Drosophila and Anopheles reveals that pairs of duplicated genes have strikingly different expression patterns.An analysis of nuclear-encoded oxidative phosphorylation genes in  In eukaryotic cells, oxidative phosphorylation (OXPHOS) uses the products of both nuclear and mitochondrial genes to generate cellular ATP. Interspecies comparative analysis of these genes, which appear to be under strong functional constraints, may shed light on the evolutionary mechanisms that act on a set of genes correlated by function and subcellular localization of their products.Drosophila melanogaster, D. pseudoobscura and Anopheles gambiae orthologs of 78 nuclear genes encoding mitochondrial proteins involved in oxidative phosphorylation by a comparative analysis of their genomic sequences and organization. We have also identified 47 genes in these three dipteran species each of which shares significant sequence homology with one of the above-mentioned OXPHOS orthologs, and which are likely to have originated by duplication during evolution. Gene structure and intron length are essentially conserved in the three species, although gain or loss of introns is common in A. gambiae. In most tissues of D. melanogaster and A. gambiae the expression level of the duplicate gene is much lower than that of the original gene, and in D. melanogaster at least, its expression is almost always strongly testis-biased, in contrast to the soma-biased expression of the parent gene.We have identified and annotated the Quickly achieving an expression pattern different from the parent genes may be required for new OXPHOS gene duplicates to be maintained in the genome. This may be a general evolutionary mechanism for originating phenotypic changes that could lead to species differentiation. The accessibility of whole-genome sequence data for several organisms, together with the development of efficient computer-based search tools, has revolutionized modern biology, allowing in-depth comparative analysis of genomes -4. In maA set of genes present in all eukaryotic genomes and expected to be subject to peculiar evolutionary constraints is represented by the genes involved in oxidative phosphorylation (OXPHOS), the primary energy-producing process in all aerobic organisms . To geneet al. [Analysis of co-regulated mitochondrial and nuclear genes, and of the transcription factors regulating the functional network they constitute, might also be a useful approach to investigate the origin of mitochondrial dysfunction in humans. Disorders of mitochondrial oxidative phosphorylation are now recognized as the most common inborn errors of metabolism, affecting at least one in 5,000 newborn children . In thiset al.  suggestsDrosophila and humans, and about 70% of the genes associated with human disease have direct counterparts in the Drosophila genome [Drosophila as a model system for understanding the molecular mechanisms involved in human genetic disease is validated by the recent identification of a Drosophila mutation causing a necrotic phenotype that mimics in detail the diseases that arise from serpin mutations in humans [Despite the long evolutionary divergence time, many key pathways that control development and physiology are conserved between a genome ,9. For en humans .D. melanogaster and other species of the genus Drosophila could provide a model system for developing and testing new algorithms and strategies for the functional annotation of complex genomes [D. melanogaster and D. pseudoobscura; the complete genome of the latter was recently made available by the Baylor Human Genome Sequencing Center. These two species are the only species of the Drosophila genus for which whole-genome sequence data exist at present [A. gambiae genome [Drosophila OXPHOS genes with those of this more distantly related dipteran (the divergence time between D. melanogaster and A. gambiae is thought to be approximately 250 million years, as compared to 46 million years between D. melanogaster and D. pseudoobscura [Anopheles from Drosophila [It has been suggested that comparisons between  genomes . To obta present -13. We ae genome  to compaoobscura ,16). Altosophila ,18, we sD. pseudoobscura and 78 A. gambiae genes representing the counterparts of D. melanogaster OXPHOS genes which, in turn, were previously identified as putative orthologs of human OXPHOS genes [Here we report the identification of 78 OS genes . We haveD. melanogaster nuclear genes encoding mitochondrial proteins that represent the counterparts of human peptides annotated in the Swiss-Prot database as mitochondrial [D. melanogaster OXPHOS genes in D. pseudoobscura and A. gambiae we performed a TBLASTN search [D. melanogaster peptides as queries. Sequences giving the best reciprocal BLAST hits were tentatively assumed to identify functional counterparts in two species if they could be aligned over at least 60% of the gene length and the BLAST E-score was less than 10-30. By these criteria, all the 78 D. melanogaster OXPHOS genes investigated have a counterpart both in D. pseudoobscura and in A. gambiae. To better compare the structure of the OXPHOS genes in the three dipteran species, we used the predicted coding sequences as queries for a search of expressed sequence tags (EST) [A. gambiae genes investigated. Although little EST information is available for D. pseudoobscura, it was still possible to predict unambiguously the exon-intron gene structure of the OXPHOS genes in this species, as well as the amino-acid sequence of their full-length products, by exploiting the high level of similarity with D. melanogaster. The results of BLAST analysis, together with the construction of phylogenetic trees that also include other genes that show lesser but still significant sequence similarity to the 78 genes assumed to be one-to-one orthologs in the three species investigated (see below), strongly suggest that the newly identified D. pseudoobscura and A. gambiae genes are the functional counterparts of the 78 D. melanogaster genes used as probes.We have previously reported  the idenhondrial . On the N search ,21 on thD. melanogaster, D. pseudoobscura and A. gambiae putative orthologous genes and their duplications when present (see below), and aligns the orthologous coding sequences (CDS), and also aligns their deduced amino-acid products with the corresponding human protein.Table D. melanogaster/D. pseudoobscura average amino-acid sequence identity is 88%, compared to 64% between D. melanogaster and A. gambiae. Figure D. melanogaster/D. pseudoobscura, D. melanogaster/A. gambiae and D. melanogaster/human gene products are compared. A multiple alignment of each cluster of homologous proteins is shown at the MitoComp website [For the products of the OXPHOS genes investigated, the  website .D. melanogaster and D. pseudoobscura, and to 70% between D. melanogaster and A. gambiae.It should be kept in mind that identity values reported in Figure c  in the three dipteran species: the amino-acid sequences of the gene products are identical in D. melanogaster and D. pseudoobscura, whereas 96% identity is preserved between Drosophila and Anopheles. Coding sequences are also extremely conserved, suggesting that the nucleotide sequence itself is subject to strong evolutionary constraints, maybe due to codon usage bias. Only synonymous substitutions (21 out of 108 codons) were found on comparing D. melanogaster and D. pseudoobscura cytochrome c coding sequences, whereas 28 synonymous substitutions and only four nonsynonymous substitutions were observed between D. melanogaster and A. gambiae (see MitoComp website [A striking example of evolutionary conservation is provided by the genes encoding cytochrome  website ).It is well known that a given function may be supplied in different species by genes that are not directly derived from a common ancestor, that is, by paralogous, not orthologous, genes. Therefore, we thought it would be interesting to compare the structural organization of the OXPHOS genes in the three species investigated, on the principle that it should be possible to infer derivation from a common ancestor, that is, 'structural orthology', if an identical or very similar overall structure was preserved. As the introns of the putative orthologous OXPHOS genes in the three species are, as expected, too divergent in DNA sequence to be aligned, we used conservation of number of introns, conservation of their location in the coding sequence, and preservation of the reading frame with respect to the flanking exons as our primary criteria.Dpse\CG5037, putatively encoding protoheme IX farnesyltransferase, whose 5' genomic sequence was impossible to find in the relevant contig assembly, all other investigated D. pseudoobscura genes show a structural organization almost identical to that of their D. melanogaster counterparts. Of the 78 Anopheles genes studied, 39 maintain the structural organization observed in Drosophila, whereas gain or loss of introns occurred in 33, and in six the location of introns is not preserved at all. In agreement with a previous report [Drosophila NADH-ubiquinone oxidoreductase acyl carrier protein (mtacp1, CG9160) [Drosophila ATP synthase epsilon chain (sun, CG9032) [Anopheles, as shown by genomic structure comparison, alignment of splice variants and EST mapping   and the  CG9032)  have vermtacp1) in the three species are characterized by the mutually exclusive use of homologous exons that are repeated in tandem   Figure , indicato humans . HoweverD. melanogaster and several other organisms the distribution of intron length has been shown to be asymmetrical, with a large group of introns falling into a narrow distribution around a 'minimal' length and the remaining showing a much broader length distribution, ranging from hundreds to thousands of base-pairs [Interspecies comparison of the introns of putative orthologous genes indicates that there is little constraint on their nucleotide sequence, which undergoes nucleotide substitutions at a rate comparable to that of pseudogenes . Howeverse-pairs -32.D. melanogaster, 96 (70.5%) of 136 in D. pseudoobscura and 87 (67.9%) of 128 in A. gambiae fall into the short-size class . The remaining introns show a broad length distribution, ranging from 151 to 4,702 bp with no clear boundary between classes.Of the introns that interrupt the coding sequence in the 78 OXPHOS genes investigated in the present study, 88 (64.7%) of 136 in s Figure . HoweverD. melanogaster, D. pseudoobscura and A. gambiae orthologs suggests that changes from the short-size to the long-size (more than 300 bp) intron class, or the converse, have been rare in the evolutionary history of these species: only seven class changes were observed comparing D. melanogaster and D. pseudoobscura introns, and six between D. melanogaster and A. gambiae , and soht zone' . Even ifht zone' , identicD. melanogaster, 19 in D. pseudoobscura and eight in A. gambiae) each of which shows significant similarity with one of the 78 OXPHOS genes reported above. When the structure of a member of a paralogous gene set indicates that it has been produced by retroposition, it seems reasonable to assume that it is derived from a pre-existing 'parent' gene. For duplicates not clearly originating by retroposition, we also assume, on the basis of the much higher level of conservation and expression, that the genes we find to be the structural orthologs in all three species are the parent ones, and in this case also we will henceforth refer to their paralogs as OXPHOS gene duplicates. The amino-acid percentage identity between the products of duplicate gene pairs ranges from 40% to 85%. For each of the OXPHOS gene duplicates, cytological localization, number of exons interrupting the coding sequence, and number of ESTs found in the D. melanogaster and A. gambiae EST databases are reported in Table Duplications, or in some instances triplications, of 24 OXPHOS genes were found. Overall, we identified 47 genes  of 16 of the 78 OXPHOS genes investigated were found in both The genomic organization of many OXPHOS duplicates shows that they were originated by retropositional events, because they are intronless, or have only very few introns that are likely to have been inserted into the coding sequence after the duplication event. In other cases, duplication apparently resulted from transposition of genomic DNA sequences or from recombinational events, as duplicate genes maintain an identical or very similar structural organization.D. melanogaster/D. pseudoobscura divergence (about 46 million years ago). On the other hand, five duplications were found only in D. melanogaster and four only in D. pseudoobscura; in these instances, if the duplication occurred before the divergence of the two species, it has been followed by loss of one of the copies in the lineage leading to the species in which the gene is no longer duplicated. On the assumption that the rate of gene duplication is constant over time, this translates to approximately 0.0014 duplications per gene per million years (4 or 5 duplications per 78 genes per 46 million years) that achieved fixation and long-term preservation in the genome. This value is about twofold lower than the 0.0023 value calculated by Lynch and Conery [D. melanogaster. However, it can be argued that the rate of long-term preservation in the genome of OXPHOS gene duplicates cannot be meaningfully compared with the general rate of preservation of duplicates in the whole genome since, while recent data suggest that in eukaryotic genomes there is preferential duplication of conserved proteins [On the basis of the presence of the duplication in both species, supported by evidence from evolutionary trees and conservation of microsyntenic gene order, it can be inferred that 15 of the duplications identified occurred before the d Conery  for the proteins , duplicaproteins , and areA. gambiae we found only four duplications and two triplications of the OXPHOS genes analyzed; of these, four involve genes also duplicated in one or both Drosophila species . A smaller number of ESTs originating from the OXPHOS gene duplicates was observed even in A. gambiae EST libraries that are normalized. Remarkably, and regardless of the mechanism of the duplication, in D. melanogaster, in which several organ-specific or developmental stage specific libraries are available, the search showed that the expression of the OXPHOS gene duplicates is strongly testis-biased, as 97 out of the 100 ESTs originating from them were found in testis-derived libraries, while only 27 out of the 1,769 ESTs originating from the parent genes were found in such libraries, the bulk of them being instead found in libraries derived from embryos or somatic tissues.The relative abundance of ESTs in a EST library may be assumed roughly to reflect the level of expression of each mRNA in the tissues from which the library was prepared. We therefore used the mRNA sequences predicted et al. [D. melanogaster genes showing ovary-, testis- or soma-biased expression. With the exception of CG7349, CG30354, CG30093 and CG12810, for which no data were presented by Parisi et al. [et al. [Our finding that the expression of the OXPHOS gene originated by duplication is strongly testis-biased is validated by the data obtained by Parisi et al.  using thi et al. , all oth [et al. .et al. [The pattern of strongly testis-biased expression of OXPHOS gene duplicates holds for a further sample of 40 duplications of genes annotated in the MitoDrome database  as encodD. melanogaster at least there is evidence for this, as FlyBase [D. melanogaster OXPHOS genes found that lethal alleles for 11 out of 19 D. melanogaster parent genes are known (see the MitoComp website [D. melanogaster OXPHOS gene duplicates - Cyt-c-d, encoding cytochrome c). Interestingly, although Cyt-c-d is adjacent to its putative parent gene, Cyt-c-p, it shows a different pattern of expression, suggesting that the two genes must be regulated at individual gene level and not at chromatin domain level , indicaD. melanogaster duplicate genes will be necessary to answer the question of whether the testis-biased expression pattern reported here is specific to the duplicates of genes encoding mitochondrial proteins, or is a more general phenomenon. According to the balance hypothesis, validated by experimental results obtained on yeast [D. melanogaster genome, at least until they evolve a new useful function. Finally, as nothing is known about the tissue-specific pattern of expression of the genes investigated in D. pseudoobscura and Anopheles, it also remains unclear whether the testis-biased expression of gene copies originated by duplication is specific to D. melanogaster, or is also to be found in other dipterans, and possibly in other organisms.A systematic investigation of the expression pattern of other on yeast , single D. melanogaster coding sequences have an average GC content higher than the genomic average [D. melanogaster OXPHOS coding sequences reported in this work and for their D. pseudoobscura and A. gambiae counterparts (68% of the codons in the OXPHOS genes end in C or G in D. pseudoobscura and 77% in A. gambiae, compared to 74% in D. melanogaster). In all three species, the coding sequences of OXPHOS gene duplicates show a lower percentage of codons ending in C or G, when compared to both the entire set of 78 orthologous OXPHOS genes and the gene subset including only their parent genes. In samples including all the OXPHOS gene duplicates annotated in this paper the aggregate percentage of C- or G-ending codons is 63%, 46% and 73% in D. melanogaster, D. pseudobscura and A. gambiae respectively, as compared with 70%, 64% and 88% in their parent genes. In D. pseudoobscura, the shift toward a higher percentage of A- or T-ending codons is also detected in the pattern of synonymous codon usage; for 12 of the 18 amino acids that are encoded by more than one codon, the most frequently used codon in the D. pseudoobscura gene duplicates is different from the one used in their parent genes .Because of the preferential use of codons ending in C or G, the  average ,45. ThisC. elegans [D. melanogaster [et al. [D. melanogaster clusters of three or more testis-specific genes are much more frequent than expected by chance. Therefore, we investigated the chromosomal distribution of the OXPHOS genes to determine whether clustering could be detected. In all three dipteran species considered, the 78 OXPHOS orthologous genes are randomly distributed on all chromosomal arms (Table D. melanogaster genes (Ucrh, encoding the 11 kDa subunit of ubiquinol-cytochrome c reductase, and CG40002, encoding the AGGG subunit of NADH-ubiquinone oxidoreductase) have a heterochromatic location.It has been reported that in many eukaryotes including yeast , C. eleg elegans , D. melanogaster ,49 and hnogaster , genes w [et al.  reportedms Table . Two D. No evidence of OXPHOS gene duplicate clustering was found either, despite the common testis-biased expression of such genes. Moreover, no evidence of clustering with other testis-specific genes was found when an EST database search for such genes was performed in the regions flanking the investigated gene duplicates.D. melanogaster X chromosome [D. melanogaster OXPHOS gene duplicates, two out of 19 in D. pseudoobscura and none (out of eight) in A. gambiae were found to be X-linked . However, as the general pattern of much lower, testis-biased expression holds even for OXPHOS and other mitochondrial gene duplicates that apparently derive from autosomal parental genes, and even for X-linked duplicates, this pattern (and the explanation of the evolutionary preservation of such genes) cannot only be due to the selective advantage of escaping X inactivation during spermatogenesis.CG9603, all euchromatic D. melanogaster orthologs maintain their localization on the homologuos D. pseudoobscura chromosomal arm . A further duplication event, occurring in the D. pseudoobscura lineage after the D. melanogaster/D. pseudoobscura divergence, probably created the Dpse\CG10396.1-Dpse\CG10396.2 gene pair.OXPHOS gene duplicates also almost always maintain the same chromosomal location and microsyntenic gene order in ra Table . On the  website , complexDrosophila species, when D. melanogaster and A. gambiae are compared the only meaningful correspondence found concerns the genes on the D. melanogaster 2L and the A. gambiae 3R chromosomal arms  in most or all the tissues where their parent genes are expressed, as judged by the abundance of ESTs derived from their transcripts in all libraries other than those derived from testis. On the other hand, OXPHOS gene duplicates have a strongly testis-biased pattern of expression, a finding validated by other authors with a different approach based on the use of microarrays [A. gambiae, although no testis-specific ESTs databases are available, a pattern of expression of almost all duplicate OXPHOS genes different from that of the gene from which they originated, and possibly limited to specific tissues, is suggested by the fact that in all EST libraries available the abundance of the sequences originated from the duplicate genes is very low when compared with that of the sequences derived from their respective parent genes.The comparative analysis of the 78 OXPHOS genes in the three dipteran species shows a high level of amino-acid sequence identity, as well as a substantial conservation of intron-exon structure, indicating that these genes are under strong selective constraints. An unexpected and intriguing result of this study is that in roarrays . In A. gD. melanogaster, the acquisition of a new, testis-biased pattern of expression may be required to maintain duplicates of certain genes in the genome. This may also allow rapid acquisition of new functions by the gene product(s), as it has recently been shown that proteins encoded by duplicated genes with a changed expression pattern often show accelerated evolution [We suggest that, at least in volution ,59. SubfNo data are at present available to support the possibility that our findings could be extrapolated to other gene sets or even to the whole genome. However, we propose that duplication of the genes encoding products that are part of multiprotein complexes may be especially deleterious, unless sequence divergence allowing only testis-specific expression of one of the duplicate copies occurs. In turn, this could facilitate the development of novel functions, which is usually assumed to be the main evolutionary advantage of gene duplication, providing a general mechanism for originating phenotypic changes that might also lead to species differentiation.D. pseudoobscura and A. gambiae, contigs from BCM [D. melanogaster OXPHOS peptides listed in the MitoDrome database [To identify orthologous OXPHOS genes and their duplications in from BCM  and scaffrom BCM  were seadatabase  as queriAmino-acid sequence identity and similarity values were obtained from pairwise alignments using the Needleman-Wunsch global alignment algorithm at the EMBL-EBI server . Multiplin silico. A. gambiae mRNAs were assembled by overlapping ESTs extracted from AnoBase [The genomic sequence of each gene was manually searched for intron-exon boundaries and the predicted mRNA sequence reconstructed  AnoBase .A. gambiae gene with the four-letter code 'agEG' followed by the last four or five digits of its Ensembl [D. pseudoobscura genes were named with the code 'Dpse\CG' followed by the Celera number of their D. melanogaster counterparts.We have named each newly identified  Ensembl  gene numD. pseudoobscura OXPHOS genes investigated here were assigned a chromosomal location where possible, using the putative chromosomal assignments available at BCM [D. pseudoobscura contigs. We also utilized the Ensembl mosquito genome server [A. gambiae annotated OXPHOS DNA sequences.The e at BCM  for the e server  to identD. melanogaster EST database, available from the National Center for Biotechnology Information (NCBI) contains ESTs from cDNA libraries obtained from different developmental stages and body parts. The relative abundance of the transcripts of duplicate or triplicate D. melanogaster OXPHOS genes was defined by counting their cognate ESTs in non-normalized cDNA libraries generated by the Berkeley Drosophila Genome Project (BDGP) [A. gambiae OXPHOS genes was defined by counting their cognate ESTs in all libraries recovered from the Anobase server [The t (BDGP)  from embt (BDGP) . ESTs fre server . Since tD. melanogaster P-insertion OXPHOS mutants is reported in the MitoComp website [The list of  website  and was  website  and from website .D. melanogaster, D. pseudoobscura and A. gambiae putative OXPHOS orthologs are shown and compared, and their deduced amino-acid products are aligned with the corresponding human protein. When paralogs of the gene exist, neighbor-joining trees derived from distance matrix analysis are also shown to visualize the evolutionary relationships between them. Additional data files available with the online version of this article are as follows. Additional data file D. melanogaster OXPHOS genes investigated and their D. pseudoobscura, A. gambiae and human counterparts. Additional data file et al. dataset [D. melanogaster, D. pseudoobscura and A. gambiae.A web-based dataset, MitoComp, contains all data on which this work is based and is available at . It incl dataset . AdditioD. melanogaster OXPHOS genes investigated and their D. pseudoobscura, A. gambiae and human counterpartsA table that reports pairwise amino-acid sequence conservation values between the Click here for additional data fileet al. datasetData extracted from the Parisi Click here for additional data fileD. melanogaster, D. pseudoobscura and A. gambiaeThe codon usage in the orthologous and duplicate OXPHOS genes of Click here for additional data file


The increasing prevalence and impact of obstructive lung diseases and new insights, reflected in clinical guidelines, have led to concerns about the diagnosis and therapy of asthma and COPD in primary care. In Germany diagnoses written in medical records are used for reimbursement, which may influence physicians' documentation behaviour. For that reason it is unclear to what respect ICD-10 codes reflect the real problems of the patients in general practice. The aim of this study was to assess the appropriateness of the recorded diagnoses and to determine what diagnostic information is used to guide medical treatment.All patients with lower airway symptoms (n = 857) who had attended six general practices between January and June 2003 were included into this cross sectional observational study. Patients were selected from the computerised medical record systems, focusing on ICD-10-codes concerning lower airway diseases (J20-J22, J40-J47, J98 and R05). The performed diagnostic procedures and actual medication for each identified patient were extracted manually. Then we examined the associations between recorded diagnoses, diagnostic procedures and prescribed treatment for asthma and COPD in general practice.Spirometry was used in 30% of the patients with a recorded diagnosis of asthma and in 58% of the patients with a recorded diagnosis of COPD. Logistic regression analysis showed an improved use of spirometry when inhaled corticosteroids were prescribed for asthma  or COPD . Spirometry was also used more often when sympathomimetics were prescribed .This study revealed that spirometry was used more often when corticosteroids or sympathomimetics were prescribed. The findings suggest that treatment was based on diagnostic test results rather than on recorded diagnoses. The documented ICD-10 codes may not always reflect the real status of the patients. Thus medical care for asthma and COPD in general practice may be better than initially found on the basis of recorded diagnoses, although further improvement of practice patterns in asthma and COPD is still necessary. There is broad agreement between existing guidelines for asthma -4 regardHowever, it remains difficult to draw a clear picture of management of obstructive lung diseases in primary care since the diagnoses of asthma and COPD often have not been separated in many surveys . This coFurthermore, systematic registration of diseases in primary care are often hampered by missing of recorded diagnoses  or discrThe aim of this study was to assess the appropriateness of the recorded diagnoses and to determine what diagnostic information is used to guide medical treatment. For this purpose we analysed associations between recorded diagnoses, diagnostic procedures and medical treatment regarding asthma and COPD in general practice.A cross-sectional observational study was performed in six computerised general practices with a total of eleven GPs in the Rhine-Neckar region in Southern Germany.All patients who had attended their physicians with asthma, COPD or other lower airway diseases from January until June 2003 were included in the study.The study was approved by the Medical Ethics Committee of the University of Heidelberg.Patients were identified by searching the electronic files for documented diagnoses concerning lower airway diseases within the time frame. The data were evaluated systematically according to the international classification of diseases (ICD-10). In detail, the practice software was checked for the ICD-10-codes of the chapter "chronic lower airway disease": J40 (bronchitis, not specified as acute or chronic), J41 (simple and mucopurulent chronic bronchitis), J42 (unspecified chronic bronchitis), J43 (emphysema), J44 (chronic obstructive pulmonary disease), J45 (asthma), J46 (status asthmaticus), J47 (bronchiectasis). Additionally, the ICD-10-codes J98 (diseases of bronchus, not elsewhere classified) and R05 , and the repeated documentation of ICD-10-codes belonging to the chapter "acute lower airway disease" were extracted in order to detect potentially existing but not detected asthma or COPD. The chapter "acute lower airway disease" includes the diagnoses J20 (acute bronchitis), J21 (acute bronchiolitis) and J22 (unspecified acute lower respiratory infection).The performed diagnostic procedures and the actual medication for each identified patient were extracted and documented manually by two independent scientific fellows (L.G., I.M.) of the department. The variables of the used protocol for documentation are listed in table Every ICD10-code specified above was extracted and written down, at maximum up to three ICD-10-Codes per patient were found. The diagnoses were clustered into three groups: asthma, COPD, and other lower airway diseases. If more than one diagnosis was documented, asthma (J45) or COPD (J44) were extracted as the leading diagnosis.The ICD-10 group J43 (emphysema) was included into the ICD-10 group J 44 (COPD). The ICD-10 groups other than J45 (asthma), J44 or J43 were merged into the group "other lower airway diseases".The performed diagnostic procedures were extracted manually. Four ways of making diagnoses have been found in the records: 1. taking medical history only, 2. taking medical history plus trial of medication, 3. taking medical history and performing spirometry, 4. taking medical history plus single measurement of PEF. The additional performance of bronchial challenge testing and chest X-ray was documented, too.Baseline data were evaluated descriptively. The t-test was used to detect differences of age. Linear logistic regression was used to test relationships between diagnosing asthma or COPD and the performed diagnostics. The relation between prescribing patterns and performed diagnostics was also examined with logistic regression analysis.Overall, 8765 patients attended the practices within the time frame. 857 (9,8%) patients had lower airways diseases .A total of 255 (2.9% of 8765) had the recorded diagnosis of asthma and 112 (1.3% of 8765) had COPD (107 with J44 = COPD, 5 with J43 = emphysema). A combination of asthma and COPD was documented in 25 (0.2% of 8765) cases.The 465 (5.3% of 8765) patients with "other lower airway diseases" included seven cases with acute bronchitis (J20), none with acute bronchiolitis J21), one case with unspecified acute lower respiratory infection (J22), 251 (2.9% of 8765) cases with not specified bronchitis (J40), 38 with simple and mucopurulent chronic bronchitis (J41), 57 with unspecific chronic bronchitis (J42), one with bronchiectasis (J47), seven with diseases of bronchus, not classified (J98), 103 (1.2% of 8765) with cough / bronchial hyperreactivity (R05), Patients with COPD were significantly older than those with asthma or other lower airway diseases. There was no case of twofold documentation of "acute lower airway disease" (J20 - J22) within the time frame. The details regarding the patient population are described in table , one casTable In about 17% the diagnosis asthma was made only on the basis of medical historySpirometry was performed in 58% of the cases with recorded COPD. In 29% the diagnosis was made only by medical history. A small percentage (7%) was diagnosed by history taking and PEF-measurement and 5% by diagnosing by therapy.Concerning the other lower airway diseases, spirometry was done in 19% in order to exclude asthma or COPD. These diagnoses were made mainly on the basis of medical history (73%).The associations between recorded diagnoses and diagnostic procedures are calculated by binary logistic regression  or 'trial of medication' (OR 8.1). The use of spirometry was not significantly associated with the recorded diagnosis asthma.Concerning the recorded diagnosis of COPD, 'trial of medication' was not associated significantly. Instead, the recorded diagnosis was based on spirometry (OR 4.4). Bronchial challenge testing was used to exclude asthma (OR 6.7). Also chest X-ray showed a significant association with the recorded diagnosis of COPD (OR 4.1).The recording of other lower airway diseases was mainly based on the medical history (OR 10.9), the other diagnostic procedures are used only in few cases (OR < 1).The documentation of the prescribed medication showed that 66% of patients with asthma and 42% with COPD received sympathomimetics table . 38% of Only a small amount of patients with 'other lower airway diseases' received anti-obstructive medicine (6–8%).The logistic regression showed that sympathomimetics were given in asthma when having shown efficacy  (OR 8.0) or when spirometry was performed (OR 2.3). Therapy with corticosteroids was significantly associated with spirometry (OR 5.2) and bronchial challenge testing (OR 3.3).Treatment of COPD with sympathomimetics was significantly associated with the use of spirometry (OR 4.1). Inhaled corticosteroids were also given more often when spirometry (OR 4.7) and Chest X-ray (OR 4.6) had been performed. Bronchial challenge testing was used to exclude asthma (OR 6.2).In asthma as well as in COPD corticosteroids are not prescribed only by trial of medication and are not given after making the diagnosis only by medical history.Prescribing of cromoglycate was associated with PEF measurement in both asthma and COPD (OR 1.7 respectively 14.1)Treatment of the other lower airway diseases with sympathomimetics respectively corticosteroids was mainly associated with testing the efficacy of therapy (OR 31.5 respectively 14.0). The prescription is lower associated with spirometry (OR 3.1 respectively 6.1) or x-ray (OR 1.9 respectively 6.2). Cromoglycates were given mostly on the basis of 'taking medical history only' (OR 12.0) and trial of medication (OR 6.2).Although guidelines recommend the use of spirometry, this was used in only in 30% of the patients with a recorded diagnosis of asthma and in 58% of the patients with a recorded diagnosis of COPD. A high number of recorded diagnoses of asthma and COPD were insufficiently diagnosed by PEF-measurement, medical history and diagnosing by therapy. On the first sight, patients with asthma seem to be treated insufficiently with anti-inflammatory therapy and patients with COPD seemed to be over-treated with steroids.However, further analysis showed a high association between prescribed corticosteroids and sympathomimetics as long term medication and performed diagnostic procedures. This improvement of diagnostic investigation when potential harmful drugs are given demonstrates that treatment of asthma and COPD by the GPs may be more consistent with guidelines than other studies have found ,13. ThisAccording to this, our study shows that a deeper look insight with analysis of diagnoses together with performed diagnostics and medication could prove a better care for people with obstructive airway diseases than a single analysis of the documented diagnoses. As a conclusion, ICD-10 codes seem not to be able to reflect the work in primary care, in particular if the difficulties of the diagnostic and therapeutic differentiation between asthma and COPD in earlier stages are taken into account ,20.Therefore it seems that the documented diagnosis due to ICD-10 often serves as a 'working hypothesis'. The needs of family physicians for practice and research would be best met by the International Classification of Primary Care (ICPC-2) , but theAnother critical point is, that the 'trial of medication' as a 'fact of life in primary care' could implicate a patients' perception of improvement which could lead the doctor to perpetuate the therapy, thus resulting in over-prescribing accompanied by an avoidable risk of side-effects. Especially this 'trial of medication' may reflect diagnostic uncertainty in general practice, which must normally have consequences for the classification and labelling. In absence of a reasonable classification system the GPs are forced to hide this uncertainty by documenting diagnoses in ICD-10-codes. As a result, GPs could feel the uncertainty nevertheless, but start seeing it as a personal shortcoming rather than a reality of medicine. Therefore this exaggeration of 'labelling with ICD-10' could also constrain a systematic quality improvement in diagnostic performance in primary care.The strength of the study was the detailed analysis with manual extraction and documentation of performed diagnostics and actual medication of each identified patient. As the ICD-10 classification made it difficult to identify patients with obstructive airway diseases in junction with performed diagnostics and therapy we searched all records manually. Because of this complexity the study had the limitation that the number of GPs was small, which reduces the generalisability of the descriptive findings, although the estimates of associations are less sensitive for this problem.The prevalence of asthma and COPD in this German sample of general practice seems to be low compared to the prevalence of about 5% in studies of other countries -27. One The conclusion of this study is that performance assessment should take the reliability problems of recorded diagnoses into account, especially if the ICD-10 classification is used. A more detailed analysis may be necessary for adequate evaluation of primary care, consequential pointing out to the fact, that treatment of obstructive airways diseases by the GP's may be more consistent with guidelines than other studies have found. Despite these findings improvement of diagnostic performance in general practice is necessary, because a routine use of spirometry is of growing importance against the background of the increasing prevalence of obstructive airway diseases.The author(s) declare that they have no competing interests.AS conceived and designed the study, wrote the report. LG and IM checked the practice software and documented data. MB helped with interpreting the data and writing the report. MW helped with interpreting the data and writing the report. JS helped with revising the report. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Malaria is a major public health problem for countries in the Southern Africa Development Community (SADC). While the endemicity of malaria varies enormously across this region, many of the countries have districts that are prone to periodic epidemics, which can be regional in their extent, and to resurgent outbreaks that are much more localized. These epidemics are frequently triggered by climate anomalies and often follow periods of drought. Many parts of Southern Africa have suffered rainfall deficit over the past three years and countries expect to see increased levels of malaria when the rains return to more 'normal' levels. Problems with drug and insecticide resistance are documented widely and the region contains countries with the highest rates of HIV prevalence to be found anywhere in the world. Consequently, many communities are vulnerable to severe disease outcomes should epidemics occur.st Southern African Regional Epidemic Outlook Forum, which was held in Harare, Zimbabwe, 26th–29th September, 2004.The SADC countries have adopted the Abuja targets for Roll Back Malaria in Africa, which include improved epidemic detection and response, i.e., that 60% of epidemics will be detected within two weeks of onset, and 60% of epidemics will be responded to within two weeks of detection. The SADC countries recognize that to achieve these targets they need improved information on where and when to look for epidemics. The WHO integrated framework for improved early warning and early detection of malaria epidemics has been recognized as a potentially useful tool for epidemic preparedness and response planning. Following evidence of successful adoption and implementation of this approach in Botswana, the SADC countries, the WHO Southern Africa Inter-Country Programme on Malaria Control, and the SADC Drought Monitoring Centre decided to organize a regional meeting where countries could gather to assess their current control status and community vulnerability, consider changes in epidemic risk, and develop a detailed plan of action for the forthcoming 2004–2005 season. The following is a report on the 1 Representatives from malaria control services in nine Southern African countries participated in the meeting: Angola, Botswana, Madagascar, Mozambique, Namibia, Swaziland, Tanzania, Zambia, and Zimbabwe.The Southern African region has a long and varied history of malaria control with periodic epidemics occurring ,2. TheseThe purpose of the meeting was: to enable malaria control services from epidemic prone countries to gather and review their control programme status and epidemiological trends for the past 3–5 years and identify and map districts they consider to be vulnerable to epidemics; to learn about advances in the science of seasonal climate forecasting and review the implications of the forecast for the forthcoming season; to learn about environmental variables pertinent to epidemic risk and readily available sources of monitoring information; to review methods of early detection using case surveillance data; and, using the WHO framework for MEWS, to develop plans of action for epidemic preparedness and response for the forthcoming season.The MEWS framework as set out by WHO consists of four components: 1) vulnerability monitoring; 2) seasonal climate forecasting; 3) environmental monitoring; and 4) sentinel case surveillance. This framework is illustrated in Figure There are many factors that increase the vulnerability of a population to malaria epidemics ,7 and inIn recent years there have been significant scientific advances in our ability to predict climate on the seasonal timescale . The skiThe availability of environmental variables pertinent to malaria transmission, such as rainfall, temperatures, humidity, and flooding, were discussed and information on where they could be obtained was provided. The two basic sources of such information are periodic summaries  available through the internet from the SADC DMC, the Famine Early Warning Systems Network (FEWS-NET) or the International Research Institute for Climate Prediction, Columbia University (IRI) websites; or directly from national meteorological services' ground-based weather observations. Generally, summary products are available free of charge, whereas the meteorological services may need to charge for raw data. Countries were encouraged to begin dialogue with their national meteorological services and discuss the more specific information requirements and support they may need.The paramount importance of developing good health information and sentinel surveillance systems was acknowledged. The process of MEWS development is seen as offering opportunities for strengthening integrated health systems surveillance. It is in itself dependent on good epidemiological data for testing and validating the relationships between the component parts. Methods of using indicators for epidemic early detection were discussed. Various indicators such as the mean × 2 standard deviations, the 'normal channel', cumulative-sum and weekly case thresholds have been tried, tested and used in a number of Southern Africa countries, and countries are encouraged to develop and use what is most appropriate and effective for their purposes. However, a number of the countries acknowledged having a poor statistical basis on which to develop and test early warning and detection indicators.Following the formal presentations setting out the MEWS components and epidemiological trends, the discussions centred around the countries' perceived control needs over the coming season and the information requirements for developing appropriate plans of action for epidemic preparedness and response. The countries represented varied markedly in their current levels of endemicity/epidemicity, surveillance and control coverage. Tanzania is for the most part a highly endemic country with an estimated 16–19 million cases per year. Botswana and Swaziland, by contrast, are currently recording cases in the low thousands and hundreds respectively. Zimbabwe's economic situation has recently compromised its control programme, and two of the countries, Mozambique and Angola, are in process of reconstructing their control programmes after recently emerging from major disruption due to long-term conflict situations. However, all of the countries did acknowledge the integrated MEWS approach as offering a useful framework for improving their epidemic planning, preparedness and response capabilities.. The participants discussed the difficulties in access to and interpretation of meteorological data. The representatives from the meteorological services expressed a willingness to engage in closer collaboration to address these issues. The participants voiced a clear need to improve the availability of the seasonal climate forecast to the epidemic prone districts. They also highlighted the need for better communication of the forecast to non-climate users. Requests were specifically made for forecasts that are more 'meaningful' to the health sector. In response, the meteorological sector pointed at the necessity to know more specifically what information the health sector requires in order to then meet this need. Forecasts could, for example, be expressed simply as the probability of the coming season being wetter or drier than the previous season, or two, or three, or n seasons; or compared to that of the last epidemic season; or as probabilities of exceeding a given threshold for the season. However, it was stressed that forecasts will always be probabilistic and not deterministic. Moreover, countries were encouraged to refer to forecast updates as the season progresses. The issues of how to communicate better the probabilities and uncertainties associated with seasonal climate forecasts were addressed more closely. While many activities in malaria control are based on probabilistic, uncertain premises , public health professionals are well aware of the limitations of their own indicators. While recognizing the potential value of advance lead-times for planning, they are understandably cautious in basing critical decisions on uncertain information from others, and the health and meteorological sectors probably need to work this through in more local collaborative settings.Based on the the climate forecast for October, November, December, and the extended forecast for January, February, March, which are posted on the DMC website One additional issue that came out strongly during the meeting was the need for broader cross-border collaboration on epidemic prevention and control as 'true epidemic' prone areas are often based on particular environmental zones rather than administrative boundaries. For example, high rainfall anomalies in Angola may ultimately find their way as increased stream-flow into Botswana and Namibia, and create extensive breeding sites for vectors. Drought, food security, or a range of other factors, may lead to migrations of people across borders from one level of endemicity to another and pose a significant increase in epidemic risk. Development of national epidemic risk maps therefore ought to reflect the situation in neighbouring countries. There are a few examples of cross-border initiatives in the region: Republic of South Africa, Swaziland and Mozambique; Republic of South Africa and Zimbabwe. Both are showing promising results.The meeting ended with the presentation of the recommendations, to be followed up within the next twelve months. The majority of the recommendations highlighted the need for stronger collaboration a) within the health sector itself; b) among the health sector and the climate-meteorological sector, and other relevant sectors; and c) among the various countries in the region. The participants committed themselves, with their partners, to developing integrated early warning systems as a decision support tool for improving epidemic preparedness and response planning. They recognized that this will be best achieved by drawing on appropriate scientific and technological advancements , by conducting operational research, and with the help of technical development and support, strengthen the capacity for improved epidemic preparedness and response in the districts at risk.The successful implementation of MEWS will depend on close cooperation among several partners: National Malaria Control Programmes must work closely with National Meteorological Services, supported by the regional Drought Monitoring Center, WMO and WHO. Opening these channels of communication will allow public health professionals and climate-environmental scientists and practitioners to incorporate more meaningful variables into the seasonal forecasts. In addition, it is necessary to exchange information with institutions dealing with vulnerable populations such as food security and refugee agencies, to develop mutually beneficial mechanisms that ensure easy access and utilization of relevant information for planning or decision-making. It was recognized that by adopting the MEWS approach for malaria control planning the overall health information and surveillance system would be strengthened as other diseases have strong climate and environmental components to their distribution, and further dialogue with Integrated Disease Surveillance and Response services would be useful. Training and capacity building requirements were discussed with WHO-AFRO regarding implementation within the national IDSR activities in the Southern Africa sub-region, and sub-regions elsewhere in Africa. In the final evaluation of the meeting participants, from both health and climate communities, considered that the meeting had provided a very useful overview of the issues and offered a good starting point for them to develop more flexible Plans of Action for Epidemic Preparedness and Response in their countries. It was recommended that a similar meeting be held each year prior to the onset of the rainy season.


Synthetic peptide arrays and libraries, in particular, have accelerated the process. Several factors have hindered or limited the applicability of various techniques, such as the need for deconvolution of combinatorial libraries, the inability or impracticality of achieving full automation using two-dimensional or pin solid phases, the lack of convenient interfacing with standard analytical platforms, or the difficulty of compartmentalization of a planar surface when contact between assay components needs to be avoided. This paper describes a process for synthesis of peptides and phosphopeptides on microtiter plate wells that overcomes previous limitations and demonstrates utility in determination of the epitope of an autophosphorylation site phospho-motif antibody and utility in substrate utilization assays of the protein tyrosine kinase, p60The overall reproducibility of phospho-peptide synthesis and multiplexed EGF receptor (EGFR) autophosphorylation site (pY1173) antibody ELISA (9H2) was within 5.5 to 8.0%. Mass spectrometric analyses of the released (phospho)peptides showed homogeneous peaks of the expected molecular weights. An overlapping peptide array of the complete EGFR cytoplasmic sequence revealed a high redundancy of 9H2 reactive sites. The eight reactive phospopeptides were structurally related and interestingly, the most conserved antibody reactive peptide motif coincided with a subset of other known EGFR autophosphorylation and SH2 binding motifs and an EGFR optimal substrate motif. Finally, peptides based on known substrate specificities of c-src and related enzymes were synthesized in microtiter plate array format and were phosphorylated by c-Src with the predicted specificities. The level of phosphorylation was proportional to c-Src concentration with sensitivities below 0.1 Units of enzyme.The ability of this method to interface with various robotics and instrumentation is highly flexible since the microtiter plate is an industry standard. It is highly scalable by increasing the surface area within the well or the number of wells and does not require specialized robotics. The microtiter plate array system is well suited to the study of protein kinase substrates, antigens, binding molecules, and inhibitors since these all can be quantitatively studied at a single uniform, reproducible interface. Clearly new technologies capable of accelerating the processes for defining the interactions between kinases and their substrates and modulators would be of great value.Phosphorylation and dephosphorylation of proteins are major mechanisms mediating signal transduction throughout the cell and are intimately involved in the regulation of cell growth, physiology, differentiation, and death. Phosphorylation is accomplished by means of kinases which when stimulated by an afferent signal transmit the signal via phosphate transfer to the next site in a pathway. In some cases phosphoprotein-protein interactions take place that modulate signal transduction, e.g. by revealing previously sequestered phosphoacceptor sites in one or both of the interacting proteins, thus creating branch points in pathways. Critical questions exist regarding the identification of the in vivo targets and inhibitors of the kinase [Two highly productive approaches have been the determination of optimal substrate motifs favored by individual kinases, by various combinatorial peptide library approaches and, the use of antibodies to study phosphorylated peptide motifs (reviewed in ,2). Synte kinase ,4. Whilein situ, accelerating the deconvolution process when the number of combinations is, or becomes more limited. Peptide arrays capable of displaying diverse functions including kinase substrate activity have been successfully produced by two methods: in situ synthesis on planar membranes or arrays of pins [A second search strategy for functional peptides employs arrays of spatially addressable peptides that can be tested  of pins -10, or aWe present data here demonstrating the applicability of an automated system for peptide design and synthesis in microtiter plates to the production of peptides and phosphopeptides. We further demonstrate the capability of these peptide arrays to be recognized correctly by specific phospho-motif antisera and to serve as kinase substrates.Previous work led to the development of a system for the automated synthesis of peptide arrays on the inner surfaces microtiter plate wells -13,  and known sequence (NAEpYLRV). Testing was begun with the monoclonal antibody 9H2 produced against a peptide containing the NAEpYLRV sequence. An array of alternating NAEYLRV and NAE(pY)LRV peptides was prepared in a microtiter plate consisting of 12 8-well strips. Monoclonal 9H2 antibody ELISA was performed using three of the strips from the middle section of the plate. It was found that the antibody reacted strongly with the peptide wells containing phosphorylated tyrosine but not with the non-phosphorylated peptide wells or control wells without peptide  with eight out of the twenty phosphotyrosine-containing sequences , or E-pY-L in its predominant form. Peptide sequence # 84 [see To assess the significance of these primary cross-reactivities a consensus table was constructed from 9H2 antibody reactive and non-reactive sequences Figure . A stron# 84 see  which la# 84 see , Figure γ1, the tyrosine phosphatases, Figure YFEEEELV, may also exist.Three of the 9H2 cross-reactive phosphotyrosines are clustered within the C-terminal end of the receptor (992–1173), are known autophosphorylation sites and are known to be recognized by proteins containing Group III SH2 binding domains   was greater than expected based on quantity of enzyme used in previously published work [To characterize the quantitative aspects of substrate phosphorylation by c-Src kinase, a panel of peptides based on known substrate specificities of c-src and related enzymes were synthesized in microtiter plate wells. Each peptide extended from the polylysine backbone by an ε-amino side-chain and by an additional C-terminal Cys unit. Thus, all of the peptides were equally available to the enzyme, with reduced steric hindrance and a uniform presentation. Subsequently, 90 μL of reaction buffer containing varying amounts of c-Src kinase were incubated for 20 min at 30°, according to the manufacturer's instructions. The wells were then washed with distilled water and assayed for the presence of phosphotyrosine by ELISA using a mixture of the broadly cross-reacting phosphotyrosine antibodies previously described in Figure EEIYGEFF  (Src,1) EEIYGEFF  (Src,2) EEIYGEFF  reactiviEEIYGEFF ,22 activhed work ,24 and wThe method for production of synthetic peptide solid-phase arrays in microtiter plates described here is capable of making high quality peptides, as seen by mass spectrometry of the released unfractionated products. The peptide synthesis method is completely automated and has been greatly simplified by the use of standard automated liquid handlers and the use of activated amino acid solutions that may be prepared and stored in advance and added just once at the beginning of the synthetic process. In the present demonstration, 96 well microtiter plates were used; but, 384, 1536, or containers of any well density compatible with the solvents and liquid handler may be used. The solid phase peptide arrays produced on a polylysine backbone were found to be of very high density, provide very low levels of nonspecific binding and steric hindrance, and participate effectively in a variety of biochemical reactions.The strategy described here for the preparation of solid phase synthetic peptide arrays in microtiter plate wells for use in multiplexed assays offers many advantages in the study of protein kinases, particularly in a research environment. In a research environment combined cycles of hypothesis generation and testing, with assay flexibility, speed, quantitative accuracy and precision are of greater concern than in large scale screening applications of large numbers using limited, previously selected variables. The industry standard microtiter plate format ensures compatibility with a vast number of assay platforms and the polylysine backbone with its extended three-dimensional display provides a highly efficient, sterically unhindered, and extremely low background display of the peptide products.L-pY-L/HB (where HL- is hydrophilic and HB is hydrophobic) is highly redundant within the cytoplasmic domain. Three of the eight antibody reactive sites have been previously identified as autophosphorylation sites and are recognized by Group III SH2 domain proteins . Specifically, these were N-methylpyrollidone (NMP), 1 M N-Hydroxybenzotriazole (HOBT) in NMP, 1 M dicyclohexylcarbodiimide (DCC) in NMP, diisopropylethylamine (DIPEA), acetic anhydride, and trifluoroacetic acid (TFA). NMP from this vendor was consistently free of basic impurities and was stored over Molecular Sieve (4A) after opening. NMP solutions were stored at -20C and allowed to attain room temperature before opening. FMOC-protected α-amino acids were purchased from Penninsula Laboratories (San Carlos, CA) and side-chain substitutions were, Asn(Trt), Asp(OtBu), Cys(Acm) or Cys(Trt), Gln(Trt), Glu(OtBu), His(Trt), Lys(Boc), Ser(tBu), Thr(tBu) and Tyr(tBu). FMOC-Arg(Pbf), FMOC- [bis(dimethylamino)phosphono))-tyrosine] , and FMO2O was used (95%, 2.5%, and 2.5%, respectively).Automated preparation of solid phase synthetic peptide arrays in microtiter plates was performed as described previously  (Saxinge4 in TFA, λmax = 495). In the second method, FMOC-L-Ala was coupled directly to the polylysine supports. The FMOC protecting group was released in 20% piperidine, harvested from multiple wells by serial transfer, and quantitated by fluorimetric scanning in a Perkin Elmer LS50B using E(301) = 7800 (1 mM/ml). Values for spectral constants and coefficients were determined from standards dissolved in the cleavage solvent. The solid phase capacity was 380 pMoles/well and the releasable capacity was 150 pMoles/well.TPX microtiter plates were oxidized at either room temperature or 60°C and reacted with polylysine. Peptide quantities were estimated by two different methods. In the first, the polylysine amino groups were modified with the cleavable linker HMPB [4-(4-Hydroxymethyl-3-methoxyphenoxy)-butyric acid] and FMOC-L-Cys(Acm) was coupled to the HMPB-substituted support as described above. Terminal amino groups were deprotected with 20% piperidine in NMP, washed with NMP and derivatized with dabsyl chloride. The dabsyl-amino acid derivative was released with 95% TFA, harvested from multiple wells by serial transfer, and measured by spectrophotometric scanning in a Beckman DU65 using an extinction coefficient of Dabsyl Chloride = 6.5 × 10A polylysine coated TPX microtiter plate was modified by preliminary reaction with FMOC-Rink Amide linker (Novabiochem) to allow peptide cleavage after synthesis using standard DCC/HOBT coupling conditions, as described above. All peptides were initiated with C-terminal Cys(Acm) to provide a constant initiation and cleavage environment. Peptides were released and deprotected by incubation for 3 hours at room temperature in 100 μL of TFA containing water, ethanedithiol and phenol, or TFA containing water and triisopropylsilane. An additional 10 uL of water and 16 hours of incubation time are required for deprotection of [bis(dimethylamino)phosphono))-tyrosine] . SolutioGoat anti-mouse IgG (phosphatase conjugated) was purchased from Kirkegaard & Perry (Gaithersburg, MD) and Upstate Biotechnology (Lake Placid, NY). Murine monoclonal antibody 9H2, purchased from Upstate Biotechnology was prepared against a synthetic peptide containing the EGFR autophosphorylation sequence at Y1173 and was certified by the manufacturer to be specific for EGF-stimulated A431 cells by Western blot. Murine antisera prepared against phosphotyrosine were 4G10 (Upstate), ab8076 (Abcam, Ltd.), and PT01L (Oncogene Research Products). Antiserum RDI-egfract-1 was prepared against isolated tyrosine phosphorylated (activated) EGF receptor from EGF-challenged murine L-cells (Research Diagnostics, Inc.). All sera were used according to the manufacturer's recommendations and assayed by indirect ELISA. The extent of reaction was determined using a phosphatase assay kit from Kirkegaard & Perry.c-src protein-tyrosine kinase, Cat# PK03 (Oncogene Research Products) according to the instructions in the manufacturer's insert. In sequence, all wells received 30 μL of Kinase Assay Buffer (0.05 M HEPES, pH 7.5 + 0.1 mM EDTA + 0.015% BRIJ 35), 30 μL of appropriately diluted p60c-src in Kinase Dilution Buffer (0.1 mg/mL BSA + 0.2% β-mercaptoethanol), 30 μL of ATP mix (0.03 M MgCl2 +0.15 mM ATP) in Kinase Assay Buffer. Plates were incubated at 30°C for 30 min, rinsed with distilled water and assayed for the presence of phosphotyrosine as described above. Each unit of p60c-src enzyme catalyzes the incorporation of one pMole of phosphate into tyrosyl residues.Peptide array substrate evaluations were performed using p60(9H2): antibody prepared against a synthetic peptide containing the EGFR autophosphorylation site at Y1173carbonyldiimidazole (CDI)dicyclohexylcarbodiimide (DCC)diisopropylethylamine (DIPEA)EGF receptor (EGFR)ethanedithiol (EDT),N-Hydroxybenzotriazole (HOBT)N-methylpyrollidone (NMP)phosphotyrosine kinase (PTK)Polymethylpentene (TPX)trifluoroacetic acid (TFA)triisopropylsilane (TIPS),Amino acid and polypeptide abbreviations were in accordance with IUPAC-IUB recommendations.WCS designed the strategy for peptide synthesis, participated in the design of the strategy for peptide synthesis validation, carried out the peptide synthesis, testing, functional analyses, and drafted the manuscript. TPC participated in the design of the strategy for peptide synthesis validation, carried out the mass spectrometry analyses and interpreted the ms results. DJG participated in the design of the strategy for peptide synthesis validation. TDV participated in the design of the strategy for peptide synthesis validation and coordinated the mass spectrometry analyses.EGRF (pY) peptide scanning array Vs 9H2 Antibody ELISA. EGF receptor phosphotyrosine and tyrosine overlapping peptide array sequences and ELISA test results for each peptide array.Click here for file


Although clinical ethicists are becoming more prevalent in healthcare settings, their required training and education have not been clearly delineated. Most agree that training and education are important, but their nature and delivery remain topics of debate. One option is through completion of a clinical ethics fellowship.In this paper, the first four fellows to complete a newly developed fellowship program discuss their experiences. They describe the goals, structure, participants and activities of the fellowship. They identify key elements for succeeding as a clinical ethicist and sustaining a clinical ethics program. They critically reflect upon the challenges faced in the program.The one-year fellowship provided real-time clinical opportunities that helped them to develop the necessary knowledge and skills, gain insight into the role and scope of practice of clinical ethicists and hone valuable character traits.The fellowship enabled each of the fellows to assume confidently and competently a position as a clinical ethicist upon completion. Bioethics is being integrated into healthcare settings more widely and systematically than ever before. In Canada, clinical ethicists are employed in many teaching hospitals and their presence is increasing in community hospitals and long-term care facilities. Although individuals who work in the field come from diverse backgrounds with a variety of skills and training, the roles that clinical ethicists fill have some commonalities. Most clinical ethicists serve as resource persons and engage in consultation services, research, education and policy development within a healthcare setting, as well as engage in organizational ethics activities .The American Society for Bioethics and Humanities has developed a set of core competencies for health care ethics consultation ,3. The SA clinical ethics fellowship is perceived by some to be one of the ways in which necessary core competencies can be acquired . CurrentThe University of Toronto Joint Centre for Bioethics (hereafter referred to as the JCB) developed and implemented a clinical ethics fellowship program to assist in meeting the identified need for clinical knowledge and skills ,3,6,11. The JCB is a collaborating centre of the World Health Organization. It was formed in 1995 and is a partnership among the University of Toronto and its affiliated hospitals. With a membership of over 160, approximately 20 of whom work full-time in bioethics, it represents the largest multi-disciplinary group of in-hospital ethicists in Canada. Its members are widely published and actively engaged in a number of locally and nationally funded ethics research projects. In addition to the clinical ethics fellowship, the JCB offers two bioethics graduate programs.The first two participants in the JCB's clinical ethics fellowship entered the program in July 2001. The second cohort of two fellows began the program in August 2002. (In September 2003, the program expanded to include three fellows, and in September 2004 grew to five fellows.) The primary purpose of the one-year fellowship program is to provide the necessary preparation individuals require for a smooth transition from academic and clinical education, training and experience to the position of clinical ethicist. The fellowship provides multi-site clinical ethics opportunities at both specialty and general hospitals, exposes fellows to a variety of multi-disciplinary approaches to clinical ethics, supports the work of the ethicists at the JCB's affiliated hospitals, and, lastly, expands and strengthens the network among clinical ethicists, both within the JCB and across Canada. To be eligible for the fellowship candidates must have a graduate degree in bioethics or a professional degree with significant bioethics training. Preference is given to candidates with previous exposure to clinical bioethics including consultation and teaching experiences.In the first two years the program of the program, each fellow rotated through four of the JCB's eight affiliated teaching hospitals, both specialty and general. As the number of affiliated hospitals has continued to grow, so too has the number of fellows. The fellowship was structured so that each fellow was concurrently assigned to two hospitals for a six-month period of time, averaging about two days per week on site at each hospital. A minimum of one day per week was spent working at the JCB where the fellows shared well-equipped office space. This functional arrangement promoted opportunities for collaboration, reflection and mutual support among the fellows. The fellows received a monthly stipend that was sufficient for covering basic costs of living.Project for Examining Effectiveness of Clinical Ethics (PEECE). PEECE was an ongoing research initiative. Its purpose was twofold: to describe the current state of affairs of clinical ethics across sites and through interviews with key stakeholders to identify benchmarks of effectiveness. Fellows participated in all aspects of the project from reviewing literature, developing a proposal, collecting and analyzing data to preparing papers for publication. Policy discussions revolved around such varied topics as sexuality in long-term care, pharmaceutical sponsorship, gift-giving in the context of professional/provider relationships and end-of-life care.Throughout the year, fellows attended and actively participated in the weekly Wednesday meetings and case conferences of the JCB's Clinical Ethics Group, as well as the weekly seminars hosted by the JCB that were open to the university community and the public. The Clinical Ethics Group is comprised of the ethicists who work at the JCB's affiliated hospitals. The focus of these weekly meetings is to develop exemplary models of clinical ethics practice in diverse healthcare settings. Activities include research and practice collaborations, sharing of ideas and resources, strategic planning and policy discussions. Fellows actively participated as full members of the group in these meetings. For example, one of the projects that fellows worked on was the conception, development, and implementation of the During the weekly case conferences, individual ethicists bring complex and challenging cases forward for broader consultation and review. For example, in the second year of the fellowship, a pressing clinical situation arose, with an accompanying set of complex ethical questions. This was the emergence of Severe Acute Respiratory Syndrome (SARS). The weekly case conference discussions during this time period focused on ethical issues such as the professional duty to care for and treat patients, limits of confidentiality and visitor restrictions. Among the many other cases that came to the case conference were situations of conflict around end-of-life treatment and defining futility, moral distress of staff providing care in the context of serious resource limitations, elder abuse in the community, and pregnancy termination for genetic anomalies. Fellows who were involved in the cases collaborated and co-presented with the hospital ethicist. Fellows also provided background literature, developed presentation materials and other resources for the Clinical Ethics Group on specific ethical issues as requested. In addition to providing a mechanism for acquiring broader consultation on a particularly challenging and complex case, the case conferences served as a quality assurance mechanism for the affiliated hospitals.The weekly meetings and case conferences were a resource for the clinical ethicists and clinical ethics fellows to receive collegial support and networking opportunities. The weekly seminars featured local, national and international speakers on a wide range of topics.Fellows were encouraged and mentored to participate in a wide range of activities at each of the affiliated hospitals. The fellows were warmly welcomed into the various institutions by the clinical ethicists, staff and patients. Participation in the preparation and delivery of formal and informal educational activities comprised the largest element of the fellowship, and occurred on at least a weekly basis and frequently more often. Educational activities included presenting at Grand Rounds on ethics topics such as clinical ethics decision-making, moral distress, and advance care planning; leading unit-based rounds on topics such as artificial hydration and nutrition at the end-of-life; facilitating brown bag lunches on topical ethics issues; teaching segments of undergraduate and graduate programs; and developing and implementing innovative curriculum for ethics committee members.Second, case consultations were another activity in which the fellows routinely engaged. Initially, fellows participated in the preparation for case consultations and then observed the consultation process as it unfolded. They provided support for the hospital ethicists by gathering background information about the case, reviewing the relevant literature and documenting the consultation in the health record. As the Fellows progressed through the program and their skills and confidence increased, they assumed more responsibility in consultations by chairing or facilitating meetings. In addition, fellows had the opportunity mentor graduate bioethics students by including them in consultations. Throughout the fellowship, fellows received immediate feedback on the progress and outcomes of the consultations from the hospital ethicist. This debriefing opportunity was invaluable for fellows, enabling them to gain insights into the context of the case, the nature of the conflict or difficulty and the unique and recurring themes that were encountered within and across consultations. Teachable moments, individual strengths and areas for further skill and knowledge development were also identified. The number of consultations varied from one site to another, but over the course of the fellowship, each fellow was exposed to a wide variety of consultation experiences. Case consultations differed in terms of their length, from a very short 10-minute conversation to up to 6 hours in a single day with continuing follow-up over subsequent days, weeks and sometimes months.Third, fellows participated in policy and guideline development, although these activities consumed less time than educational and consultation duties. For example, one fellow developed guidelines for the administration of blood and blood products to pediatric Jehovah's Witness patients. She then took the draft to focus groups consisting of various stakeholders both internal and external to the hospital and redrafted the guidelines based on this input.Fourth, fellows participated in clinical ethics research and research ethics board activities. An example of such research was a chart audit conducted by a fellow to examine how consent and capacity issues were being addressed in a particular facility. Several practice concerns were identified and subsequently a facility-wide educational program was implemented. In addition, the fellows engaged in a variety of other scholarly activities including writing, presenting and publishing on ethics-related topics in a variety of forums, which allowed the fellows to develop a comprehensive understanding of a wide variety of strategies for building a sustainable, integrated and accountable ethics program. These experiences, which built professional knowledge, skill and confidence, laid the foundation for the fellows in developing their professional identity as clinical ethicists. Observing the hospital ethicists in action, the fellows realized that these clinical ethics roles were developed over time and with effort. This helped to shape realistic goals and expectations for the early phase of a clinical ethics career.In July 2001, Paula Chidwick and Laurie Hardingham were the first fellows accepted into the JCB's clinical ethics fellowship program. Dr. Chidwick holds a PhD in Philosophy and prior to entering the fellowship program completed an ethics internship at Sunnybrook and Women's College Health Sciences JCB. She has taught bioethics at the University of Toronto. Laurie Hardingham is a registered nurse who has worked in a variety of healthcare settings. She has comprehensive academic education in philosophy, completing a Masters in Philosophy and doctoral course work in philosophy. She has taught philosophy and ethics at the University of Calgary and Mt. Royal College, as well as planned and coordinated the Provincial Health Ethics Network in Alberta.Karen Faith and Dianne Godkin were selected for the 2002/2003 clinical ethics fellowship program. Karen Faith completed a Masters in Science majoring in bioethics through the Collaborative Program at the Joint Centre for Bioethics and the Institute of Medical Sciences, University of Toronto. After completing her degree in bioethics, Ms. Faith was a part-time ethics consultant to several healthcare organizations. Previously, she was a social worker who worked in the area of mental health. Ms. Faith has taught at York University, Seneca College and Centennial College in Toronto. Just prior to beginning the clinical ethics fellowship, Dianne Godkin completed a PhD in Nursing. During her doctoral studies she focused on ethics and gerontology, particularly in the areas of end-of-life decision-making and advance care planning. While studying at the University of Alberta, she taught an interdisciplinary graduate course in health ethics and was an observer on a healthcare ethics committee. The objectives that the fellows set out to accomplish during the fellowship included gaining expertise in the clinical consultation process, further developing their teaching and researching skills, increasing their confidence in working through difficult ethical situations as they unfold and expanding their multi-disciplinary network of contacts.The fellowship helped prepare the fellows to make the transition to clinical ethicists by providing real-time clinical opportunities. Although there were opportunities to attend lectures, seminars and conferences and to participate in research projects and the activities of research ethics boards, the focus of this fellowship was clinical practice.Generally, bioethics education is largely theoretical, focusing on academic course work in philosophy and ethics, as well as other disciplines, at the graduate level. Practical clinical experiences for individuals wishing to pursue a career as a clinical ethicist have been very limited historically and offered only sporadically. In this fellowship, ethical challenges unfold and are addressed within the day-to-day experiences of hospital life. Although hypothetical or retrospective cases studied in the classroom are useful in applying theory to clinical cases, the value of experiential knowledge gained when cases are encountered in the here and now involving real people with tangible consequences cannot be overstated. One fellow recalls a case involving a family having a very difficult time coming to terms with the imminent death of a loved one. The family was adamant that "everything be done", a phrase that often is bandied about in these sorts of discussions and requires considerable exploration. In this case, "everything" was defined by the family to include CPR and admission to intensive care. The fellow attended a meeting with the family and the healthcare team to discuss the plan of care, but the patient, although capable, was too ill to attend. The fellow had not met the patient. The description of the patient by the healthcare team was completely different from that given by the family. The fellow was uncomfortable with the decisions made without directly hearing the patient's voice. It was not until the fellow met with the patient and the physician alone, that she began to understand the situation. Seeing the physical frailty, but clear thinking and comprehension of the patient fuelled her wish to see that the patient received the care that she desired. It mattered what decisions were made, the situation was no longer hypothetical, but was real and the stakes were high.Through their daily work and interactions with staff in the various hospitals, the fellows became familiar with the fast-paced clinical environment and culture, the healthcare providers' values and practices and the complexity and diversity of ethical issues. Given the unpredictability of when consultation requests would surface, fellows found themselves needing to be flexible and accommodating, often leaving writing or research activities to respond to requests for consultation. Fellows could be called to the intensive care unit, coronary care unit, emergency department, or hospital boardroom at any time and some of these consultations required an immediate response. Consultations of a less emergent nature were scheduled for a later time and often included meetings with the healthcare team, families and patients. Fellows carried a pager so that they could be reached immediately.Other learning opportunities included the following: developing and implementing an ethics program through participation in strategic planning activities; raising the profile of ethics in a hospital using a variety of networking, public relations and communication strategies; reaching out to those who questioned the value of ethics programs by establishing an ongoing presence on units that were struggling with a particular ethics issue; building trust and establishing credibility with healthcare professionals by recognizing, understanding and responding first to their most urgent needs; identifying opinion leaders in the organization and integrating them into the ethics program; building bridges with senior management; and supporting the work of ethics committees as well as other hospital committeesThroughout the year, the fellows each worked with a number of clinical ethicists with varied approaches, backgrounds, training and expertise. As a result there were numerous and ongoing opportunities to develop a multiplicity of skills. Through the observation and mentoring of the clinical ethicists, fellows honed their mediation, communication and negotiating skills. They developed political, practical and conflict resolution skills in both observing and responding to conflicts pertaining to patient care decisions They learned to use wisdom or judgment, particularly in establishing credibility, gaining trust and responding to challenges regarding their role and duties. For example, when a fellow witnessed a clinical ethicist's role being challenged by a senior hospital staff member, the clinical ethicist modeled a respectful but assertive approach, demonstrating both good judgment and clarity of purpose. They acquired skills in the recognition, prevention and management of moral distress and moral residue. Many of the clinical ethicists shared personal experiences of morally distressing situations and modeled the need for broad consultation through the JCB consultation group and debriefing with colleagues as a way to cope with stress. The development of this last skill has proven invaluable as the role that moral distress and residue play in the clinical setting becomes increasingly acknowledged and better understood ,16.The skills that were nurtured and developed during the fellowship mirror the ethical assessment skills, process skills and interpersonal skills that have been identified as core competencies for ethics consultation ,3 As theThe fellows observed that the scope and practice of the clinical ethicists included four primary areas of focus: building capacity, acting as a resource, organizational ethics and scholarly work. The goals of capacity building within the organization included promoting ethical sensitivity and discernment, increasing ethics knowledge and skills and enhancing ethical behavior in the delivery of healthcare. This was accomplished through formal and informal educational activities, committee work, consultations and daily interactions with staff. As a resource, clinical ethicists were called upon to do ethics consultations, provide information and share expertise in various areas of ethical concern. Clinical ethicists' organizational ethics activities were diverse and included the development of policy, guidelines and procedures, collaborative initiatives with other departments and professionals and strategic planning. As well, all of the clinical ethicists were engaged in scholarly activities such as research, writing and publishing, presenting at conferences and teaching at universities and colleges.As a result of working with clinical ethicists in a variety of healthcare settings with different educational backgrounds, the fellowship experience offered a broad perspective on the role and scope of clinical ethics practice. Because clinical ethics is a relatively young field that continues to evolve and define itself ,7,17,18,By observing and participating with the clinical ethicists in their daily activities the fellows identified certain important character traits for this role, such as humility, respect for others, self-knowledge, self-awareness and courage. Although other character traits were also observed, the fellows agreed that these particular traits were both necessary and desirable and thus worthy of emulation in their own practice.The fellows observed that clinical ethicists who modeled humility recognized that their role was neither that of judge nor moral expert, but as a member of the team who was able to engage in a collegial process of deliberation and ethical decision-making. As well, with humility came the recognition that one ethicist cannot be knowledgeable in all areas and that it was essential to build up a network of colleagues from different educational backgrounds with whom to consult. Similar traits such as self-knowledge and self-awareness involved the ability of the ethicist to recognize his or her strengths and limitations. The extent to which the ethicist demonstrated self-knowledge and self-awareness influenced their own self-care practices and ability to manage work demands and work related-stress and thus avoid burnout. Fellows observed ethicists maintaining an attitude of respect toward the opinions of all concerned parties; they ensured that each individual's voice was heard and his or her perspective considered. When clinical ethicists upheld an ethical position in the face of considerable opposition the fellows concluded that ethicists modeled courage. The traits deemed important by the fellows reflect many of the character traits that are considered to be prerequisites to successful healthcare ethics consultation ,11. FurtThrough their fellowship experiences in a variety of ethics programs at differing stages of development, the fellows recognized certain elements that appeared to contribute to an effective clinical ethics program. First, a clinical ethics program needs to be integrated throughout the organization. Integration was key in building capacity from bedside to boardroom and dispelling myths about the role of ethics and ethics programs. Embedding ethical considerations into all aspects of decision-making is achieved through an understanding of how ethics can be a resource for the staff when they face ethical dilemmas. Indicators of a well-integrated ethics program included a clear understanding of the program by staff, visibility within the organizational structure and accessibility of the ethicist to staff, patients and families.Second, a sustainable ethics program requires organizational support and a commitment through the provision of a dedicated budget for ethics including administrative support, adequate physical space and resources, as well as support for continued education. Organizational commitment can be demonstrated through a clearly defined and stable reporting structure and the clinical ethicist's participation in decision-making at the management level. Such organizational commitment allows the ethicist the resources and time to provide the services that support excellence in patient care and to help staff when faced with ethical issues. The clinical ethicist needs to have clear goals and parameters for the work and establish reasonable expectations in order to provide an effective service, reducing ethicists' moral distress and burnout.Third, clinical ethicists cannot work in isolation and need the support of a network of colleagues both within and outside of the field of ethics, especially when confronted with complex or unusual cases in new and emerging areas. One of the roles of clinical ethicists is to act at the same time as both trusted organizational insider and as an objective neutral outsider. Clinical ethicists are best able to succeed in this capacity when they develop collaborative relationships with other service providers in the healthcare settings for example, risk management, pastoral care and social work. Fellows observed that this network of support included the JCB clinical ethics group as well as key professionals knowledgeable in areas of bioethics relevant to the specialized areas of health care. For example, one clinical ethicist had particular expertise in pediatric settings and was called upon often by colleagues when an ethical challenge concerned the care of neonates or children.Fourth, the clinical ethicist's ability to see beyond the initial presenting problem was a crucial skill in the case consultation process. As the clinical ethicist entered into the situation the scope of inquiry often broadened and new and larger, and sometimes quite different, questions emerged. For example, when called in by staff for a consultation, the fellows often observed that upon discussion with the patient or family a different problem was brought to light. Fellows observed that ethicists that kept the dynamic nature of the consultation in mind usually had more successful consults.is" before contemplating its future [is and the clinical ethicist's role, as well as acquiring the necessary knowledge, skills and character traits. The one-year practical learning experience in clinical ethics was perceived by the fellows as an excellent way for them to begin to understand what it means to be a clinical ethicist and to develop core competencies to succeed in that role. However, as the field is evolving quickly with new issues emerging, sometimes quite unexpectedly, it is unlikely that one would ever feel fully prepared to independently step into the position of clinical ethicist. The fellows in the second cohort learned this lesson first-hand, when Severe Acute Respiratory Syndrome (SARS) struck Toronto and dramatically transformed the work environment in the hospitals in which they served [Christine Harrison challenges those engaged in bioethics to consider what "bioethics s future . The cliy served . RotatioAfter the first year of the program, a position became available for a one-year senior clinical ethics fellowship. Laurie Hardingham accepted that position, and during the senior fellowship year, she worked in one teaching hospital, concentrating on ethics consultations, increasing educational opportunities for staff and strengthening the clinical ethics program in that hospital. She was also available to mentor and advise the new first year fellows, supporting the fellowship program. The senior fellowship allowed her to develop a greater understanding of how to integrate ethics throughout an organization and develop the ability to more effectively utilize organizational structures and resources in the clinical ethics program.The hospital ethicists that the fellows assisted had many organizational commitments, were involved in numerous projects and could be called upon at a moment's notice for consultations. As the areas of focus for clinical ethics services varied significantly between hospital settings, fellows were required to review and research literature on many complex and different ethical, clinical and legal topics. To meet the demands of working in a fast-paced healthcare environment with rapidly changing needs, fellows were also faced with the challenges of being available, flexible and accommodating. Being introduced to several hospital settings at the beginning of each rotation presented the fellows with the additional tasks of quickly familiarizing themselves with and acclimatizing to new organizational rules and procedures, staff and institutional cultures. Being a fellow also brought in practical considerations such as taking leave from previously held positions, adjusting to a considerable reduction in pay and relocating to Toronto.The fellows were exposed to stylistic and theoretical differences in the way clinical ethics was practiced when working with ethicists who entered the field through diverse academic and clinical backgrounds. The potential does exist for such differences to become a barrier to learning and building trust within the clinical ethicist/fellow relationship and the fellows who experienced this learned about developing working relationships with ethicists whose priorities differed. For example, when the hospital ethicists also had responsibilities as physicians or nurses in addition to clinical ethics responsibilities, the perspectives could differ on which activities receive attention first. Therefore, it is essential that support be made available in the form of advocacy and mediation for the fellows should such a conflict arise. In this program, such support is available through the program's coordinator at the Joint Centre for Bioethics.Not unlike the field of bioethics itself, the Joint Centre for Bioethics Clinical Ethics Fellowship program is evolving with each successive year and will ultimately be judged by how well graduates are integrated into the healthcare community and the contributions they make to the field. The fellows concur that none of them would have felt sufficiently prepared to take on the considerable responsibilities, complex role demands and inevitable moral distress that are inherent in the position of clinical ethicist without the fellowship. Participation in the fellowship was instrumental in helping the fellows develop the necessary clinical ethics skills, knowledge and character traits required for them to assume a role as a clinical ethicist in a healthcare setting. As well, through the fellowship, they cultivated a support network for the future.Since completing the fellowship, each of the first four fellows has obtained a position as a clinical ethicist in a healthcare setting. Because of their fellowship experiences, they embark on their new careers with a realistic picture of clinical ethics, demonstrated core competencies and a strong network of ethics support and expertise to draw upon in the future. Although other educational models for clinical ethicists exist, a clinical ethics fellowship that is applicable to individuals from a variety of backgrounds (i.e., not limited to clinicians or philosophers only) appears to be a viable educational option and one that ought to be further developed and more formally evaluated.JCB – The University of Toronto Joint Centre for BioethicsSARS – Severe Acute Respiratory SyndromeThe author(s) declare that they have no competing interests.PC contributed substantially to the conception and design, analysis and interpretation of data, drafting of the article and revising it critically and gave final approval for the version to be published.KF contributed substantially to the conception and design, analysis and interpretation of data, drafting of the article and revising it critically and gave final approval for the version to be published.DG contributed substantially to the conception and design, analysis and interpretation of data, drafting of the article and revising it critically and gave final approval for the version to be published.LH contributed substantially to the conception and design, analysis and interpretation of data, drafting of the article and revising it critically and gave final approval for the version to be published.The pre-publication history for this paper can be accessed here: