives. Future research should address these aspects of risk as well as explicitly investigating assumptions about risks of specific diseases. Research that explicitly assesses general public risk perceptions may be valuable in determining the need for absolute risk information in specific disease contexts. Providing baseline absolute risk information in certain disease contexts may lead to improved patient treatment decision-making.The author(s) declare that they have no competing interests.LAH conceived and carried out the survey. MHR and CEW provided medical decision making context for interpretation and discussion. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: