Asthma and sickle cell disease are common conditions that both may result in pulmonary complications. We hypothesized that children with sickle cell disease with concomitant asthma have an increased incidence of vaso-occlusive crises that are complicated by episodes of acute chest syndrome.A 5-year retrospective chart analysis was performed investigating 48 children ages 3–18 years with asthma and sickle cell disease and 48 children with sickle cell disease alone. Children were matched for age, gender, and type of sickle cell defect. Hospital admissions were recorded for acute chest syndrome, cerebral vascular accident, vaso-occlusive pain crises, and blood transfusions . Mann-Whitney test and Chi square analysis were used to assess differences between the groups.Children with sickle cell disease and asthma had significantly more episodes of acute chest syndrome (p = 0.03) and cerebral vascular accidents (p = 0.05) compared to children with sickle cell disease without asthma. As expected, these children received more total blood transfusions (p = 0.01) and chronic transfusions (p = 0.04). Admissions for vasoocclusive pain crises and exchange transfusions were not statistically different between cases and controls. SS disease is more severe than SC disease.Children with concomitant asthma and sickle cell disease have increased episodes of acute chest syndrome, cerebral vascular accidents and the need for blood transfusions. Whether aggressive asthma therapy can reduce these complications in this subset of children is unknown and requires further studies. Lung disease is a major cause of cardiopulmonary disability and mortality . ProgresAsthma is the most common chronic disease of childhood occurring in 6% of the general population. Moreover, the African American population has the highest prevalence (8%), emergency department (ED) visits, hospitalizations and risk of mortality of any ethnic population in the United States [The African American population is at significant risk for the occurrence of both diseases simultaneously but little is known of the affect of asthma on individuals with sickle cell anemia. In this study, we examined hospitalized children with sickle cell disease with concomitant asthma establishing whether there was an increased rate of acute chest syndrome or other complications compared to patients with sickle cell disease without asthma in those presenting with vaso-occlusive pain crises.We performed a 5-year retrospective chart review of 48 children with asthma and sickle cell disease and compared them to a control group of 48 children with sickle cell disease alone. The 48 children with sickle cell and asthma represented all patients with complete medical records with both diseases that were cared for at our center. These children had no hospitalizations for sickle cell crises at other facilities. Children in the control group were matched for age, hemoglobinopathy and gender. Sickle cell disease was determined by high performance liquid chromatography and isoelectric focusing analysis and grouped into phenotypes of hemoglobin SS or hemoglobin SC.Children were included in the asthma group if they had medical record documentation of a discharge diagnosis of asthma (ICD-493) and had been prescribed asthma medications.Vaso-occlusive pain was defined as pain that could not be explained by injury or infection requiring hospital admission and treatment with intravenous pain medication. The criteria for a diagnosis of acute chest syndrome included respiratory distress, hypoxemia, and a new infiltrate on chest x-ray that required hospitalization and a transfusion of packed red blood cells. A cerebral vascular accident diagnosis was based on the new onset of an acute neurological syndrome with a focal neurological finding on examination associated with ischemic changes (images compatible with stroke) on a brain MRI or computed tomography scan.Inpatient admissions were recorded from March 1997 to March 2002 for episodes of acute chest syndrome, vasoocclusive pain crises, cerebral vascular accidents, total blood transfusions, exchange transfusions and chronic transfusions (monthly blood transfusions).Exclusion criteria included any child who had incomplete documentation of their hospital records or those who had moved into or out of the Milwaukee area during the 5-year study period. Six children were excluded because they moved from Milwaukee. The study was approved by the Investigational Review Board.The Mann-Whitney test was used to evaluate differences between the incidents of vasoocclusive pain crises, acute chest syndrome, total blood transfusions and cerebral vascular accidents. Chi-squared analysis was used to evaluate the number of exchange and chronic transfusions. Statistical significance was given to p values 0.05 or less.or hospital admissions for asthma. Of these children, 13 (76%) had been prescribed only albuterol. Twelve (25%) had at least 1 ED visit and at least 1 hospital admission for asthma. Twenty-six (54%) subjects had only hospital admissions for asthma, while 15 children had only been treated in the ED for acute asthma. Of 14 children with a severity category documented, 9 had mild intermittent asthma, 1 each with mild persistent and moderate persistent asthma, and 3 with severe persistent asthma. No patient had documented bronchoprovocation with methacholine and only 3 had documented spirometry with 1 consistent with airway obstruction, 1 with reversibility of airway obstruction, and one with poor technique and unreliable results. Two patients (age 3) were too young for spirometry. Four subjects had been seen in the Asthma and Allergy clinic for consultation and the diagnosis of asthma reaffirmed. Twenty-one (44%) subjects had a primary family member with asthma. Only 5 children were born preterm  and none were diagnosed with bronchopulmonary dysplasia.All subjects in the asthma group had asthma recorded in their medical record with symptoms consistent with asthma. All had been prescribed albuterol; while 28 (58%) had been prescribed controller medications . Most patients had been prescribed combination therapy with more than 1 controller medication or had been switched from 1 controller to another. There was no reliable means to confirm adherence to the prescribed asthma drug regimen. Seventeen (35%) subjects had not had prior ED The cases and controls were well-matched for age, gender and type of hemoglobinopathy Table . The casPatients with sickle cell disease and asthma had significantly more episodes of acute chest syndrome, cerebral vascular accidents, blood transfusions and chronic transfusions as compared to the control group Table . AdmissiNo patient with SC disease in either group had a history of a cerebral vascular accident. Only 1 patient with SC disease had acute chest syndrome, while 3 children in the asthma group had acute chest syndrome with one child experiencing 2 episodes.Despite the high prevalence of these two diseases that affect the African American population, there is paucity of research investigating patients with concomitant asthma and sickle cell disease.In this study, we discovered that children with both asthma and sickle cell disease are significantly more likely to develop severe complications of sickle cell disease including acute chest syndrome and cerebral vascular accidents compared to children with sickle cell disease alone. The significance of these findings relates to the hypothesis that appropriately aggressive treatment of asthma in children with sickle cell disease may diminish the frequency of pulmonary complications.Patients with reactive airway disease and sickle cell disease have a lower transcutaneous oxyhemoglobin saturation . The lowCerebral vascular accidents affect 10% of the children with sickle cell disease often with devastating long term implications. The Cooperative Study of Sickle Cell Disease reported that recent and recurrent episodes of acute chest syndrome are risk factors for cerebral vascular accidents . LikewisOur study demonstrates that pediatric patients with sickle cell disease and asthma are more likely to require acute and chronic blood transfusions. It is not surprising that these patients needed more frequent transfusions since treatment for cerebral vascular accidents and severe acute chest syndrome is blood transfusions. Although SS type of sickle cell anemia is more severe than SC type, the small numbers of patients with SC prevent us from making broad conclusions regarding the degree of risk based on hemaglobinopathy.Two studies have demonstrated increased airway hyperreactivity with reversibility in patients with sickle cell disease without known asthma ,13. ThirA randomized, placebo-controlled trial of 43 episodes of acute chest syndrome in 38 children revealed that intravenous dexamethasone prevented clinical deterioration in mild to moderately severe episodes of acute chest syndrome . Mild anLimitations of our study include those inherent in a retrospective analysis including selection bias, measurement bias and confounding factors. The children who received transfusions for acute chest syndrome likely represent the more severe form of disease. In contrast, those patients deemed to have asthma were primarily being treated as if they had mild to moderate asthma. Only 3 had been diagnosed with severe persistent asthma and none were on daily or every-other-day oral steroids. Importantly, even individuals with mild intermittent asthma can experience severe asthma exacerbations. Other sources of potential error that deserve recognition include the diagnosis of asthma (whether over-diagnosed or under-diagnosed), medication compliance and unknown admission to other hospitals. Although a strict definition of asthma was lacking, the history gleaned from the medical records supported an asthma diagnosis. Most patients with asthma are not cared for by asthma specialists and frequently the diagnosis is made on clinical grounds without formal pulmonary function testing. Additionally, spirometry was not routinely performed during ED and hospital admissions. Although other EDs and hospitals in the metropolitan area evaluate, treat, and admit pediatric patients with asthma exacerbations, the concomitant diagnosis of sickle cell disease likely prompted evaluation at the children's hospital.In this small series, children with a history of asthma and sickle cell disease developed acute chest syndrome and cerebral vascular accidents more frequently than children with sickle cell disease without asthma. The implications of this retrospective study are wide ranging and should lead to further prospective investigations. Whether aggressive asthma therapy in patients with sickle cell disease and asthma reduces the incidence of serious complications is unknown. The potential gains are far reaching and could make enormous impacts on the morbidity, quality of life and mortality of many patients.The author(s) declare that they have no competing interests.MEN conceived the study, participated in its design and coordination and drafted the manuscript. JL participated in data collection. MCZ participated in coordination and manuscript preparation. JPS participated in design, coordination and manuscript preparation. KJK participated in design and coordination.


Problem-based learning (PBL) is gaining popularity as a teaching method in UK medical schools, but statistics and research methods are not being included in this teaching. There are great disadvantages in omitting statistics and research methods from the main teaching. PBL is well established in Australian medical schools. The Australian experience in teaching statistics and research methods in curricula based on problem-based learning may provide guidance for other countries, such as the UK, where this method is being introduced.All Australian medical schools using PBL were visited, with two exceptions. Teachers of statistics and medical education specialists were interviewed. For schools which were not visited, information was obtained by email.No Australian medical school taught statistics and research methods in a totally integrated way, as part of general PBL teaching. In some schools, statistical material was integrated but taught separately, using different tutors. In one school, PBL was used only for 'public health' related subjects. In some, a parallel course using more traditional techniques was given alongside the PBL teaching of other material. This model was less successful than the others.There are several difficulties in implementing an integrated approach. However, not integrating is detrimental to statistics and research methods teaching, which is of particular concern in the age of evidence-based medicine. Some possible ways forward are suggested. Problem-based learning (PBL) is a method of teaching and learning which is widely used in the education of medical students ,2. In prThis project was conceived at a meeting of statisticians from UK medical schools, which is held every year at the Burwalls conference centre, University of Bristol. In 2002, one of the topics for discussion was problem-based learning (PBL). Several things became apparent during this discussion. First, most of the statisticians present had no idea what problem-based learning was. Second, in the few UK medical schools where PBL was in use, statistics and research methods were not being taught through this medium. Statisticians in some of these schools felt excluded. Third, the new medical schools which were starting up in the UK were adopting PBL as their method of teaching.As far as I know, only four of the older UK medical schools have adopted a PBL curriculum at the time of writing: Liverpool, Manchester, Glasgow, and St. George's. St. George's is a special case, as it has two medical courses. The five-year undergraduate entry course is taught in the traditional way, with a small 'case-based learning' element added on. The new four-year Graduate Entry Programme (GEP) is based on the McMaster model, a PBL course which takes graduates in any subject . In noneNew medical schools, such as Anglia, Peninsular, and Hull-York, are being set up as PBL courses . This isThe adoption of PBL in new medical schools may indicate the possibility that PBL will become more widely adopted in UK medical schools. If this were to happen, there would be a real danger of statistics and research methods being marginalised in the medical curriculum. It is difficult enough to persuade medical students of the importance of these topics and it would be even more so if it were taught outside the mainstream of the course. More importantly, in the era of evidence-based medicine these topics should be central to the medical curriculum, not on the sidelines.During a sabbatical visit to Australia, I spent one to three weeks each at most of the medical schools. I interviewed educationalists and statisticians at each of these to ask how statistics and research methods were taught to medical students in their problem-based learning curricula. I had no prior knowledge of how this might be done, so the interviews were open in nature, rather than structured. I enquired about statistics as a subject in its own right and the wider principles of research, critical reading of research, and evidence-based medicine. I then synthesised the information collected to provide a picture of the current situation.. I am particularly interested in how statistics is taught in problem-based learning programmes. I am presenting this material at the forthcoming International Biometrics Conference in Cairns and preparing a paper for possible publication. I would be very interested to learn how these matters are ordered in [your university] . . . Are you the right person to ask? If not, could you suggest someone who would be?' I obtained helpful replies from all three universities.I visited the following universities with medical schools: the University of Western Australia, Perth, Flinders University, Adelaide, Monash University, Melbourne, the University of Melbourne, the Australian National University, Canberra , the University of Sydney, the University of Newcastle, and the University of Queensland, Brisbane. I omitted the University of Adelaide, the University of New South Wales, and James Cook University, Townsville, but was able to get information from them by email. I identified potential informants from the university website and emailed them as follows: 'I am interested in the teaching of statistics to medical students. Last year I visited several medical schools in Australia, but unfortunately I did not manage to visit [your university] on this trip, a serious omission. I have written a report on my experiences, which is available on one of my websites at I went to Australia in search of the fully integrated teaching of statistics and research methods as part of the PBL tutorials. I did not find it anywhere. I did find three different models:• Material integrated but separately taught,• A parallel course,• PBL used only for 'public health' related subjects.In addition, one school  did not use problem-based learning and one (James Cook) taught virtually no statistics. More information about the individual medical schools is available see .This approach was used or planned at the University of Sydney, the University of Melbourne, and the Australian National University. However, of these only the University of Sydney had actually put this into practice. The University of Melbourne and the Australian National University were about to implement what was essentially the Sydney model.In this model, statistics and research methods are taught by PBL and the PBL triggers are integrated with the PBL problems for other parts of the course, but the material is not taught in the same tutorials or by the same tutors as anatomy, biochemistry and physiology. There will be a separate set of triggers for the statistics, etc., presented in a separate tutorial, and by separate tutors.I asked why the main PBL tutors could not do this. As I understood it, the function of a PBL tutor is to facilitate and guide the group, not to impart knowledge. I had no problem, at least that I was aware of, in acting as PBL tutor when students were working with triggers designed to elicit questions about anatomy, biochemistry, and physiology, subjects of which I know virtually nothing. Besides, many of these tutors must routinely read journals which bristle with P values, t tests, correlation coefficients, etc. They must be familiar with the terms, if nothing else. Answers to this included:• the tutors themselves refused to do it,• in the early years of the course we need expert tutors,• many tutors are still rooted in the old paradigm and are reluctant to embrace EBM and it was the view of my informants that experience round the world shows that it is difficult to teach EBM principles.The team at the University of Sydney were on the whole positive about their course.This approach was used at Monash University, the University of Queensland, Flinders University, the University of Newcastle, and the University of Adelaide.In this approach, a non-PBL course is given separately from the main PBL course. This may consist of any combination of lectures, seminars, practicals, web pages, or text handouts. Usually there is an attempt to link this to the PBL cases by using examples related to the case of the week. For example, the case of the week might be asthma and the parallel course could include a critique of a paper reporting a trial of a treatment for asthma.There are several problems with this approach to teaching statistics and research methods. As noted in the Background, the subject may seem peripheral to the main thrust of the medicine course. Student feedback tends to give a much lower approval to parallel courses than to the main PBL teaching. Finally, teaching is dependent on the cases chosen by the PBL teachers, who may change the cases or reorder them at little or no notice. This can make statistics teaching, which is much more dependent on the order of presentation than most subjects in the medical curriculum, extremely difficult.Most people involved in these courses were unhappy with them, the exception being a group project in the Flinders course.This approach was used at the University of Western Australia.This was an unusual model, found at only one university. PBL teaching had been initiated by an enthusiast, after a period spent at McMaster University. She was a member of the public health group and persuaded her colleagues to introduce PBL. However, only about one third of the course is taught this way, anatomy, biochemistry and physiology are taught traditionally. The consequence of this approach is that the tutors are drawn from the population medicine area and so are quite happy to teach statistics, research methods, and EBM. The triggers can be chosen as population-oriented problems, rather than being restricted to the patient case.People I spoke to were very positive about this course, not surprisingly as they saw themselves as the educational leaders in their institution.I did not to find anywhere in Australia a single instance of truly integrated teaching of statistics and research methods through PBL. I have to conclude from this that there are considerable difficulties. I did, however, find an instance of unintegrated PBL teaching, at the University of Western Australia, where anatomy, biochemistry and physiology have continued to be taught in a more traditional manner.What are these difficulties?• If tutors are drawn mainly from the laboratory disciplines, they may be unsympathetic to the population and clinical foci of EBM and its core subjects. We need strong advocates to persuade them of the importance of these topics.• If tutors are drawn from the clinical staff, they may be unsympathetic to the idea of EBM as a core activity. Converting the existing clinical teachers to the new paradigm of EBM may take a generation, but this will be a problem whatever teaching method we use.• Tutors may be ignorant of the principles and details of statistics. This may be true. My own experience as a PBL tutor has been that lack of knowledge has seldom been a problem. It is not the function of a PBL tutor either to impart knowledge or to explain concepts. Persuading tutors of this should be part of their general PBL training.• The changing patterns of cases as the course develops is a particular problem for statistics. This is undoubtedly true.• The nature of the subject does not lend itself to PBL. It would certainly be difficult to deduce the calculations required for a t test from a problem based on a patient with asthma. However, we must consider what we actually want to teach. I do not think there is much point in teaching undergraduate medical students to analyse data, even using computers. What we need to teach them is how to understand research publications, the evidence on which we hope their future evidence-based practice will be based. We can certainly do this using triggers such as published papers, as the University of Western Australia has demonstrated.• The patient case is not suited to teaching these subjects. This is true, though I do not think it is beyond us to devise cases which lead to research and evidence questions. However, we must persuade our colleagues that the patient vignette is not the only type of problem which we can use.We should not see only the difficulties, however, but also the opportunities. If we can integrate statistics and research methods into PBL, there may be great advantages. One consequence of integration would be that the subject would not be marginalised or seen as separate. It would be just one aspect of medicine. Indeed, one of the features of a PBL course is that the distinctions between the different subjects should blur.A second advantage would be that students would be learning statistics and research methods in the contexts in which we hope that they will apply them: the interpretation of clinical data and the assessment of research evidence. The relevance of the subject should be very clear and during their professional careers they would be more likely to be able to recall and use this material when needed.So how can we do it? There must be an advocate for statistics and research methods at the start of the preparations for PBL, who is able to argue convincingly for the inclusion of these subjects. They must form part of the matrix of topics and problems. This is not an easy task and I have been told of great difficulty experienced at this level, as other teachers sought to exclude these subjects so that their own discipline could have more time. This is a natural human reaction, of course. I think that statistics and research methods are central to medical education, and would argue for more of them, at the expense of some anatomy if necessary.We must get away from the idea that the problem must be a patient vignette. There are some statistical topics which can be covered quite conveniently in this way, such as measurement error, coefficients of variation, reference intervals, sensitivity and specificity, etc. After all, when my GP looks at my serum cholesterol on his practice computer, it has by the side of it a 95% reference interval. However, problems could equally be a published paper. We use these routinely in the teaching of statistics, research methods, and critical appraisal.We could use a paper as a trigger for non-research methods topics. For example, a paper on an asthma trial could trigger questions about asthma as well as about randomisation. This may lead to too many questions being raised by the trigger. Another possibility would be to have such a trigger immediately following a case vignette problem on the disease in question. The patient vignette would raise the questions on anatomy, biochemistry, pharmacology, etc., associated with the disease. The following problem using a research paper would then raise only the research methodological questions. The fixed resource sessions in this week would then be devoted to these.We may also, for variety, link research publications to the patient vignette by devices such as newspaper articles which the patient presents to the physician , or say that in a patient problem the clinician has already found a Cochrane review.Fixed resource sessions could include lectures, but I am very reluctant to suggest formal lectures in statistics and research methods for medical students. I would prefer to offer open question and answer sessions, where students can ask the statistician lecturer to explain anything they are unsure of. For example, if the PBL trigger is a randomised controlled clinical trial with results presented in terms of P values, we might be expecting this trigger to lead to questions such as 'why randomize?', 'what does P < 0.05 mean?', and 'should patients be told they are in a trial?'. Students should have made some attempt to answer these before the fixed resource session.There are several difficulties in implementing an integrated approach. However, not integrating is detrimental to statistics and research methods teaching, which is of particular concern in the age of evidence-based medicine. I remain optimistic that we can incorporate statistics and research methods into a fully problem-based curriculum. I think that if we do not, medicine will be the poorer for it.The author(s) declare that they have no competing interests.J M Bland is the sole author.The following is a typical example drawn from the teaching at St. George's Hospital Medical School. The students work in a small group with a tutor to act as facilitator. The students are given the following information:'You are a member of the on-duty Trauma Team in the Accident and Emergency department one Monday evening, when you are alerted to the imminent arrival of an "RTA (road traffic accident) patient". As she is being wheeled in to the Emergency Room, the paramedic accompanying her reports on the circumstances:'The patient is called Janet Phillips, she appears to be a medical student in her early 20s, and was on a Pelican crossing when she was struck by a car which had gone out of control. Janet had been thrown some distance from the car by the force of the impact. She is confused, in shock, has superficial lacerations to her face and severe pain in her pelvis and legs.'At the accident site her airway was cleared, oxygen administered, a traction splint applied to her right leg, and she was immobilised on a long spinal board.'The driver had only minor injuries but his breath smelled strongly of alcohol, and he was now in police custody.'In the Accident and Emergency Department Janet is given a pelvic clamp to arrest her internal bleeding.'The students are told that they should address four general themes in their discussion: basic and clinical sciences, patient and doctor, community and public health, and professional development. In the first tutorial, they are asked to work through the following steps:1. Clarify any terms and concepts in the scenario with which they are not familiar.2. Define the problem(s) and issue(s) raised by the scenario.3. Analyse the problems and issues, seeking explanations or hypotheses.4. Agree on specific questions (learning objectives) for each of the four general themes to which you need answers.They then work individually on tackling the questions agreed upon. In thesubsequent tutorial they discuss their findings and decide how the problems and issues raised by the scenario could be resolved. They may then be given further information about the patient, which might raise further topics for their research. When a problem has raised all the points required, a fresh problem is presented to the students.The pre-publication history for this paper can be accessed here:'The Australian Universities visited'. This provides a descriprtion of teaching at the universities visited as part of this project.Click here for file


Caenorhabditis elegans and the pathogenic soil bacterium Serratia marcescens.Co-evolutionary arms races between parasites and hosts are considered to be of immense importance in the evolution of living organisms, potentially leading to highly dynamic life-history changes. The outcome of such arms races is in many cases thought to be determined by frequency dependent selection, which relies on genetic variation in host susceptibility and parasite virulence, and also genotype-specific interactions between host and parasite. Empirical evidence for these two prerequisites is scarce, however, especially for invertebrate hosts. We addressed this topic by analysing the interaction between natural isolates of the soil nematode Our analysis reveals the presence of i) significant variation in host susceptibility, ii) significant variation in pathogen virulence, and iii) significant strain- and genotype-specific interactions between the two species.C. elegans, the high specificity is observed among isolates from the same population, such that it may provide a basis for and/or represent the outcome of co-evolutionary adaptations under natural conditions. Since both C. elegans and S. marcescens permit comprehensive molecular analyses, these two species provide a promising model system for inference of the molecular basis of such highly specific interactions, which are as yet unexplored in invertebrate hosts.The results obtained support the previous notion that highly specific interactions between parasites and animal hosts are generally widespread. At least for  By definition, parasites have a negative effect on host fitness. Since parasites usually show a shorter generation time than their hosts, they are also able to adapt rapidly to newly arising host genotypes. Both characteristics together select for hosts with efficient counter-adaptations. Subsequently, parasites are favoured if they can circumvent these host countermeasures. Such interactions may result in a co-evolutionary arms race, consisting of repeated cycles of the emergence of new parasite offences and host countermeasures. Hence, parasite-host interactions can lead to extremely rapid evolutionary change ,2. As suPotamopyrgus antipodarum versus Microphallus [Bulinus globosus versus Schistosoma [Daphnia magna and its microparasite Pasteuria ramosa [Drosophila melanogaster and its parasitoid Asobara tabida [Bombus terrestris and the trypanosome Crithidia bombi [Macrocyclops albidus and the cestode Schistocephalus solidus [Co-evolutionary arms races between hosts and parasites  are in many cases assumed to be determined by negative frequency dependent selection. In particular, rare parasite and host genotypes should be at an advantage because commonness facilitates evolution of host or parasite counter-adaptations, respectively ,5. Such ophallus  or Bulinistosoma ), the asa ramosa , betweena tabida , betweenia bombi , or betw solidus . ClearlyCaenorhabditis elegans (Nematoda: Rhabditidae) by the Gram-negative bacterium Serratia marcescens (Enterobacteriaceae). C. elegans has recently been established as a model to study parasite-host interactions and in particular the genetics of host defence [Bacillus thuringiensis [C. elegans encounters under natural conditions have not yet been unambiguously identified. The ubiquitous soil-dwelling bacteria Pseudomonas aeruginosa, B. thuringiensis and S. marcescens are all likely candidates [S. marcescens, recently adopted as a model to study the genetic basis of virulence [In this study, we evaluated differences in host resistance and parasite virulence, both defined in a broad sense and reciprocally as the effect of an infection on host condition . In particular, we tested the presence of genetic variation and also the presence of strain- and genotype-specific interactions during the infection of the nematode  defence -17. It i defence ,19. Abou defence ,19. Theyngiensis . The parirulence ,21, as airulence , thus beC. elegans strains with 5 different S. marcescens strains plus one control . The C. elegans strains were isolated from Münster, in Northwest Germany, and belong to four different microsatellite genotypes [S. marcescens strains originate from different locations around the world. The interaction between the two species was examined with the help of a survival assay, in which the survival of individual worms was monitored in the presence of a defined concentration of bacteria [In our main experiment, we compared the consequences of infection of eight different natural enotypes . The S. bacteria . The surχ2 = 0.904, d.f. = 35, P > 0.999). In the control (worms with dead S. marcescens Db11), only 12 out of 625 were not found in the category "alive" (1.92%). Of these, 9 were morbid and 3 were dead. The recorded number of live worms per strain did not differ significantly from 100% . It also did not differ significantly among the worm strains . These results show that the experimental set-up itself does not cause significant levels of dead or morbid worms and that it does not have a different effect on different worm strains.80 out of a total 3840 cases (2.08%) had to be excluded because of errors during automated worm-transfer (either no worm or more than one worm per well), resulting in between 75 and 80 usable data points per combination of worm and bacterial strains. The number of valid cases did not differ significantly among these factor combinations , the observed differences are most likely determined genetically. Most interestingly, the interaction between specific worm and bacterial strains seems to differ across the table. For instance, C. elegans strain MY10 is more susceptible to S. marcescens strain Sma13 than to ATCC274, whereas the opposite is true for C. elegans strain MY20 . However, they are both significantly worse than the respective saturated models . The latter test examines whether the model employed considers a sufficient number of factors or factor combinations to explain the variation found in the data. The results suggest that the model is not sufficiently complex. We decided against employing more complex models (e.g. consideration of host genotype nested in host strain in a single model), because the response variable is ordinal with only three categories , such that a larger number of predictor variables in the model would most likely lead to highly increased random error in the regression analysis. Thus, as an alternative, we analysed the data using association tests.In general consistency with these observations, ordinal logistic regression (OLR) analysis indicates a significant effect of the factors bacterial strain, worm strain or genotype, the interaction between the two, and also experimental run on the treatment response Table . The twoC. elegans strains is due to differences in genotypic composition. The remaining results indicate that the significant effect from one of the factors on the treatment response is independent of the significant effect from one of the other factors. This finding is consistent with the presence of an interaction effect from the factors bacterial strain and nematode strain/genotype, as above suggested by OLR.Two-way associations were analysed with the LRT. The results show a significant effect of either of the different factors on worm condition Table . The relχ2 = 0.093, d.f. = 3, P = 0.996). In the control treatment of this experiment, all animals were alive.In the second experiment, we specifically addressed the presence of an interaction between two bacterial strains  and four host strains , the latter belonging to two different host genotypes. For this experiment, all factor combinations were included in each 96-well plate and in one experimental run. Only 7 out of 384 cases had to be excluded for the reasons given above (1.82%), resulting in 46 to 48 data points per factor combination. Again, the number of valid cases did not differ among factor combinations . Moreover, they were not significantly worse than the respective saturated models , suggesting that they contained sufficient details to explain the observed variation.The second experiment confirmed the presence of variation in host resistance and pathogen virulence, although the overall level of virulence was lower than in the main experiment Fig. . SubsequSubsequent performance of association tests generally corroborated the OLR analyses: The different predictor variables had a significant effect on the treatment response  genetic differences in virulence among natural S. marcescens strains, and also iii) strain- or genotype-specific interactions between the two. The first of these points is generally consistent with our previous results on the presence of strain-specific differences in resistance of C. elegans towards Bacillus thuringiensis [C. elegans strains from different locations across the world, whereas in the present study all strains derive from the same place (the town of Münster in Northwest Germany) [C. elegans, because the S. marcescens strains considered did not come from the same location.We here provide evidence for the presence of i) genetic differences in resistance among natural ngiensis . HoweverGermany) . PreviouGermany) . Our preC. elegans and S. marcescens in the wild must currently be considered unclear. To date, it is unknown whether the two species indeed co-exist under natural conditions, even though it is strongly suggested by the fact that both – especially S. marcescens – are common soil inhabitants [The observed strain- and genotype-specific interactions represent an important precondition for negative frequency dependent selection. As such, they may contribute to the emergence of co-evolutionary arms races ,4. The rabitants ,23. If tC. elegans-S. marcescens interactions as a side effect. For example, the C. elegans strains may have adapted differently towards environmental toxins. If the underlying detoxification mechanisms are also employed in the defence against pathogens, then this may result in the observed differences in resistance. Such mechanisms could indeed be of relevance in the interaction with S. marcescens, for which at least one toxin (hemolysin ShlA) was previously suggested to contribute to pathogenesis in C. elegans [The situation is clearly different if the two species do not share the same natural habitat. In this case, the observed specificity must be the result of independent adaptations of parasite and host strains to other environmental conditions. Pleiotropy of such adaptations should then have produced the specific  elegans . MoreoveC. elegans and Bt toxin, the main virulence factor of B. thuringiensis [M. albidus and the cestode S. solidus [D. magna and its microparasite P. ramosa [C. elegans in response to S. marcescens [Drechmeria coniospora [B. thuringiensis [C. elegans, this nematode may in the future provide a valuable model system to dissect the molecular basis of specificity in invertebrate-pathogen interactions.Whatever its origin, the finding of high specificity in the interaction has further implications. The molecular basis of highly specific resistance is currently unexplored in invertebrate hosts. It could be due to the presence of different alleles of a certain cell surface protein targeted by specific parasite effector molecules. Such cell surface proteins have been suggested to be important for the interaction between ngiensis ,25. As a solidus  or the w. ramosa . The prercescens , the funniospora , and alsngiensis . ConsideS. marcescens. This bacterium is considered to be an opportunistic pathogen with a broad host range [C. elegans also mediate virulence in other hosts , whereas other genes do not [S. marcescens in humans, where this bacterium has become a growing health problem, primarily in nosocomial infections [Similarly, the observed highly specific virulence was previously unknown for st range . Hence, s do not . This alfections .C. elegans and its potential microparasite S. marcescens, our study provides evidence for i) genetic variation in host susceptibility and parasite virulence, and also ii) strain- and genotype-specific interactions between the two. These two factors represent an important precondition for frequency dependent selection and as such for the emergence of co-evolutionary arms races. Such highly specific interactions were previously unknown for C. elegans or S. marcescens. Moreover, they have not as yet been reported for invertebrates other than molluscs and arthropods. At least for C. elegans, the observed variation was found among strains from the same population, such that it could indeed be of relevance for evolutionary changes under natural conditions. Taken together, these findings suggest that there is widespread potential for co-evolutionary interactions in animal hosts. Both C. elegans and S. marcescens represent important model organisms in biological research for which a diversity of manipulative techniques is available. Therefore, the association between these two species may in the future provide a valuable tool for the comprehensive analysis of such highly specific interactions.Based on the analysis of natural isolates of the nematode C. elegans strains with 5 different natural S. marcescens strains plus one control. The C. elegans strains were isolated by HS and co-workers in 2002 from Münster, North-West Germany [C. elegans strains had all been cryo-preserved within 5 generations after isolation [We compared eight different natural  Germany . They ar Germany . Some of Germany . Mainten Germany . These Csolation . They wesolation .S. marcescens strain Db11 was originally isolated by H. Boman [C. elegans strain N2 when tested on solid agar, whereby virulence varied in the following order (from high to low): Sm2170 > ATCC274 > Db11 > Sma3 = Sma13 [8 per ml. As a control, we used heat-killed bacteria of strain Db11 (incubation at 70°C for 15 min). These dead bacteria were previously shown to have lost their deleterious effects on C. elegans [The H. Boman ; the strH. Boman ; and strH. Boman , Sma3, SH. Boman . They we = Sma13 . Note, h elegans .C. elegans and S. marcescens was assessed using a simple survival assay [S. marcescens strains and the control, randomised across the plate. Only one C. elegans strain was examined per plate. Eight plates were analysed in parallel, each with one of the eight different C. elegans strains. Five runs of this set-up were performed, whereby the order in which strains were analysed during each run was randomised. This set-up resulted in a maximum of 80 data points per bacterial strain – worm strain combination.The interaction between al assay . For thi7 bacterial cells per well at the start of the survival assay. This concentration was found in a pilot study to permit detection of differences in survival among worm strains after 24 h. After this time period, the condition of the worms was recorded using the following three categories: i) alive , ii) morbid , iii) dead .For a specific run, 50 μl NGM solution (without any bacteria) were first added to each well of the 96-well plates using a multi-channel pipette. Thereafter, individual worms were transferred to each well using the COPAS automated worm-sorter (Union Biometrica Inc.). For worm-sorting, we used synchronised L4 stage worms. Success of worm transfer was monitored. If no worm or more than one worm was transferred to a particular well, then it was excluded from further analysis. After worm transfer was completed, 50 μl NGM solution with pathogens were added to each well with a multi-channel pipette. These 50 μl contained 45 μl NGM solution and 5 μl of bacteria in LB with an OD of 0.1, resulting in a total of approximately 1 × 10N = 40) using differential interference contrast microscopy and a fluorescent microscope .After completion of the experiment, we re-assessed the interaction between two bacterial strains (Db11 and ATCC274) and four worm strains . The general set-up was the same as above. In this case, a total of four 96-well plates were studied at the same time. In contrast to the above experiment, each 96-well plate contained both the different worm and bacterial strains, randomised across the plate. This set-up results in a maximum of 48 data points per factor combination. In addition to the above, we included two 96-well plates as a control. These contained heat-killed Db11 bacteria and the four worm strains randomised across plates (48 data points per worm strain). After exposure to the pathogens, we confirmed that worms were indeed infected with the bacteria by analysis of some of the animals . Based on the hierarchical order of the categorical response variable , we used an ordinal logistic regression analysis (OLR) ,37. For For the main experiment, the lack of fit test was significant, indicating that the chosen model may not be sufficiently complex to explain the variation in the data (see results section). Therefore, we additionally employed association tests based on the inferred frequency counts for the different factor combinations. We specifically assessed the association between the treatment response (condition of the worms) and either of the following factors: bacterial strain, worm strain, worm microsatellite genotype, and run. The significance of the association was inferred using the likelihood ratio test (LRT) ,38. We fCMH, Cochran-Mantel-Haenszel test; LB, Luria broth; LRT, likelihood ratio test; NGM, nematode growth medium; OD, optical density; OLR, ordinal logistic regression.HS designed the study, carried out the experiments, analysed the data and wrote the first draft of the manuscript. JJE participated in the design of the study, the interpretation of the data and revised the manuscript.


The medical specialities chosen by doctors for their careers play an important part in the workforce planning of health-care services. However, there is little theoretical understanding of how different medical specialities are perceived or how choices are made, despite there being much work in general on this topic in occupational psychology, which is influenced by Holland's RIASEC  typology of careers, and Gottfredson's model of circumscription and compromise. In this study, we use three large-scale cohorts of medical students to produce maps of medical careers.Information on between 24 and 28 specialities was collected in three UK cohorts of medical students , in applicants  or entrants  and in final-year students . Mapping used Individual Differences Scaling (INDSCAL) on sub-groups broken down by age and sex. The method was validated in a population sample using a full range of careers, and demonstrating that the RIASEC structure could be extracted.Medical specialities in each cohort, at application and in the final-year, were well represented by a two-dimensional space. The representations showed a close similarity to Holland's RIASEC typology, with the main orthogonal dimensions appearing similar to Prediger's derived orthogonal dimensions of 'Things-People' and 'Data-Ideas'.There are close parallels between Holland's general typology of careers, and the structure we have found in medical careers. Medical specialities typical of Holland's six RIASEC categories are Surgery , Hospital Medicine (Investigative), Psychiatry (Artistic), Public Health , Administrative Medicine (Enterprising), and Laboratory Medicine . The homology between medical careers and RIASEC may mean that the map can be used as the basis for understanding career choice, and for providing career counselling. Medical careers begin as undifferentiated, and postgraduate training ends with most doctors specialised for a specific area of practice. Relatively little is known about the transition from the medical student, who can be seen as a relatively undifferentiated, totipotent 'stem doctor' ,2, potenUK medical education requires undergraduates to study a wide range of medical specialities, and most students will have sampled many of the broad areas of practice by the time they qualify. As a result, it is often assumed that students do not make their career choices until after they have finished at medical school, remaining agnostic about their final speciality choice until that time. However, not only medical school entrants (e.g. ), but evMuch research into medical careers does not take into account the broader research literature on non-medical careers see -25), or , or 25])circumscription and compromise in career choice.The present study takes its origins in three separate sets of theoretical approaches, each of which examines different aspects of careers. None of these approaches, however, concerns medical careers specifically. Neither are they restricted to career choice in adulthood. Furthermore, at least one of them is specifically developmental, emphasising the processes by which career choice occurs and changes. The best place to begin this brief theoretical review is with the work of Gottfredson , who ideCareers differ in their demands, requiring different amounts of intellectual ability, manual skill, long-term commitment, or willingness to work in particular environments, and can be better suited to particular personalities, aptitudes, and physical dispositions. Individuals also differ, having different aptitudes, interests and abilities. Career choice therefore involves people considering the entire range of careers and then circumscribing those which they regard as broadly acceptable, making their eventual choices within that subset.negative, meaning that careers are rejected because they do not have attributes which are consonant with the person making the choice, rather than positively chosen for their special suitability.An important practical point highlighted by studies such as Gottfredson's is that choices tend to be compromise. Because of various practical constraints, certain careers are restricted in the number of people they can accommodate or they are unsuitable in other terms, such as their geographical location or the remuneration they can provide. The eventual career chosen is one that 'satisfices,' [Once circumscription has taken place, a number of possible careers still remain. The second stage of choice is sfices,'  being remap of careers. In her 1981 paper she provides an example a two-dimensional representation of 129 occupations which have been scored in terms of 'Prestige level' (high vs low) and 'sextype rating' (masculine vs feminine). When careers are mapped into this space, the process of circumscription involves drawing an area within which careers are acceptable to a person, being neither too masculine nor too feminine, nor being too high in terms of their prestige and hence effort required, nor too low, and hence insufficiently rewarding. A primary concern of the present study is the nature of the map underlying medical careers, and on which circumscription eventually takes place.Implicit in Gottfredson's conceptualisation is the concept of a Journal of Vocational Behavior published in 2000  the structure of careers and career preferences, ii) the correlations of careers with ability and personality, and iii) the developmental processes by which career choices are made. The question for medical education is the extent to which these approaches are appropriate for understanding medical career choice. If they are valid, then that will allow the much broader research literature from career choice in general to inform the more specific area of medical career choice. Underpinning the models of both Ackerman and Gottfredson is Holland's picture of a relatively simple, two-dimensional career map, onto which ability and personality can project, and on the basis of which career choices can develop. We therefore have two main objectives in this paper; firstly, to use data on career preferences from three separate cohorts of medical students, both at the time of application and in their final year at medical school, in order to derive a map of medical careers. And second, to assess the extent to which this specific map of medical careers is homologous to Holland's more general map of a broad range of careers.career preference, but rather in career similarity. If a student has a preference for career A and career B, but has no interest in career C and D, it follows that career A is probably relatively close to career B on the map, and career C is relatively close to career D, whereas careers A and B are likely to be more distant from careers C and D. A matrix of similarities between all possible pairs of a large number of careers from a large number of students then allows one to construct the underlying map . Thearbitrary to rotation. Turning the map through any angle does not change any of the distances between pairs of objects, and therefore the axes of the map cannot be known – in the case of a geographical map, there is no indication of the north-south and east-west axes. The problem of the arbitrariness of dimensions can be circumvented by means of a variant of MDS known as INDSCAL  [Although conventional MDS can reconstruct the underlying map showing the relations between a number of objects, the map itself is Scaling) ,45. ThisTwo different types of data have been used in the present study. The bulk of the analysis looks at data collected during studies of medical student selection and training, and can be used to map medical careers. A subsidiary, but important, analysis looks at a large convenience sample of people of different ages who were taking part in a survey about careers in general, and were asked about their interest in a range of careers, of which only a few were medical. These latter data allowed us both to calibrate specific medical careers in the context of the general Holland typology, and to validate the INDSCAL methodology for deriving a map of careers.The data were collected during three longitudinal studies of medical student selection, the first of which began in the autumn of 1980, looking at students who had applied for entry to medical school in 1981 ,47, the For the present paper, the analysis of the 1981 and 1986 cohort data considers data on all applicants who replied to our questionnaires, whereas the 1991 cohort, which was very much larger, considers only those questionnaire respondents who entered medical school . In Students who entered medical schools in 1981, 1986 or 1991  were followed up as final-year students in 1986 (or 1987), in 1991 (or 1992) and 1996 (or 1997). Students still in medical school were identified through their medical schools, and questionnaires sent to those medical schools. Response rates were 65%, 50%, and 56% in the follow-up of the 1981, 1986 and 1996 cohorts of students in their final year ,51.The questionnaires used in the study, both at application and in the final year, were detailed, typically covering 16 sides of A4, and the results reported here concern only one of the questions asked. Career preferences were assessed by a question which used the rubric,"Below is a detailed list of specialities in which a medical career can be pursued. Please indicate your attitude towards each speciality as a possible career. If you either know nothing about a speciality, or have no opinions about it at all, simply leave that answer blank".not to go into this".A list of specialities followed, each of which was rated on a five-point scale, for which the categories were, "Definite intention to go into this", "Very attractive", "Moderately attractive", "Not very attractive", and "Definite intention The list of specialities varied a little over the different surveys, becoming slightly more extensive as the years passed. The original list was based on the questionnaire distributed as part of the Royal Commission on Medical Education of 1968   and by sex. It asked about the suitability of twenty-four different careers for the person. Twenty careers were derived, as far as possible, from Holland's RIASEC classification, with at least three in each of the six categories. In addition there were four categories which were medical . The rubric was,"Below is a list of careers. Please indicate for each one how much you think it might have been suitable for you as a career".Each career was rated on a five-point scale, ranging from 'Extremely suitable', through 'Very suitable' and 'Quite suitable', to 'Not very suitable' and 'Completely unsuitable'. The subjects were a convenience sample obtained from amongst friends and relations by a first year lab class at University College London, each student being responsible for obtaining a group of twelve subjects, stratified by age and sex.INDSCAL analysis was carried out using the ALSCAL program within SPSS 10.1. Data in each subset were broken down into four groups by age and sex, age referring to mature vs non-mature students in the medical student samples  and to subjects aged <42 or ≥ 42 in the general population sample.The raw data, which were collected on a 5 point Likert scale, were transformed into Euclideanbased dissimilarities for all combinations of career pairs, using the PROXIMITIES program in SPSS. Four different dissimilarity matrices were produced , and these matrices provided the basis for the INDSCAL analysis that involved minimisation, in Euclidean space, of the discrepancies between the career dissimilarities and the corresponding interpoint distances on the map. The loadings of each career on the two extracted dimensions were then plotted onto the figures to provide the maps.The dimensionality of MDS/INDSCAL analyses can be assessed, in a manner analogous to that used in factor analysis in which eigenvalues are plotted against components. In MDS one plots a measure of 'stress'  against the number of dimensions which have been extracted. If too few dimensions have been extracted then the stress is high, the model not accounting adequately for the richness of the data. The optimal number of dimensions is typically indicated by a sudden 'dog-leg' in the stress plot.We consider firstly the general population sample since it both validates the method which we will subsequently use for the medical student samples, and also helps calibrate the axes.The questionnaire was completed by 1044 subjects, 49.2% of whom were male, and 46% aged over 42. Multidimensional scaling used the INDSCAL method, with the four groups comprising older and younger males and older and younger females. The stress plot indicated that there were two major underlying dimensions in the data, as Holland's typology would suggest . TheOf some importance, given the arbitrariness of the Holland hexagon to rotation in conventional MDS, is that the INDSCAL analysis clearly sets one axis as running from R to S, with the other axis orthogonal to that, running from I and A to C and E. These are similar to the Things-People and Ideas-Data dimensions shown in figure applicants in the 1981 and 1986 cohorts, and all entrants in the 1991 cohort), and were 330, 376 and 1437 for the final-year students in the 1981, 1986 and 1991 cohort studies. The INDSCAL analyses were restricted to those subjects for whom complete career information was available; this consisted of 538 applicants and 312 final-year students in the 1981 cohort, 1118 applicants and 301 final-year students in the 1986 cohort, and 1638 entrants and 1437 final-year students in the 1991 cohort. See Sample sizes for the medical student studies were 1135, 2032 and 2973 for the students in the 1981, 1986 and 1991 cohorts , separately for the combined applicant data and the combined final-year data from the three cohorts. The stress formula attempts to quantify the discrepancies between the fitted distances in the model and the observed dissimilarities among the career ratings, with larger values indicating poorer fit. It is obviously the case that the more dimensions are extracted, the better the fit of the model and, hence, the lower the stress value. However, it is also the case that a greater number of dimensions complicates interpretation and may lead to overfitted and unstable solutions. The stress levels with 1,2,3,4,5, and 6 dimensions were .352, .174, .112, .077, .059 and .048 for applicants, and .390, .174, .112, .082, .064 and .052 for final-year students. For the final-year students, it is clear that two dimensions are necessary, and that there is little advantage of adding extra dimensions. The applicant data are slightly less clear and although there is still no doubt that at least two dimensions are necessary there is a suggestion that a third dimension may be of value. Subsequent scrutiny of models with three dimensions suggested that the third dimension was contributed almost entirely by one or two specialities such as forensic medicine, which have a high public and media profile, but which form only a small proportion of medical personnel. It was, therefore, felt to be safe to extract two dimensions, particularly since Holland's typology provided an i.e. >21 yrs old; or typical post-school entry, at ≤ 21 years old). INDSCAL analyses can clarify the underlying dimensions within data as long as the sub-groups are likely to vary along those dimensions. It should be noted that many studies have found sex differences in medical career interest  and age , and yost . In each of the six analyses, the male subjects put more emphasis on the Things-People dimension whereas the female subjects put more emphasis upon the Data-Ideas dimension . In the 1981 cohort there is also a suggestion that younger subjects put more emphasis on the Things-People dimension, and older subjects on the Data-Ideas dimension for differentiating careers, but the effect is smaller in the 1986 cohort, and barely visible in the 1991 cohort, suggesting a possible change in the way these groups perceive medical careers. In interpreting these analyses it should be noted that although the absolute size of the various groups was more than adequate for the INDSCAL analyses, the group weights for the mature candidates  in the 1981 and 1986 finalyear data were based on very small samples, ranging between 5 and 17 participants, and may, therefore, be somewhat unstable.The maps shown in figures The primary objectives of this study were to use the empirical method of individual differences scaling to derive maps of the underlying perceived structure of medical career specialities, and to assess the extent to which those maps are similar to those described by Holland in his hexagonal representation of the RIASEC groups of careers. That this method is a valid way of deriving Holland's structure in general is seen in figure all of the four medical specialities are seen by the general public as being primarily concerned with Ideas rather than with Data, as surely befits medical careers.The general population sample also rated four medical specialities, with Surgery and Anaesthetics at one extreme, and Psychiatry at the other, and these medical specialities differed principally along the R-S dimension. That Surgery and Anaesthetics are more concerned with Things, and Psychiatry is more concerned with People fits well with the reduction of Holland's hexagon to the two dimensions of Things-People and Ideas-Data. It is also worth noting that The MDS analyses demonstrate that the representation of the various medical specialities by the medical student samples can be captured within a two-dimensional space, as Holland had suggested. The maps shown in figures From scrutinising figures Surgeons can be seen as the engineers of medicine, solving problems at high levels of mechanical and technical proficiency, with an emphasis upon practical skills, craftsmanship, and immediate and effective results.The core of Hospital Medicine  is diagnosis, achieved by carrying out appropriate investigations. Physicians typify the model of the 'scientist-practitioner', investigating symptoms and signs and relating them to the underlying pathophysiology of the patient.Psychiatrists, and also General Practitioners, have a more artistic approach to medicine, seeing, interpreting and responding imaginatively to a range of medical, social, ethical and other problems. The emphasis in many ways is on the uniqueness of the patient, the ideas that they are expressing, and the psycho-social theories and concepts which are necessary for interpreting the individual.Although most medicine is concerned with individual patients, the remit of Public Health is primarily social in the sense of applying medicine to society as a whole, treating the 'body politic'. It is noteworthy that in the maps, Public Health is not only at the Social end, but also closer to Data than to Ideas. Public Health manages social and community health by the appropriate analysis of data.The management of hospitals and health-care requires the creative skills of the business executive, the lawyer and the personnel director to achieve a smoothly running system. People, both patients and carers, are at the heart of any health-care system, and therefore administrative medicine is at the People end of the dimension.The running of efficient systems in haematology, histopathology or chemical pathology requires many of the attributes shared with the accountant or the banker, including the willingness to develop, implement and follow standard procedures within a complex system. The emphasis is inevitably upon the things that do the measurements, and upon the data collected, rather than the ideas or people behind the data and the technology.The analyses in this paper suggest that in our groups of students there is a broad similarity between preferences for medical careers and the typology found by Holland in careers in general, suggesting that the structures are homologous. Although our study has been restricted to medical students in the UK, our findings are likely to be generalisable, given that the patterns are found in three separate cohorts studied over a decade, and across medical school applicants and final-year students. Just as Holland's typology is found in most studies of careers, over a period of three decades and in many countries, despite a wide range of changes in society, in education, and in the nature of jobs and careers themselves, so we would predict that our typology of medical careers will be robust to such changes. To put it more strongly, we would predict that despite enormous changes in every aspect of medicine over two and a half millennia, just as Hippocrates recognised that surgery is different in many ways from other branches of medicine, and that not every doctor wishes or is able to be a surgeon, so the same applies today and will probably continue to apply as long as medicine is practised. That is likely to be so primarily, as Ackerman has suggested, because Holland's typology is underpinned by wide-ranging, broadly defined individual differences in aptitude and personality  which arIt may at first be felt that our approach to mapping careers is fundamentally different to that of Gale and Grant ,59, who INDSCAL Individual differences scalingMDS Multidimensional scalingRIASEC Realistic-Investigative-Artistic-Social-Enterprising-ConventionalSpeciality abbreviations in figures ADM Administrative MedicineANS AnaestheticsARM Armed ForcesBMS Basic Medical SciencesDRM DermatologyENT Ear, Nose & ThroatFRN ForensicGER GeriatricsGPlrg GP Large Group practiceGPsml GP Small practiceGPsng GP Single handedGUM Genito-urinary medicineIND Industrial MedicineINF Infectious diseasesLAB Laboratory MED Internal MedicineO&G Obstetrics & GynaecologyO&T Orthopaedics & TraumaOPH OphthalmologyPED PaediatricsPHM Pharmaceutical MedicinePSY PsychiatryPTH PathologyPUB Public HealthRAD Radiology/ RadiotherapyRDL RadiologyRDT RadiotherapyRES ResearchSRG SurgeryThe authors declare that they have no competing interests.ICM had collected the data in the various surveys over a number of years. ICM and KVP jointly decided how to do the statistical analysis, and KVP was responsible for the programming and data analysis. ICM wrote the first draft of the paper, which was revised by KVP, with both authors being responsible for the final draft.The pre-publication history for this paper can be accessed here:Additional analyses of dataClick here for file


While studies of non-model organisms are critical for many research areas, such as evolution, development, and environmental biology, they present particular challenges for both experimental and computational genomic level research. Resources such as mass-produced microarrays and the computational tools linking these data to functional annotation at the system and pathway level are rarely available for non-model species. This type of "systems-level" analysis is critical to the understanding of patterns of gene expression that underlie biological processes.FunnyBase that has been used to store, annotate, and analyze 40,363 expressed sequence tags (ESTs) from the heart and liver of the fish, Fundulus heteroclitus. Primary annotations based on sequence similarity are linked to networks of systematic annotation in Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and can be queried and computationally utilized in downstream analyses. Steps are taken to ensure that the annotation is self-consistent and that the structure of GO is used to identify higher level functions that may not be annotated directly. An integrated framework for cDNA library production, sequencing, quality control, expression data generation, and systems-level analysis is presented and utilized. In a case study, a set of genes, that had statistically significant regression between gene expression levels and environmental temperature along the Atlantic Coast, shows a statistically significant (P < 0.001) enrichment in genes associated with amine metabolism.We describe a bioinformatics pipeline known as FunnyBase can be accessed at . Data and source code are available by request at jpaschall@bioinfobase.umkc.edu.The methods described have application for functional genomics studies, particularly among non-model organisms. The web interface for  Fundulus heteroclitus (killifish) [Investigating patterns of gene expression using mouse and human microarrays has produced insights into cancer ,2, cardillifish) ,29.Fundulus heteroclitus are distributed along the eastern coast of North America which has one of the steepest thermal clines in the world: northern populations have environmental temperatures more than 12°C below southern populations across 12 degrees of latitude. Migration among populations is sufficient to minimize random genetic drift [Fundulus species can be used to discern adaptive changes [F. heteroclitus an ideal species to investigate adaptive variation in gene expression.The killifish ic drift  but not ic drift ,32. Popu changes ,33,34. TMicroarrays from diverse EST collections offer opportunities to address many biological problems, but to effectively use this information often requires a locally generated bioinformatics approach. Tools like the TIGR Gene index  and UnigFunnyBase to meet these functional genomics needs. FunnyBase provides functional information for >40,000 ESTs from the teleost fish Fundulus heteroclitus, provides the means to quickly process, evaluate, and store annotation based on similarity searches of public resources, and integrates these data with species-specific clustering and microarray analysis. Perhaps ironically, the greatest challenge for functional annotation based on similarity searches is an overabundance of data. There are a number of databases to chose from, and often the single best hit from a given database search is not the most informative. FunnyBase implements a strategy to make maximum use of systems-level functional information from Gene Ontology (GO) [We have developed ogy (GO)  assignmeogy (GO) . SpecifiFunnyBase can be systematically analyzed in the context of biological functions. We present a case study to illustrate how assessment of systems-level annotation can identify statistically significant functional differences among sets of genes.Microarray data using genes annotated in FunnyBase , metabolic or signal pathway , or biological function . Sequence data from the first module and functional annotation from the second are matched using database similarity searches (BLASTX and BLASTN). E-values, bit scores and local alignments are stored in the 'Similarity Data' table for all significant matches. One of the strengths of wissProt , NCBI UnwissProt , and NCBwissProt ) to provFunnyBase annotation is accessible through the web or through local SQL queries and data-mining scripts.The third module, Microarray Production & Analysis provides a list of unique genes to be printed and integrates expression data from microarray experiments with the Hierarchical Annotation module. This provides functional annotation for expression data. Fundulus cDNAs was (1) generate a high quality unidirectional cDNA library, (2) normalize the library, (3) randomly pick colonies and amplify by PCR the cDNA within the vector, (4) sequence and identify PCR products, and (5) after approximately every 1,000 clones, subtract these from the normalized library and repeat steps 3–5. Details for all protocols are provided at  and were used in the Comparative Functional Genomic course at Mount Desert Island, ME 2000.The overall strategy used to isolate and sequence thousands of FunnyBase are identified by a number series: unique sequence number, array number, plate number and well identifier (example: 23434_125_001_H04). The remaining 6,966 un-submitted sequences failed to meet one of the sequence quality parameters. Two criteria are used for defining "good" sequences: 1) >100 bp of sequence with Phred score >20 or 2) form an overlapping cluster with other sequences. Of the 19,937 sequences processed with the current version of FunnyBase, 17,893 (90%) passed one of these quality parameters. In earlier iterations of FunnyBase, visual inspection and later a sequencer-specific quality measure equivalent to a Phred score of 15 were used as filters resulting in 3,603 of the first 5,760 sequences (63%) and 13,922 of the next 15,168 sequences (92%) meeting quality standards, respectively. Re-sequences account for 5,668 sequences, and 4,625 of these were submitted.We sequenced 46,433 ESTs and 40,043 of these are available in the dbEST database at NCBI  as of June 26, 2004. Sequences in FunnyBase system has a number of integrated quality control steps. First, a Ctenophore cDNA (NCBI: accession number: CN992733) that is unlike anything else in GenBank is used as a control. Controls are placed in 96-well plates in wells corresponding to the plate number and two orientation wells (A5 and F9). Sequences from 96-well plates are automatically scanned for these controls so that the identity and orientation are confirmed and a report is generated for manual review. Secondly, all clones used for microarray production are re-sequenced. This is necessary because individual cDNAs are cherry picked, re-grown and re-amplified, and each of these steps has the potential to introduce or magnify an error. For example, for a 6,000 gene array, a 5% error rate would result in 300 incorrect clones. Re-sequenced array plates are compared using pair-wise BLAST [One of the most important steps for producing microarrays from cDNA libraries is being able to associate the bacteria containing the cDNAs of interest with the EST annotation. High-throughput procedures are highly prone to tracking errors including: loading plates into an automatic sequencer in the wrong order, orienting symmetric plates in the wrong direction, or mislabeling of plates. The ability to identify these types of mistakes requires controls for identifying plates and plate orientation. The se BLAST  against EST projects generate a number of redundant sequences due to the random selection of cDNAs from tissue libraries Table . ClusterFunnyBase contains a total of 40,043 EST sequences from F. heteroclitus heart and liver. Clustering with CAP3 yields 3,776 clusters that contain 30,688 ESTs (77%). The remaining 8,991 ESTs (23%) are singletons. By storing the results of clustering with annotation, FunnyBase easily identifies these genes and aids in the selection of genes to be used for library subtraction with the goal of picking less common transcripts.The 10 annotated clusters with the most sequences are listed in Table FunnyBase have two or three-to-four sequences  were annotated as compared to 333 of 1,131 (30%) of confirmed high quality singletons. The lower percent of annotated singletons suggests that these are either rare fish-specific genes, or represent otherwise divergent, likely non-coding (5' or 3' UTR), regions.Of the 12,776 unique ESTs , 3,877 (30%) were annotated. The distribution of e-values for these annotations is shown in Figure Danio rerio UniGene, Oncorhynchus mykiss Unigene, Oryzias latipes Unigene, or GenBank NR. FunnyBase includes locally parsed copies of these databases in a relational format. Thus, all searches are done locally and annotation features beyond the FASTA description can be queried. Consensus sequences from the Fundulus EST clusters as well as high quality single unique sequences (singletons) are used as query sequences for BLAST searches against these public databases. The use of consensus sequences, when available, allows sequences that do not contain regions of significant similarity with known protein  to be annotated if they are members of an annotated cluster. All hits with e-value less than 0.001 and their associated alignments are stored in the database and tracked with any associated functional annotation. Users can specify a custom level of significance when assessing the validity of homology based annotation. This record, which goes beyond storing a certain number of 'best hits', is critical because in many cases additional results may have a negligibly lower alignment score, but provide much more useful functional data.Annotations for ESTs are based on similarity using BLASTX or BLASTN  to sequesystematic functional annotations below). Human Unigene provided an additional 311  annotations. SwissProt provided an additional 32 annotations  with 743 fewer annotated sequences than the NR. However, SwissProt is uniformly well annotated as compared to NR where informative functional annotation can easily be buried by numerous uninformative hits at similar e-values. Besides increasing the number of annotations, comparing the annotations from multiple databases ensures that mistakes in the curation can be detected and information such as alternative gene names can be compiled from multiple sources.The use of multiple databases increases the total number of annotated ESTs Fig.  as compaFunnyBase includes locally parsed representations of public databases such as SWISS-PROT in a relational database format. These databases provide additional information that cross-references other public resources such as GO, KEGG or OMIM [In conjunction with performing similarity searches by BLAST,  or OMIM  that areFunnyBase links annotated genes to enzymes in KEGG pathways based on enzyme commission (EC) numbers. These pathway associations are stored and queries can readily identify genes from a given pathway that show specific patterns of expression. For visual inspection of the pathway, the web interface  links directly to the graphical KEGG pathways in which a gene occurs.KEGG  is a uniFunnyBase, 588 (14%) participate in one or more pathways defined by KEGG. These 588 ESTs represent 105 different pathways. Table FunnyBase for the 10 pathways associated with the largest number of distinct sequences (contigs or singletons). The extent that a given pathway is represented in FunnyBase can be used to identify metabolic differences among tissues [Of the 3,877 annotated ESTs in  tissues  or in diFunnyBase includes two non-trivial steps to make the best possible use of GO terms. First, many GO annotations are lost if only the single 'best hit' from a homology search is considered because GO annotation is applied most often to a few model species such as human that may not appear as the single 'best hit' in a list of BLAST results. FunnyBase identifies the gene name associated with the 'best hit' BLAST result and then uses all GO annotation associated with hits from the complete BLAST results that have the same gene name as the 'best hit' and an e-value of e < 10-12. The goal of this approach is to identify annotation associated with a single 'best hit' gene based on results that may come from multiple species (orthologous genes) and therefore may have varying degrees of sequence similarity due to phylogenetic distance, but to avoid the problem of selecting an inconsistent set of GO terms arising from gene families that share regions of sequence similarity but may have different functions.The Gene Ontology project (GO) has produced a structured vocabulary in the form of an acyclic directed graph that biologists can use to annotate genes in a systematic manner . FunnyBaRNA polymerase II transcription factor activity, enhancer binding (GO:0003705) rather than the more general parent term transcription regulator activity (GO:0030528). However, in functional genomic analysis, significant patterns of expression may exist at the more general level of functional description. FunnyBase takes advantage of the connected parent-child relationship of GO terms provided by using the relational database version of GO available for download at  to identify such relationships. These data are used to extract the tree of more general GO terms related to those provided by public databases. A FunnyBase script then re-annotates genes with this more complete set of GO terms.Secondly, GO annotation in public databases tends to annotate sequences with only the most specific GO term available, for example Of the 3,877 annotated genes, 1,912 (54%) are assigned one or more GO terms with a total 6,728 GO assignments being made directly based on information in public databases such as SwissProt. Using parent-child GO term relationship backtracking, an additional 34,112 GO term assignments were made, resulting in a final count of GO assignments of 36,024 excluding the most general terms that divide GO into three categories. Thus, on average, 19 GO terms are assigned to each of 1,912 annotated genes.Fundulus clusters that were similar to a sequence in Human RefSeq , 1,818 (76%) had distinct gene annotations. This method of clustering clusters by similarity to well-annotated reference sequences provides a method to more accurately define the number of unique genes represented by an EST set.Humans have approximately 30,000 expressed genes, yet there are over 1,000,000 human UniGenes (NCBI). Clearly, these clusters of cDNAs greatly overestimate the number of unique genes. Similarly, FunnyBase has multiple clusters for the same gene: 15 apolipoprotein I, 10 cytochrome oxidase I, and 53 vitellogenin clusters. To provide a more precise estimate of the number of unique genes, consensus sequences were queried against the 27,695 sequences from the Human RefSeq  databaseFunnyBase can be integrated with microarray data in a rigorous manner, we used a data set based on a microarray of metabolic genes printed from ESTs annotated in FunnyBase [Amine Metabolism (GO:0009308) is assigned to 14% of the 62 statistically significant genes but only 3% of the non-significant genes (those that do not show significant regression with temperature). A Fisher-exact test indicates these frequencies (14% vs. 3%) represent different underlying distributions (p < 0.001). Specifically, genes involved in amine metabolism are overrepresented in the set of genes that show regression with temperature as compared with the remaining sequences. This significant increase is found for two other non-mutually exclusive GO terms: amino acid and derivative metabolism, and amino acid metabolism (p < 0.05). Other GO terms show a reverse trend although none were statistically significant. For example, ion transport (p = 0.08) and cell growth (p = 0.16) had few genes with a clinal variation in expression. These data suggest that the functions of genes influence whether they are affected by ecologically interesting patterns of expression . These PCR products (PCR-cDNAs) were denatured and hybridized to single stranded plasmids from the cardiac cDNA library. Taking advantage of Cot values, the most abundant cDNAs were annealed to the more abundant PCR products and were removed selectively by hydroxyapatite-column chromatography. The single-stranded plasmids in the flow-through were converted to double strands using the Sequenase DNA polymerase . DH10s E. coli (BRL) were transformed with these double-stranded plasmids by electroporation. The number of recovered plasmids and the resulting complexity of the normalized library depended on the duration of hybridization or Cot values. Two normalized libraries were made using either a 12 or 24 hour hybridization. The library from the 12-hour hybridization yielded 250,000 plasmids. The library from the 24 hour hybridization yielded 3,000 plasmids and had a greater representation of rare mRNAs and greater frequency of non-recombinants.Normalization of cDNA libraries reduces the differences among expressed genes to less than 10-fold among rare and abundant mRNAs ,45. Norm4)2SO4, 2.25 mM MgCl2, 2% (v/v) DMSO, 0.1% (v/v) Tween 20). Two-step thermal cycle conditions were used . PCR products were purified manually in 96 well format using Sephadex G-50 in a deep well plate with a 0.2 microfilter  or robotically using AmPure  and EvolP3 96 pipetting liquid handling system .Characterization of cDNAs  used 96 well plates and octopipettes. To characterize cDNAs, 96 individual bacterial colonies from the normalized library were randomly chosen, and each was grown in 1.25 ml of Superbroth in 2 ml-96 well plates. After 18 hours of growth, two 250 ul bacterial glycerol stocks were made and stored in 96 well plates at -80°C. One microliter of these bacterial growths was used for PCR reactions using forward and reverse plasmid specific primers: . PCR reactions had 0.2 mM dNTPs, 10 pmoles of each primer, 1 unit of Promega Taq (0.2 ul), and reaction buffer with detergents and DMSO , 16 mM (NHPCR products were sequenced from the 5' end (relative to the mRNA) on an ABI 373 or ABI 3730 sequencer using ABI "Big Dye" reaction mix. We typically used 1/16 the amount of reaction mix, yielding 300 to 400 unambiguous bases. Sequences were purified using biotin primers and streptavidin coated magnetic beads (for the ABI 373) or Agencort CleanSeq (for the ABI 3730).Ctenophore cDNA #5, a random cDNA with no similarity to any sequence in GenBank). Two wells (#40 and #67) always contained the marker cDNA, and thus any misloading or mislabeling of sequencing lanes was identifiable. The third marker cDNA was placed in a well that corresponds to the plate number . 2) After the production of 12 plates, one row (8 wells) from each plate was re-sequenced. Thus, 8/96 or ~8% of all sequences and their locations were confirmed. 3) cDNAs used for microarrays were re-sequenced. These measures are important to ensure that the correct and known cDNAs are printed.We used three procedures to verify that the correct sequence was associated with each cDNA. 1) Each 96-well plate had three wells with a "marker cDNA" . After a 24 hour hybridization, genes in the library that bound to these biotin-labeled PCR products were removed with the use of magnetized, streptavidin coated beads. DH10s Computational work was done on an Apple G5 dual 2 GHz processor system with 4 GB of RAM. Data are stored in a MySQL database, perl scripts were used extensively for parsing and loading data, and PHP was used on an APACHE web server to construct the user interface. Additional programs available from their authors are mentioned within context. Software and databases are described in Table F. heteroclitus cardiac library encoding essential proteins for cellular metabolism isolated from over 40,000 expressed sequences . These 384 cDNAs were amplified with amine-linked primers and printed on 3-D Link Activated slides  using GeneMachine OminGrider, and blocked following slide manufacturer protocols. The suite of 384 amplified cDNAs was printed as a group in four spatially separated replicates. Four hybridization zones of these four replicate arrays were printed per slide, with each zone set separated by a hydrophobic barrier. Samples were hybridized twice; once with Cy3 and once with Cy5 resulting in overall technical replication of 8-fold per sample.Microarrays were printed using a select 384 cDNAs from RNA was extracted from tissue homogenate in a chaotropic buffer using phenol/cholorform/isoamyl alcohol and RNA quality was assessed using the Agilent 2100 Bioanalyzer . RNA for hybridization was prepared by amplification using a modified Eberwine protocol  using thFunnyBase and provided statistical analyses of database. MFO initiated, designed protocols and provided sequences for F. heteroclitus' EST project. JDV optimized robotic interfaces for sequencing and sequenced ESTs. JLR and KJK sequenced ESTs. GJW collaborated on bioinformatics and database development. JAW provided microarray data and analyses. DLC initiated F. heteroclitus' EST project and developed the database and annotation schemes for FunnyBase. All authors read and approved the final manuscript.JP designed, scripted and implemented