The purpose was to examine how General Practitioners (GPs) use clinical information and rules from guidelines in their decisions on drug treatment for high cholesterol values.Twenty GPs were presented with six case vignettes and were instructed to think aloud while successively more information about a case was presented, and finally to decide if a drug should be prescribed or not. The statements were coded for the clinical information to which they referred and for favouring or not favouring prescription.The evaluation of clinical information was compatible with decision-making as a search for reasons or arguments. Lifestyle-related information like smoking and overweight seemed to be evaluated from different perspectives. A patient's smoking favoured treatment for some GPs and disfavoured treatment for others.The method promised to be useful for understanding why doctors differ in their decisions on the same patient descriptions and why rules from the guidelines are not followed strictly. The medical decision examined in our study is whether or not to initiate drug treatment for high cholesterol values. The topic has been the focus of much debate on the grounds that the proportion of individuals with elevated cholesterol values is high in most Western populations, and that the costs for treating all these people with drugs life-long would be enormous, with a marginal benefit in risk reduction for the majority of them -3. The cThus, the decision-making task can be carried out as follows. The first step is to decide whether the patient case is an instance of secondary prevention, diabetes or FH, and if not, to estimate the numerical risk for coronary disease within ten years. A risk above 20% would justify pharmacological treatment, given that life style intervention has been tried for a sufficiently long period.In this study we address the question of how General Practitioners (GPs), who manage most of the cholesterol testing and treatment in Sweden, make such decisions when guidelines are not physically available to them. We will try to highlight the decision-making by examining how it is affected by clinical variables describing the patient and by medical knowledge and decision rules on behalf of the doctors. The reason for studying decisions without access to written guidelines is that as experienced GPs (in the case of three of us), we have found that this is how decisions on cholesterol treatment are usually made. Furthermore, in a previous study concerning the ability of GPs to make numerical estimates of future cardiovascular risks, we asked the GPs if they had access to any tool for making numerical risk estimates at their clinic . Only niOur first set of research questions concerned how different kinds of information about the patient  relate to the decision to prescribe a drug or not to do so. First, we estimated the importance of the individual information categories by counting the total number of times they had, according to the coding of the verbal protocols, been valued in a positive or negative direction in relation to the decision at hand. Second, in order to get an idea about why different doctors reach different decisions when presented with identical case information, we made the following analyses. For each of the patient cases separately, the subgroup who decided to prescribe was compared with those with an opposite decision regarding how often they valued different information categories. Third, to further understand how the participants differed in their judgments, we examined which kinds of specified information about a patient  are the most likely to lead to disagreement, i.e. to be judged in a positive direction by some participants and in a negative direction by others.Disagreement about the evaluation of data on a given variable may result from different cut-off values, e.g. for the cholesterol variable. A certain value can be considered high for one participant and thus speaking for drug treatment. The same cholesterol value may be considered almost normal by another participant and thereby speaking against drug treatment for the same patient. The age variable may also be associated with disagreement due to different cut-off values. A higher age is generally associated with a higher risk, but there is a lack of evidence for the potential benefit of treating the oldest age groups, and this may introduce different cut-off values for different doctors.Disagreement may also be caused by what might be called different perspectives. If we take smoking as an example all doctors should recognize smoking as a factor associated with an increase in future cardiovascular risk, and should accordingly make statements with a positive directionality for drug treatment. On the other hand, some doctors in some situations may regard actions aimed at smoking cessation as more beneficial than cholesterol reduction, which may give smoking a negative directionality in relation to drug treatment. Overweight can be regarded in the same way, i.e. as an indicator of drug treatment or as indicating change of life style as preferable to drug treatment. Thus, there are two alternative treatment philosophies – drug treatment or life style change – which in turn may be associated with opposite evaluations of the same data in relation to drug treatment. To the extent that these philosophies in fact are associated with different evaluations, one may regard them as different perspectives where certain data (e.g. smoking) are seen from different angles, as risk indicators or as entities that could be changed through patient's own efforts (i.e. by changing life style) as a means to treat the his or her health problem. The latter perspective may also be associated with somewhat moralistic evaluations, e.g. that overweight or smoking is the patient's own choice or own "fault", which in turn would decrease the inclination to initiate drug treatment. Some evidence for this conjecture comes from a CJA study by Evans et al . They inOur second set of research questions concerned the use of rules, and the concept of risk as shown in the verbal protocols. Six patient cases were chosen that included two high-risk patients (secondary prevention or diabetes) for whom the guidelines can be transferred to a simple decision rule . Our question was how frequently such decision rules were in the verbal protocols and their content in relation to practice guidelines for elevated blood lipids. For the remaining four cases (primary prevention) no such simple guideline-based rule can be applied and instead, a numerical risk calculation is suggested. We examined the extent to which references to risk estimates were made in the think-aloud protocols. For both secondary and primary prevention cases we were interested in determining how the decisions corresponded to what is indicated by guidelines and risk algorithms.In sum, our research questions concerned: Importance of information (which categories of information about the patients seem to be most important for the decisions?). Patterns of importance for "Yes" and "No" decisions . Disagreement (which categories of information give rise to disagreement?). Use of rules (their frequency and contents). Risk estimation .Our approach to analyse think-aloud protocols in a medical decision task for the relative importance of different information categories and the amount of disagreement in the evaluation of these categories has not been tried before as far as we know. We believe that the results can be useful for understanding why doctors reach different decisions in response to the same patient cases and why they often do not act in accordance to guidelines. This knowledge should be useful as an aid to design guidelines and teaching.The 20 participants received the same six patient cases and the order of the cases was the same for all participants. Cases with "Yes"- and "No" decisions as the recommended treatment according to the guidelines were mixed as evenly as possible. Ten of the participants were randomly assigned to a condition where, in addition to thinking aloud, they also rated their willingness to prescribe a drug at regular intervals during each case. As was described in a previous paper , this grTwenty GPs working in the southern Stockholm area participated. There were 10 males and 10 females. Their ages varied between 34 and 60 years (M = 48.3) and they had practiced between one and 22 years (M = 11.4) as specialists in family medicine. A total of 36 doctors were contacted by telephone. They were selected so as to have a relatively even distribution across different districts in the area and according to gender, but the selection was not random. Twenty-four agreed to participate, but before the session four of these later declined to participate.Six clinical cases were selected from an original set of 40 authentic cases with cholesterol values above normal at least 5.5 mmol/L). The original set was used in a Clinical Judgment Analysis design with a different sample of doctors and is described in Backlund et al . Two of .5 mmol/LThe different kinds of clinical information presented on the six successive screens were divided time-wise in the same way for all six cases. The order in which this information was presented was arranged so as to be as realistic as possible in relation to clinical practice . Table The study was conducted at the doctor's office or in a room nearby. All visits and recordings were made by one of the authors (LB). The cases were presented on a computer screen (Software Question Asker™ (QA) . In the The study was approved by the local ethics committee.Each case ended with a screen with the following question: "Would you prescribe a cholesterol-lowering drug for this patient?" The participant responded by clicking on one of two response alternatives, "Yes" or "No".The sessions were tape-recorded. A secretary then transcribed the recorded sessions into a written, word-by-word format. The protocols were segmented into statements. The next step was to categorize the statements into one of ten categories concerning the general characteristic of the statement (cp. Cognition Categories): Attention, Evaluation, Rule, Explanation, Action pharmacological treatment, Action non-pharmacological treatment, Action other, Want of information, Rating  and Other. The set of categories is described in more detail in Backlund et al . Each stTwo of the authors (LB and YS) independently coded the protocols from the first six participants. Reliability was computed separately for directionality  and for information category (one of 21) as the percentage of statements that were coded into the same directionality/information category. For these first six participants, the inter-judge reliability was 92% for directionality and 94% for information category. The reliability measures were considered to be satisfactory and therefore only one of the authors (LB) performed the remaining coding.The original set of 21 different information categories was reduced to eleven. When we ranked the information categories with regard to the frequency of positive or negative evaluations there was a great leap between weight (frequency 14 and rank eleven) and triglycerides (frequency four and rank twelve). We therefore excluded triglycerides and information categories with fewer evaluations. Examples of such excluded categories were information about physical examination of the heart and lungs  and test results concerning liver or thyroid function. The remaining eleven information categories were Cholesterol, LDL (low density lipo-protein), HDL (high density lipo-protein), weight, smoking, CHD (coronary heart disease), diabetes, hypertension, heredity, sex and age.Table F  = 8.80, p < .01, as was Information category × Case interaction, F  = 11.80, p < .01. . Thus, as could be expected, the different information categories were evaluated unequally often, and the pattern of relative importance differed in the six individual patient cases. All other main effects and interaction effects were also significant with p < .01.Figure In the following, the six patient cases will be analysed separately. This may allow us to detect possible differences in the pattern of importance between different information categories for participants who decided to prescribe a drug and those who made the opposite decision. For each case the number of evaluative statements was the dependent variable in a 2 (Decision: Yes/No) × 11 (Information category 1–11) × 2 (Direction: positive/negative) ANOVA, with the first as a between-group variable and the latter two as within-group variables. The statistical effects are summarized in Table The main effect of Decision was not significant in any of the cases, indicating that there was no evidence of an association between the number of evaluative statements and decision outcome. For four of the cases the effect of Direction was significant, indicating that positive and negative statements were of unequal frequency. Except for case SH , positive statements were more frequent than negative ones. For all six cases the different information categories were evaluated unequally often (i.e. main effect of Information). A significant Decision × Direction Interaction, indicating that the decision to prescribe or not to prescribe was associated with different distributions between positive and negative statements, was found in only two of the cases. Direction × Information Interaction was significant or nearly significant for all five cases, suggesting that the distribution of positive and negative directionality was unequal across the different information categories.The most interesting part of these analyses, however, is whether different decisions were associated with different evaluative patterns across the information categories. Statistically, this corresponds to two- or three-way interaction effects including Decision and Information.A significant Decision × Information interaction for a case would indicate that participants with a "Yes"-decision had their number of evaluative statements differently distributed across Information categories compared to participants with a "No"-decision, regardless of whether the direction was positive or negative. The three-way interaction includes the directionality of the statements as well. As can be seen from Table Case IS represents a 67-year-old female with hypertension as a central risk factor in addition to her cholesterol elevation. She also had a modest heredity. As Figure Case TW Figure  represenCase AR Figure  represenThus, an analysis of the response patterns for these three cases suggests that the "Yes" and "No"-groups differed not only in how much they evaluated the central risk factor(s) (in addition to cholesterol elevation) as favouring drug treatment but also in that the "No"-group seemed to have identified at least one information category as evidence against treatment. As regards the remaining cases, the evaluative pattern for case PU (young woman with severe heredity for CHD) could be interpreted in a similar way, with the patient's (young) age as an argument against drug treatment, whereas case GM (diabetic case) did not invite any such interpretation. For Case SH, no comparison between response groups could be made as all participants decided not to prescribe.We defined agreement as the degree to which the same information about a patient case was evaluated with the same directionality. We hypothesized that disagreement would be more common for information about lifestyle-related factors like smoking and weight than would be the case for medical conditions like hypertension and diabetes. There was only one case with a clear overweight and one case where the patient smoked, and the number of evaluative statements concerning these two information categories was therefore rather low. Regarding case TW's smoking, there were 21 statements with a positive direction and ten with a negative direction. For case AR's overweight there were three positive and six negative statements. For hypertension, each of the cases had either only positive or only negative directions . The same pattern was found for diabetes and CHD, except that for diabetes two statements concerning case GM were negative compared to 24 statements with positive directionality, and for CHD (case AR) one statements was negative whereas 31 were positive. Thus, with minor exceptions the participants agreed on the evaluations of these three information categories. The data were consequently in line with our hypothesis.There were few evaluative statements concerning the sex of the cases. With one single exception all statements were negative and concerned female cases, which is in line with the known lower risk for female patients to suffer from cardiovascular diseases. A corresponding tendency towards positive evaluations of the male cases was not clearly demonstrated in this material, which could suggest a possible bias in how sex is evaluated as a risk factor. As far as the age variable is concerned, there were both positive and negative statements (i. e. disagreement) for four of the six cases, which was in accord with our expectations. Among the information categories concerning laboratory values, cholesterol was evaluated most often by far, with a fairly even distribution of positive  and negative  statements. For at least four of the cases, there were approximately the same numbers of positive and negative evaluations of the same cholesterol value. In other words, according to our definition there is evidence of disagreement among participants in the evaluation of the different cholesterol values.At the level of individual participants, there were eleven instances where doctors made both positive and a negative evaluation(s) of one case. Four of these eleven concerned smoking, two cholesterol, two LDL and one each of hypertension, coronary heart disease and diabetes.A total of 32 statements (i.e. not more than 1.6 per participant) were coded as rules. According to our judgment, 18 of these 32 statements were derived from or were compatible with the guidelines  and twelve of these 18 were a more or less directly referring to secondary prevention or diabetes (e.g "He has angina pectoris and should be below 5 in cholesterol"). Examples of other contents for the statements coded as rules were age limit for cholesterol treatment, importance of looking for secondary hypercholesterolaemia, the role of LDL/HDL ratio, priority of smoking vs. pharmacological treatment, the desired blood pressure value for diabetics blood pressure and the cut-off value for ten-year risk for primary prevention. For two of the patient cases , the guidelines allow a simple decision rule to be applied. Of the 32 instances of reference to a rule, 24 were in connection with these two patient cases.For the four primary prevention cases, IS, TW, SH and PU, a number of statements referring to numerical risk estimate (guidelines say 20% within the next ten years) could have been expected. Only two participants referred to numerical risk estimates.We discuss first how the doctors evaluated the available information in relation to the decision to be made . When each case was analyzed separately, there was some evidence of different patterns of information use shown by prescribers and non-prescribers. The non-prescribers seemed to evaluate central risk factors with a positive directionality less often than prescribers, and they also appeared to identify at least one information category that was given a negative directionality. This is compatible with theories that describe decision-making as search for arguments or reasons for one or the other decision alternative ,17.against treatment were used as arguments for treatment by other doctors. We interpret this finding as showing that prescribing and not prescribing doctors evaluate given information from different perspectives, i.e., from different viewing angles that will put different aspects of the given information in the foreground and background, respectively [Paradoxically, the information categories that some doctors used as arguments ectively . If we tThe use of life style factors as arguments for prescription decisions was further illustrated in a separate analysis based on the same verbal protocols that also include a task where the doctors were asked to describe freely how they usually reason when they meet patients with high cholesterol values . The proDisagreement was also shown for the age variable. Age is generally considered as positively and monotonically related to risk for future cardiovascular events. At the same time, the guidelines make the reservation that the benefit of giving drugs to very old people is unclear. As far as young patients are concerned, the perspective of ten-year risk appears to be too narrow. The recommended procedure is to increase or project the age to 60 years in order to estimate the risk ,5. For dCholesterol was another variable that was ambiguous which could be explained in part by the selection of patient cases. Four of the six cases had cholesterol values in the range of 5.0–6.5 mmol/L, which is often labeled as a mild elevation. This might have formed the basis for negative evaluations, i.e. when a value was close to normal a decision to refrain from drug prescription could have been favored. A few participants also commented that the cholesterol values were lower than they had expected, or lower than those of their own patients.Ambiguity in the decision situation due to seeing the situation in terms of different treatment perspectives (relevant for life style factors) or different ideas about the optimal cut-off points  could possibly be reduced by clearer guidelines, which in turn accentuate the need for more research on a number of issues. These issues include the role of life-style factors for coronary heart disease, as well as how patients should be motivated to change their life style, and cost-benefit outcomes of using drug treatment of patients in different age groups and with different cholesterol values.We will now consider the second set of research questions addressed in the present study, viz., the extent to which the participants used certain rules as a basis for their decisions. Based on the verbal protocols, the frequency of statements classified as a rule was rather low, on average 1.6 per participant, and most of the rules concerned secondary prevention. Part of the explanation for the low number of rules might be that the participants were uncertain about the contents of the guidelines and were therefore less willing to talk about them. However, from our separate coding for knowledge of guidelines content referred to above , the conThe low frequency of statements containing a reference to the risk concept could be explained in the same way, since the participants had no immediate access to an aid for calculating risk and were possibly unsure about the general content of such an aid . Another reason for the low number of rules might be that the instructions did not encourage the participants to explain their decisions, but simply to state aloud their thoughts about the presented information, which is generally considered as the best method to ensure that the verbal protocols reflect the cognitive processes of interest . A thirdFrom the view of evidence-based decisions and quality of care, we can say that many of the cases were difficult and that a considerable spread in the decisions was to be expected. In fact, most of the participants found it difficult to decide about several of the cases, which was evident from interviews after the sessions. On the other hand, the only case (SH) with a mild risk (5–10%) was correctly judged by every participant as not being a candidate for drug treatment. Case AR with angina pectoris represents a decision situation where the guidelines could justify pharmacological treatment in a straightforward manner, and 17 out of 20 chose to prescribe. The presence of diabetes in Case GM could similarly justify drug treatment, but this was the choice for only half the participants. The reason could be that the recommendations concerning diabetes as a risk factor in parity with coronary heart disease is rather new. The Swedish guidelines were published in 1999 and the study was conducted in 2000.One limitation of the present analyses is that most of the conclusions are based on pooled data from groups of participants, while the principal interest is in strategies at the individual level. For example, the opposing evaluations of the same patient data could only be demonstrated between doctors due to the low number of patient cases. After completion of the six cases, the participants were asked to relate in their own words how they usually reason regarding pharmacological treatment when confronted with patients with high cholesterol values. In a forthcoming paper these narratives will be analyzed at the individual level as "scripts" for dealing with cholesterol treatment. We will then have a better understanding of how knowledge and guidelines in this area of medicine are represented in memory, and how these cognitive structures are related to actual decisions and to the individual doctor's think-aloud protocols from processing the cases.In this study we have used a new method to analyse a medical treatment decision. Verbal protocols were coded with respect to how different patient variables seemed to favor or not to favor the decision to prescribe a drug or not. The method promised to be fruitful for understanding why doctors reach different decisions in response to the same patient descriptions and why guidelines are not followed.The author(s) declare that they have no competing interests.All authors participated in the design of the study. LB carried out the data collection. LB and YS performed the coding of the protocols. LB performed the rest of the data analyses and drafted the manuscript. All authors participated in the discussion of the draft. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The objective of the study was to compare the prevalence and severity of musculo-skeletal pain between two socioeconomically contrasting areas in Oslo, Norway, and to explore possible explanatory factors.Questionnaire survey, carried out as part of The Oslo Health Study in 2000–2001. Data from 821 persons (40 and 45 year old) living in a less affluent inner city area  were compared with 854 persons living in an affluent area of the city . Bivariate comparisons (chi square test) and multiple regression analyses were performed to investigate differences between the samples.61 % in east and 56 % in west (p < 0.05) reported pain/stiffness in muscles/joints during the last four weeks. 30 % in east versus 19 % in west (p < 0.001) reported extensive pain. The between area difference in extensive pain was partially explained by physical inactivity, mental health problems and being of non-Western origin.Musculo-skeletal pain is reported by 55–60 % of middle aged persons in Oslo during a four week period, and must be considered a normal phenomenon. Poor social conditions, inactivity, mental health problems and being an immigrant imply increased risk of more severe symptoms with a concomitant demand of health care. In spite of this, social health inequities still exist, and recent analyses from Oslo even indicate an 30 years . Life ex30 years . Well-knSome claim that future research on this topic should concentrate exclusively on interventions . In Grea.The data collection was part of the Oslo Health Study, a joint collaboration between the Oslo City Council, the University of Oslo and the Norwegian Institute of Public Health, which was conducted from May 2000 to September 2001. All residents born in 1924/25, 1940/41, 1955, 1960 and 1970 (n = 41353) received the three-page main questionnaire by mail, as an invitation to participate in a health screening. At the screening station a simple clinical examination and a blood test were performed, and the questionnaire was handed in. Two supplementary questionnaires were given out: one identical for all age groups, and one in four different versions. Participants were asked to fill in the supplementary questionnaires at home and return them by mail. Two reminders were sent to non-respondents. An overview of all topics covered in the questionnaires (in English) can be obtained from In the present study we analysed data from persons born in 1955 and 1960, who lived either in the inner eastern part of Oslo or in the outer western part (see below). We used data from the main questionnaire as well as from the age specific supplementary questionnaire.The variables included from the main questionnaire were: marital status, educational level, employment status, disability pension, social assistance, country of origin, physical exercise, alcohol intake, smoking habits, general health status, mental health problems, and musculo-skeletal disorders. Country of origin was recoded as Western  or non-Western  .Below is a list of various problems: Have you suffered from any of the following during the last week, including today? Put a cross for every problem. Choices: Not troubled, slightly troubled, quite a lot troubled, much troubled . The values were summarised and divided by the number of answers, and a mean value of 1,85 or more was used as a marker of mental health problems [Mental health problems were assessed by the following question: problems .Have you suffered from pain and/or stiffness in muscles and joints in the course of the last four weeks? Choices: Not troubled, somewhat troubled, very troubled  for the alternatives neck/shoulders, arms/hands, upper back, lower back, hips/legs/feet and elsewhere. The values were summarised and divided by the number of answers. A mean value of 2 or more was used as an indicator of extensive pain/stiffness in muscles or joints [Musculo-skeletal pain was explored by the following question: r joints .The variables from the age specific supplementary questionnaire included were: own income, household income, muscular pain/stiffness last 4 weeks, duration of muscular pain/stiffness, satisfaction with health care, and belief in own coping ability.st 2000, west had 67296 inhabitants and east 80 668. . We have chosen to compare these two areas, because they are strongly contrasted regarding living conditions.Oslo's local authority districts can be ranked according to: level of income, education, employment, disability pension, housing standard, number of non-western immigrants, and mortality ,12. AccoStatistical analyses were performed using SPSS version 11.0. Bivariate comparisons of categorical variables were examined by the chi square test. Multiple regression analyses (stepwise) were performed to estimate the explanatory power of independent variables. A 5 % level of significance was chosen.The main questionnaire was completed by 821 forty- and 45 year olds living in east (50.7 % women) and 854 living in west (62.9% women), corresponding to a response rate of 39.0 % in east and 43.9 % in west. Some returned the questionnaire without attending the health screening, meaning that 1348 persons completed the supplementary questionnaires.There was no significant difference regarding full time employment, and frequent use of alcohol was more common in west. For all other socioeconomic and lifestyle variables, as well as general and mental health, east came out poorer Table .The proportion having experienced muscular pain/stiffness during the last four weeks, being very troubled by muscular pain/stiffness in various body parts, or reporting extensive pain/stiffness was higher in east. No difference was found regarding pain duration. Participants in west were more satisfied with health care and more confident in own coping ability Table .Female gender, living in east, low education, low own income, non-Western country of origin, no hard exercise and mental health problems were all correlated to extensive muscular pain/stiffness . Non-western origin was the most important predictor of extensive pain/stiffness in men  and mental health problems in women . When we performed the analyses for respondents of Western and non-Western origin separately (data not shown), mental health problems were the most important independent predictor for extensive pain/stiffness for both groups .In both areas around 60 % reported pain/stiffness in muscles/joints during the previous four weeks: 61.4 % in east and 55.9 % in west, a statistically significant difference of little clinical relevance. We do not know the prevalence among non-respondents, but as The Oslo Health Study implied a comprehensive data collection on many topics, it is unlikely that muscular problems in particular should influence response rate extensively. In a questionnaire survey we carried out in the same areas in 1994  approximately 55 % in both areas reported musculo-skeletal pain during the last four weeks . In anotIt is thus important that the proportion reporting to be very troubled was significantly higher in east regarding all body regions. This might be due to a higher prevalence in east of specific musculo-skeletal diseases, like rheumatoid arthritis, fibromyalgia, etc. The Oslo Health Study asked about fibromyalgia and osteoporosis: In east 49 persons reported fibromyalgia and nine osteoporosis, compared to 19 and five in west. In previous studies we found no difference in prevalence of rheumatoid arthritis  or osteoBlank and Diderichsen found social inequities in both frequency and intensity of a variety of common symptoms in a Swedish population . Their rThe response rate in our material is low . Total response rate in The Oslo Health Study was 46.5 % for 45- year olds, 43.7 % for 40- year olds and 46 % for all age groups. Non-attendance does not occur randomly. Analyses of the impact of self-selection on the Oslo Health Study have shown that the following sub-groups were under-represented among the attendees: unmarried or divorced, males, persons with low education, low income groups, receivers of disability benefit, inner city dwellers and those not born in Norway . But wheWe consider it a strength to use geographical area as a marker of socioeconomic position, and not for example individual education or income. Residential areas are distinct and easy to handle for authorities and politicians, and the majority of health care resources are allocated at area level. That inhabitants in affluent areas are healthier than in less attractive areas, is hardly a surprise, but which are the mechanisms behind the differences? There may be a certain amount of selection: The financial disadvantage of disabled people make it more likely that they live in poorer areas. In our material, far more people of non-western origin lived in east compared to west. As being of non-Western origin showed a strong independent correlation with severe muscular pain, this selection contributed significantly to the between area difference observed. A less healthy physical environment, less healthy lifestyle, and the psychological impact of being poorer than other people, are also possible explanatory factors . Some auAs our study is cross-sectional, causal interpretations cannot be made, we can only describe associations between socioeconomic measures and the health inequities observed. Several studies have shown that education and income can not explain the difference in self-reported health between socioeconomic contrasting areas -22. Our The present study shows that even in Norway today the perception and impact of a health problem  is related to a person's socioeconomic situation. Self-reported health status is known to correlate with mortality, and it is a person's perceived health problems which influence the demand for health care. Significantly more persons living in a non-affluent area of Oslo reported extensive pain, compared to persons in an affluent area. Inactivity, poor mental health, and being a non-Western immigrant implied increase risk of severe symptoms.The authors declare that they have no competing interests.The two authors planned and carried out the data collection together. MB carried out the data analyses and drafted the manuscript. Boyh authors approved the final manuscript.


The level of phylogenetic congruence between the individual genes has been investigated utilizing Bayes factors. We also explore how changes in the substitution models affected the observed incongruence between partitions of our data set.The typical antbirds (Thamnophilidae) form a monophyletic and diverse family of suboscine passerines that inhabit neotropical forests. However, the phylogenetic relationships within this assemblage are poorly understood. Herein, we present a hypothesis of the generic relationships of this group based on Bayesian inference analyses of two nuclear introns and the mitochondrial cytochrome Terenura antwrens, the wing-banded antbird (Myrmornis torquata), the spot-winged antshrike (Pygiptila stellaris) and the russet antshrike (Thamnistes anabatinus) are sisters to all other typical antbirds. The remaining genera fall into two major clades. The first includes antshrikes, antvireos and the Herpsilochmus antwrens, while the second clade consists of most antwren genera, the Myrmeciza antbirds, the "professional" ant-following antbirds, and allied species. Our results also support previously suggested polyphyly of Myrmotherula antwrens and Myrmeciza antbirds. The tests of phylogenetic incongruence, using Bayes factors, clearly suggests that allowing the gene partitions to have separate topology parameters clearly increased the model likelihood. However, changing a component of the nucleotide substitution model had much higher impact on the model likelihood.The phylogenetic analysis supports both novel relationships, as well as traditional groupings. Among the more interesting novel relationship suggested is that the Myrmeciza antbirds and the Myrmotherula antwrens obviously need taxonomic revisions. Although, Bayes factors seem promising for evaluating the relative contribution of components to an evolutionary model, the results suggests that even if strong evidence for a model allowing separate topology parameters is found, this might not mean strong evidence for separate gene phylogenies, as long as vital components of the substitution model are still missing.The phylogenetic results are in broad agreement with traditional classification of the typical antbirds, but some relationships are unexpected based on external morphology. In these cases their true affinities may have been obscured by convergent evolution and morphological adaptations to new habitats or food sources, and genera like  The typical antbirds (Thamnophilidae) is a speciose family within the furnariid radiation (sensu ) of the In traditional classifications, the antpittas  and antthrushes (Formicariidae) were grouped together with typical antbirds in an even larger family. However, the support for the expanded antbird family was indeed weak, and both morphological -6 and moMyrmotherula to Formicivora )However, when independent evidence is lacking and incongruence occurs between individual data partitions, it may be difficult to determine whether particular partitions are better estimates of the species tree than others. Researchers might favor the "total evidence approach" for this particular reason (even though the argument for not combining data partitions with significant levels of incongruence have strong merits). However, the degree of incongruence between individual gene trees could be used to determine whether the phylogenetic conclusions should be based on the combined data set, or only those parts that are similar among the different partitions. A commonly used approach for analysing combined data with maximum likelihood is to assume a single (the same) substitution model for all of the combined genes . A sb and the two nuclear genes (myoglobin and G3PDH), but all combinations of the three genes were examined. However, limitations in the substitution models might be the most important explanation to observed incongruence between data partitions, rather than an intrinsic phylogenetic incongruence  eTamnistes and Pygiptila), antvireos and Herpsilochmus antwrens in clade B  and the "large" antshrikes  are two examples where our results are congruent with traditional classifications. The suggested relationships between the Hypocnemis and Drymophila antbirds, and the Herpsilochmus antwrens and the antvireos (Dysithamnus), respectively, have also been proposed previously based on molecular data )The posterior probabilities of trees and parameters in the substitution models were approximated with MCMC and Metropolis coupling using the program MrBayes . The gen1 compared to another M2, given the data X, and is calculated as the ratio of the model likelihoods, B12 = f(X|M1)/ f(X|M2). The model likelihoods f(X|Mi) are difficult to calculate analytically but can be estimated by using the output from an MCMC  for typical antbirds, and Irestedt et al. [1] for families. Abbreviations: AHMN = American Museum of Natural History, New York; FMNH = Field Museum of Natural History, Chicago; LSUMZ = Louisiana State University, Museum of Natural Science; NRM = Swedish Museum of Natural History; ZMCU = Zoological Museum of the University of Copenhagen. References: (1) Irestedt et al. [1]; (2) Fjeldså et al. [45]; (3) Johansson et al. [65]; Fjeldså et al. [66].Click here for file


In many of the world's poorest countries, dying is often accompanied by avoidable pain and other distressing symptoms. How can we improve care at the end of life? It must be made a public health priority Unfortunately, governments in these countries usually give care at the end of life a low priority compared with preventive and curative services . This prAs three physicians in Jamaica, Uganda, and Rwanda, we believe that providing quality care at the end of life should be seen as a global public health priority. By using relatively low-cost palliative care approaches and community-based strategies, thousands of terminally ill patients in Africa and the Caribbean could be relieved of their pain and suffering.In the countries where we work, the burden of cancer and HIV/AIDS is overwhelming. In Africa about 2.5 million people die annually from HIV/AIDS, and more than 0.5 million die from cancer ,4. SepulIn Rwanda, as in most other African countries, infectious diseases are still rife. Health professionals are often faced with the terrible dilemma of having to choose between saving lives and easing the suffering of the dying. Indeed the authorities usually believe that any investment in palliative care would be at the expense of providing life-saving treatments for those suffering from curable, often infectious illness.In many Caribbean countries, while the scourge of water- and insect-borne infectious diseases is largely under control, the prevalence rates of HIV in the adult population are some of the highest in the world . In JamaPrevention efforts—including health promotion, education, and screening—and treatments aimed at cure or prolonging life are key strategies needed to reduce the burden of HIV/AIDS and cancer in resource-poor countries . HoweverAt present, access to treatment where we are working is essentially controlled by the ability of the patient to pay. Thus, only about one in 200 people with HIV in Uganda are able to obtain antiretroviral medicines . FurtherGiven that prevention isn't taking effect in many places, and curative services are poorly available or inappropriate, we believe that the provision of palliative care  in the CThe WHO has defined palliative care as an approach that improves the quality of life for patients and their families facing the problems associated with life-threatening illness, through the prevention and relief of suffering. This is done through early identification, careful assessment, and treatment of pain and other problems—physical, psychological, and spiritual. Dying is regarded as a normal process, and death is neither hastened nor postponed . The phiEffective and relatively cheap methods exist for controlling pain and other symptoms. For example, the World Health Organization (WHO) has outlined a relatively cheap way of relieving cancer pain in about 90% of patients, which could be extended to patients with HIV/AIDS . Sadly, Several studies in East Africa have looked at the experience of dying, the quality of care at the end of life, and patients' unmet needs ,11,12. RIn the Caribbean, patients' needs at the end of life appear to be similar to those of patients in many East African countries. A qualitative study in Grenada, in the Eastern Caribbean, showed that people preferred to die at home rather than in hospital and—in the absence of pain relief and much-needed counseling, information, and financial support—they took solace in spiritual comfort . In JamaThis fictional case scenario gives an impression of the sorts of problems that patients face at the Hope Institute, Kingston—Jamaica's first public hospice.A 50-year-old woman is diagnosed with inoperable lung cancer. Because of brachial plexus involvement, she experiences severe pain and weakness of her arm. She is treated at Kingston Public Hospital with palliative radiotherapy, which helps the pain for a few months. But then the pain returns, and she requires a high dose of slow-release morphine for pain control.She lives in the mountains, and her house is a two-and-a-half-hour bus ride from Kingston, the capital city. Unfortunately, the public pharmacy in Kingston is unwilling to dispense more than a week's supply of morphine at any one time, because they have limited supplies (there is a shortage of the drug in Jamaica) and because they think the patient's dose is unacceptably high. So she has to make the exhausting five-hour round trip every week.Her husband's health has also recently declined, and the woman's sister now has to care for the patient and her husband. The family now has the financial means to afford only one small meal a day, and they rely on donations from their church community in order to survive.Because the family's savings dwindle, and the public pharmacy faces further shortages of morphine, the woman with cancer requires multiple admissions to the hospice in Kingston over the last six months of her life in order to get suitable analgesia.Uganda has made palliative care for patients with AIDS and cancer a priority in its National Health Plan . In 1993Hospice Uganda provides community-based care principally to patients suffering from HIV/AIDS and cancer. Almost all patients coming to the hospice have pain, and a great deal of attention is focused on good pain management. Uganda is only the third African country to have made morphine available and affordable to its patient population. Because of the dearth of legal prescribers , in May 2004, Uganda changed the statute. This allowed midwives to prescribe pethidine, and allowed clinical palliative care nurses and clinical officers who are specially trained and registered to prescribe morphine.How was Uganda—an African country with a relatively under-funded health service—able to provide a palliative care service? A national program using a public health approach to reach those in need was established following principles outlined in the WHO's National Cancer Control Guidelines . These gFour other African countries—Botswana, Ethiopia, Tanzania, and Zimbabwe—have made the development of home-based care a priority in dealing with the HIV/AIDS epidemic . BotswanBy using strategies such as providing access to an essential short list of relatively cheap generic medications, and other methods recommended by WHO, it has now been proven that palliative care in the African context is affordable and achievable ,7,14.We believe that, following the Ugandan and Botswanan models, palliative care should be integrated into national government strategies. In order to begin to show governments the importance and economic justification for developing a palliative care health policy, it is clear that needs assessments are an essential first step. It is likely to be much less expensive to provide community-based care with family and community support at the end of life than to burden already overcrowded hospital wards with patients suffering end-stage disease. There is a long tradition, both in Africa and in the Caribbean, of caring for the disabled, the mentally ill, and the young and elderly sick at home.Both start-up and sustainable funding are enormous issues that will need to be addressed by local governments, international funding agencies, and charitable bodies. Advocating palliative care to decision makers, providing training programs for health professionals, and making medications available and affordable are important challenges.Research in individual countries is needed to assess whether the above recommendations are suitable locally. Hospice Africa Uganda is advocating to other African governments and assessing other African countries where local laws and customs may dictate the most suitable way to provide palliative care together with government support. Partnerships and a public health approach to palliative care must be the way forward.African Palliative Care Associationapca@hospiceafrica.or.ugRepresenting Kenya, South Africa, Tanzania, Uganda and Zimbabwe E-mail: Hospice Africa (Uganda)info@hospiceafrica.or.ug; E-mail: anne@hospiceafrica.or.ugResource and Training Centre PO Box 7757, Kampala, Uganda Tel: +256 41 266 867 / 510089; Fax: +256 41 510087—residence E-mail: Centre for Palliative LearningHospice Association of the Witwatersrand PO Box 87600, Houghton, Johannesburg 2041, South AfricaHospice Informationhttp://www.hospiceinformation.info. Click on “Training” to search for courses and conferences in palliative care and bereavement. Requires member's password to access this part of the website but membership is free to people in developing countries—contact hospice information at + 44 (0)870 903 3 903 (telephone), + 44 (0)20 8776 9345 (fax), or info@hospiceinformation. Information is also circulated quarterly by E-mail to members under the title of e-Choices.At Palliative Care in Resource-Poor Settingshttp://hab.hrsa.gov/tools/palliative/chap19.html.A freely available overview of HIV/AIDS palliative care, written by Kathleen Foley, Felicity Aulino, and Jan Stjernswärd. At Living Well with HIV/AIDShttp://www.fao.org/DOCREP/005/Y4168E/Y4168E00.HTM.A freely available manual on nutritional care and support for people with HIV/AIDS, by the Food and Agriculture Organization of the United Nations. At Cancer Pain Relief: A Guide to Opioid Availabilityhttp://www.medsch.wisc.edu/painpolicy/publicat/cprguid.htm.A section of this guide, by the WHO, is freely available at


SPG4 (spastin) gene, which encodes an AAA ATPase closely related in sequence to the microtubule-severing protein Katanin. Patients with AD-HSP exhibit degeneration of the distal regions of the longest axons in the spinal cord. Loss-of-function mutations in the Drosophila spastin gene produce larval neuromuscular junction (NMJ) phenotypes. NMJ synaptic boutons in spastin mutants are more numerous and more clustered than in wild-type, and transmitter release is impaired. spastin-null adult flies have severe movement defects. They do not fly or jump, they climb poorly, and they have short lifespans. spastin hypomorphs have weaker behavioral phenotypes. Overexpression of Spastin erases the muscle microtubule network. This gain-of-function phenotype is consistent with the hypothesis that Spastin has microtubule-severing activity, and implies that spastin loss-of-function mutants should have an increased number of microtubules. Surprisingly, however, we observed the opposite phenotype: in spastin-null mutants, there are fewer microtubule bundles within the NMJ, especially in its distal boutons. The Drosophila NMJ is a glutamatergic synapse that resembles excitatory synapses in the mammalian spinal cord, so the reduction of organized presynaptic microtubules that we observe in spastin mutants may be relevant to an understanding of human Spastin's role in maintenance of axon terminals in the spinal cord.The most common form of human autosomal dominant hereditary spastic paraplegia (AD-HSP) is caused by mutations in the  Kai Zinn and colleagues use loss- and gain-of function mutations to study the Drosophila homologue of a gene mutated in human autosomal dominant hereditary spastic paraplegia Overexpression of Spastin in muscles erases their microtubule networks, consistent with the idea that Spastin is a microtubule-severing protein.In this study, we describe the phenotypes arising from mutation of the spastin mutations, and found that they produce recessive phenotypes affecting the larval neuromuscular system. The Drosophila neuromuscular junction (NMJ) uses glutamate as its neurotransmitter and employs ionotropic glutamate receptors homologous to vertebrate AMPA receptors (We made loss-of-function (LOF) eceptors . It is oeceptors .During the period from larval hatching through the third instar stage, the number of boutons at each NMJ increases by up to 10-fold in order to keep pace with the growth of its muscle target. New boutons are added by a process of budding . As thesspastin mutant larval NMJs. Boutons are more numerous than in wild-type larvae, and synaptic transmission is impaired. These changes could result from alterations in synaptic microtubule dynamics, because we find that microtubule bundles are depleted from the distal boutons of NMJs in spastin-null mutants. This is surprising, because the fact that Spastin overexpression destroys microtubule networks might lead one to expect that its removal would increase the number of microtubules. Morphological and microtubule phenotypes are seen only for a total gene deletion, indicating that complete loss of Spastin function is required to alter synaptic microtubules in the fly system. The phenotypes we see are quite different from those described in a recently published study of perturbation of Drosophilaspastin using RNAi methods  and pan-muscle (24B) GAL4 driver lines (EP element and the driver were saved. About 2% of lines (131) exhibited lethality or reduced viability with one of the drivers, and 62 of these were lethal or semilethal with both drivers. The T32 insertion on the third chromosome conferred complete lethality when crossed to either driver, and produced a neuronal-driver-dependent axonal phenotype (see below).We identified  one end . An EP eer” line . We geneer lines . Those lT32 element, we cloned a genomic DNA fragment adjacent to the insertion site and used it to identify a full-length cDNA encoding a 758-aa protein that is a member of the AAA ATPase family  gene that is mutated in the most common form of AD-HSP . The AAA domains of the two proteins are 67% identical. The other region that is conserved between the Spastins (34% identity) corresponds to aa 233–404 of the fly sequence. The same region is also weakly related (26% identity) to human Spartin, the product of the SPG20 gene mutated in Troyer syndrome, a form of “complicated” HSP  in wild-type embryos -GAL4, which is expressed in neuronal precursors and neurons  to demonstrate that the phenotypes we describe for the null mutant are due to loss of Spastin.To evaluate Spastin's functions during development, we generated several deletion mutations from the utations . Because10-12spastin and 17-7spastin have behavioral phenotypes, but they eclose at normal frequencies and are fertile (see below). In contrast, most homozygous 5.75spastin pupae do not eclose. 5.75spastin adults have very severe behavioral phenotypes, and both sexes are sterile. These results suggest that the 10-12 and 17-7 alleles are hypomorphic, and that the 5.75spastin phenotype represents the null condition. RT-PCR analysis of cDNA from 10-12spastin and 17.7spastin animals indicated that low levels of truncated spastin transcripts are still produced (data not shown). These may direct synthesis of proteins initiated from internal ATGs that could retain partial function, since they include the entire conserved AAA domain.Flies homozygous for spastin mutations. However, we saw striking morphological changes in the NMJs of 5.75spastin third instar larvae. In 5.75spastin larvae than in Canton Sw− (WCS) control larvae.  Other NMJs are affected in a similar manner  (10-12spastin and 17-7spastin mutants had bouton numbers that did not differ significantly from controls.The number of Ib boutons per muscle 4 NMJ was increased by 1.6-fold relative to y 23 °C) G, and thy 23 °C) E. This my 23 °C) D. The cly 23 °C) . HypomorUAS-spastin cDNA insertion. This was difficult because of the early lethality produced by expression of Spastin from most drivers. UAS-spastin animals bearing pan-neuronal (Elav-GAL4), motoneuronal (OK6-GAL4), or pan-muscle (24B-GAL4 or G14-GAL4) drivers did not survive to larval stages at 23 °C, and few larvae appeared even at 18 °C. However, third instar larvae in which Spastin expression from the cDNA was conferred by spinster(spin)-GAL4, a weak driver that functions in both neurons and muscles (UAS-spastin to Elav-GeneSwitch (GS)-GAL4, a driver line bearing a neuronally expressed GAL4 derivative that is only active in the presence of the progesterone analog RU486 , to depolarization of the innervating nerves. Average EJP amplitudes in the null were reduced to 78% of the levels in control (WCS) larvae (p < 0.003). We also examined the average amplitude and frequency of responses to single vesicles of spontaneously released neurotransmitter (“mini” EJPs [mEJPs]). mEJP amplitude was increased slightly, to 117% of WCS levels (p < 0.03). There was no significant change in mEJP frequency (p = 0.3).To evaluate whether 5.75spastin mutants, QC was reduced to 67% of WCS levels in these larvae (p < 3 × 10−6). 17-7/spastin5.75spastin larvae had EJP amplitude, mEJP amplitude, and QC values intermediate between those of spastin-null and control larvae. QC in these transheterozygotes was also decreased significantly, to 78% of WCS levels (p < 0.002). The changes in EJP amplitude and QC observed in 5.75spastin mutants were completely rescued by spin-GAL4-driven Spastin expression. Average QC in rescued larvae was not significantly different from wild-type (p > 0.1), and was 30% greater than in 5.75spastin mutants , a measure of the number of vesicles released per evoked event, was calculated by dividing the EJP amplitude by the average mEJP amplitude. Because the evoked EJP was decreased and the mEJP increased in 17-7/spastin5.75spastin larvae were temperature sensitive. While the average QC in transheterozygotes was reduced to 78% of controls at 18 °C, this effect was exacerbated at higher temperatures. At 29 °C, QC was 54% of wild-type  overexpressing Spastin in neurons, and found that synaptic transmission in these larvae was not significantly different from wild-type (data not shown).We also observed that the electrophysiological phenotypes of the hypomorphic ild-type F. Howeve5.75spastin pupae were able to eclose at room temperature compared to 94% of heterozygotes, and the adults that emerged had severe movement defects  flies and flies homozygous for T32 , flew well enough to distribute themselves along the sides of the column. Interestingly, for those hypomorphs that did fly out to the sides, their distribution paralleled that of the controls, suggesting that flight responses in the column were relatively normal in this subpopulation of the mutants .In summary, d longer , indicatDrosophila Spastin affects microtubule networks, we overexpressed it in embryonic muscles using the G14-GAL4 or 24B-GAL4 drivers, and then visualized muscle microtubules in late stage 16 embryos with an anti-β3-tubulin antibody that preferentially stains polymerized tubulin .We quantified Futsch distribution by dividing the patterns of Futsch staining in boutons into three classes: continuous (bundles or splayed bundles), looped, and diffuse or undetectable. In 5 larvae B, there staining D. These spin-GAL4 or the RU486-induced, neural-specific Elav-GS-GAL4 drivers. Rescued larvae had Futsch staining patterns very similar to those seen in WCS . Thus, our results indicate that microtubule bundles are selectively depleted from the distal boutons of NMJs in larvae lacking Spastin protein. boutons B. Loopedspastin gene, which encodes an AAA ATPase, are the most common cause of pure AD-HSP. We identified the Drosophila Spastin ortholog  toward diffuse patterns or the absence of detectable staining. This effect is most pronounced at terminal boutons, and is rescued by neuronal expression of Spastin  are then moved distally into the boutons of the NMJ as it grows.Based on these findings, we suggest that the depletion of microtubules in the distal boutons of Drosophila does not strongly affect outgrowth, since the embryonic CNS axon ladder develops in a normal manner and motor axons reach their appropriate targets in spastin mutants. Furthermore, axonal and muscle microtubules are not detectably altered in spastin-null embryos. Severing of microtubules in vivo, however, may usually involve the actions of multiple severing proteins. In addition to Spastin, the Drosophila genome encodes three AAA ATPases whose AAA domains are closely related to that of vertebrate Katanin-60. These are Katanin-60, CG1193, and an ortholog of mammalian Fidgetins, CG3326 , (2) spastin RNAi larvae have reduced NMJs and an increase in synaptic transmission, and (3) loss of Spastin from neurons produces an increase rather than a decrease in stable microtubules in the NMJ.After this manuscript was submitted for initial review, a paper appeared on perturbation of chniques . In direspastin mutations that delete part or all of the coding region and on rescue of null mutant phenotypes by neuronal expression from a transgene. Our results show that spastin is not an essential gene: even spastin-null flies can eclose and live for several days, and spastin hypomorphs, which would be expected to more closely resemble most RNAi-perturbed flies, eclose at normal rates and have lifespans and behavior that do not greatly differ from wild-type  from the Phylip package, based on alignment to the PFAM AAA consensus.For the overexpression screen, approximately 6,000 new spastin excision lines, alleles 10-12, 17-7, and 5.75 were generated via imprecise excision of EP T32 using SbΔ2-3. All alleles were homozygous viable, and their deletions were mapped by PCR and sequencing of larval or adult genomic DNA. Allele 5.75 causes sterility in both sexes.For the spastin rescue construct, the UAS-spastin cDNA construct was made by subcloning a 2.9-kb BglII fragment from GH11184 into the BglII site of pUAST (spastin cDNA up to 350 bp after the stop codon (excluding 681 bp of the 3′ UTR) and including 28 bp of polylinker sequence from the pOT2 plasmid at the 5′ end. The construct was injected at approximately 300 ng/μl into KiΔ2-3 embryos and several transgenic lines recovered; experiments described here used the Chromosome II insertion line 8-3-5.For the of pUAST . This frspastin-null phenotypes by spin-GAL4-driven expression was assayed by crossing UAS-spastin/CyOKr-GFP;5.75/TM3SerAct-GFPspastin to 5.75/TM3SerAct-GFPspin-GAL4/CyOKr-GFP; spastin. The numbers of Ib boutons in rescued larvae 5.75/spastin5.75). For larval overexpression experiments, the 213-3MHC-GS-GAL4 driver was used to induce muscle-specific expression of UAS-spastin at 23 °C in the absence of RU486. Expression induced by this driver was confirmed in a separate cross using UAS-EGFP.Rescue of scue see G. This wescribed . The numspastin cDNA encoding aa 136–416 (pGEX-T32PvuII), 1–167 (pAcG2T-T32BamRIa), and 380–758 (pAcG2T-T32BBA) were subcloned from GH11184 into bacterial (pGEX) or baculovirus (pAcG2T) expression vectors. Expressed protein (in the form of inclusion bodies for T32PvuII) was injected into guinea pigs  and the antiserum tested on en-GAL4/+2/+ embryos. Antisera against T32PvuII (86EX) and T32BBA (1239) both showed strong staining in the Engrailed pattern; thus, both recognized overexpressed Spastin. 86EX was purified by incubation with membrane-bound pGEX-T32PvuII protein and subsequent elution with 100 mM glycine (pH 2.5) followed by neutralization with 3M Tris (pH 8.8). pAb1239 was affinity purified using the immunogen bound to Affi-gel10 beads , followed by preabsorption with 5.75spastin larval fillets.Regions of the z-series projections with a Zeiss  510 inverted confocal microscope and 63×/1.4 n.a. or 100×/1.2 n.a. PlanApo objectives. Only larval segments A2 and A3 were analyzed. Individual boutons were defined as a Syt-positive area encircled by Dlg-positive staining ; 5.75spastin mutant, 68 ± 2.4 (38); spin rescue, 50 ± 4.9 (26); mutant sibling with spin driver chromosome, 86 ± 4.6 (8); Elav-GS rescue, 70 ± 3.0 (19); and mutant sibling with Elav-GS driver chromosome, 110 ± 9.3 (21). p < 0.02 by one-way ANOVA for all paired comparisons.Stage 16 embryos were fixed and stained using standard methods  with mAbning see , or a Syning see . All typning see  were as 2, 20; NaHCO3, 10; HEPES, 5; Sucrose, 115; Trehalose, 5; and CaCl2, 1 (Sigma). Larvae were visualized with a 5×/0.10 n.a. Olympus  objective on an Olympus BX50WI microscope. EJPs were evoked by pulling the cut end of the innervating segmental nerve into a heat-polished suction electrode and passing a depolarizing pulse sufficient to depolarize both motoneurons (Grass SD9 stimulator). For each experiment, 10–15 single EJPs evoked at 0.2 Hz were recorded, and then spontaneous mEJPs recorded for 1 min afterwards. Only recordings with resting membrane potential below −60 mV were acquired. The average resting membrane potential for control (WCS) larvae was −72.2 mV, and did not differ significantly from any of the experimental groups. Average muscle input resistance in control larvae was 8.9 MΩ, and differed significantly only from the input resistance determined for 5.75spastin /17-7spastin transheterozygotes (7.5 MΩ p < 0.04). Recordings were performed using an Axon Instruments  Axopatch 200B amplifier with CV203BU headstage operating in current clamp mode. The signal was low-pass filtered at 5 kHz, digitized through an Axon Instruments Digidata 1322A 16-bit acquisition system, and recorded using Axon Instruments Clampex 8.2 software. Mean EJP amplitude was determined by averaging all single EJPs with Axon Instruments Clampfit 8.2, and corrected for nonlinear summation according to Intracellular recordings were obtained at 18 °C, using sharp microelectrodes  filled with 3M KCl, from body wall muscle 6 (segments A3 or A4) of filleted third instar larvae, following standard methods . Larvae Eclosion rates were determined by counting numbers of empty versus full (dead) pupae on the sides of bottles in which flies had been allowed to lay for comparable time periods. The flight test assay was performed at room temperature using an opaque cylinder  coated on the inside with fresh mineral oil. Flies of a given genotype were dumped through a hole in the center of a lid at the top. The cylinder was divided into bins along its height, and the number of flies per bin counted. Flies of different genotypes were age-matched; more than 200 flies were counted for each. The climbing assay was performed on 4–5 d old flies maintained individually in vials. Climbing velocity for each fly was measured by transferring it to an empty vial, banging it to the bottom, and then measuring either the time required to reach the top of the vial or the maximum distance it climbed in 30 s, whichever came first. Three trials were performed, and the best speed was used. For lifespan tests, flies were maintained at 25 °C, transferred every 3 d to fresh food vials, and their lifespan noted.Figure S15.75spastin mutant , (B) neuronally rescued ,spastin and (C) neuronally overexpressing  larvae. The clustered, smaller, and more numerous boutons observed in mutant NMJs  are absent in neuronally rescued larvae, which resemble controls  and Syt (magenta) are shown for (A) WCS; see D. Spasti(1.4 MB TIF).Click here for additional data file.Figure S2spin-GAL4 rescue crosses, raised at 18 oC. These genotypes were (1) spin-GAL4/UAS-spastin;5.75spastin (spin Rescue), (2) spin-GAL4/CyOKr-GFP;5.75spastin , (3) spin-GAL4/UAS-spastin;5.75spastin/TM3SerAct-GFP , and spin-GAL4/5.75/TM3SerAct-GFPCyOKr-GFP; spastin .Behavioral tests were performed on flies from the four genotypes arising from the spastin mutants (0%) from these crosses  reached the top of the vial in the prescribed 30 s time limit, compared to 8% for Rescue flies (n = 75), and 100% for both spastin/+ controls (n = 39 and 21). Twenty-seven percent of mutants (5.75[R]) did not climb at all, compared to only 4% of the Rescue flies and 0% of the spastin/+ controls. Thus, although both genotypes in the mutant background (homozygous for 5.75spastin) were much weaker than either 5.75spastin heterozygous control, Rescue flies showed improved climbing ability compared to the mutants.(A) Climbing behavior. None of the spastin mutants  was significantly rescued by spin-driven expression of spastin , although lifespans were much shorter in 5.75spastin homozygotes than in heterozygous spastin/+ controls .(B) Similar to the results in (A), mean lifespan in (218 KB PDF).Click here for additional data file.Video S1Flies are shown moving in a vial.WCS fly are shown. Note the rapid rate at which they walk, as well as exhibiting climbing, jumping and flying behaviors. When still, their legs are controlled, and they are able to walk upside-down (out-of-focus fly near end of segment) for prolonged periods without falling.Segment 1: Wild-type. One female and one male 5.75spastin female is shown. Leg weakness is obvious, particularly for the mesothoracic and metathoracic legs, both when walking and standing still. She climbs poorly, and when rotated so that she is upside-down, is unable to maintain a hanging position. No wing movement or jumping is observed.Segment 2: Mutant. One spin-GAL4/UAS-spastin;5.75spastin flies, Spastin expression via the spin-GAL4 driver partially rescues the movement defects seen in 5.75spastin mutants. Two males are shown, followed by one female. Note their improved leg steadiness, velocity, and hanging ability. These flies can also jump spontaneously. The female appears to be less fully rescued; however, she is able to walk upside-down for prolonged periods, and exhibits wing movement.Segment 3: Rescue. In (7.4 MB MOV).Click here for additional data file.


Algorithms and information, fundamental to technological and biological organization, are also an essential aspect of many elementary physical phenomena, such as molecular self-assembly. Here we report the molecular realization, using two-dimensional self-assembly of DNA tiles, of a cellular automaton whose update rule computes the binary function XOR and thus fabricates a fractal pattern—a Sierpinski triangle—as it grows. To achieve this, abstract tiles were translated into DNA tiles based on double-crossover motifs. Serving as input for the computation, long single-stranded DNA molecules were used to nucleate growth of tiles into algorithmic crystals. For both of two independent molecular realizations, atomic force microscopy revealed recognizable Sierpinski triangles containing 100–200 correct tiles. Error rates during assembly appear to range from 1% to 10%. Although imperfect, the growth of Sierpinski triangles demonstrates all the necessary mechanisms for the molecular implementation of arbitrary cellular automata. This shows that engineered DNA self-assembly can be treated as a Turing-universal biomolecular system, capable of implementing any desired algorithm for computation or construction tasks. Engineered DNA self-assembly to produce a fractal pattern demonstrates all the necessary mechanisms for the molecular implementation of arbitrary cellular automata This organization is information-based: DNA sequences refined by evolution encode both the components and the processes that guide their development into an organism—the developmental program. For a language to describe this carefully orchestrated organization, it is tempting to turn to computer science, where the concepts of programming languages, data structures, and algorithms are used to specify complex organization of information and behavior. Indeed, the importance of universal computation for autonomous fabrication tasks was recognized in von Neumann's seminal work on self-reproducing automata, where he postulated a y object . If algoany algorithm can in principle be embedded in, and guide, a potentially aperiodic crystallization process. In this “algorithmic self-assembly” paradigm, a set of molecular Wang tiles is viewed as the program for a particular computation or molecular fabrication task  function: at each time step, each cell is computed as the XOR of its two neighbors. Beginning with a row of all ‘0's punctuated by a single central ‘1,' snapshots of the cellular automaton's state at successive time steps may be stacked one on top of the other to produce a space–time history identical to Pascal's triangle  modulo 2Whereas execution of a cellular automaton occurs perfectly and synchronously, molecular self-assembly is asynchronous and may have many types of errors. To be successful, an implementation of cellular automata by molecular tiling must address four challenges: (1) The abstract tiles must be translated into molecules (molecular tiles) that readily form 2D crystals. (2) Molecular tiles must be programmed with specific binding domains that match the logic of the chosen abstract tiles. (3) The binding of molecular tiles must be sufficiently cooperative to enforce the correct order of assembly and prevent errors. (4) Assembly of molecular tiles must occur on a specified nucleating structure, and spurious nucleation must be suppressed. These properties are necessary and sufficient for implementing not only the XOR cellular automaton, but also any other 1D cellular automaton. All four have been shown individually: several types of DNA Wang tiles have been designed and shown to grow into micron-scale 2D periodic crystals ; the intPreventing the types of errors mentioned above may seem impossible. For example, if a single binding domain is strong enough to hold a tile in place  may associate at a given site at a rate,  to zero . For randization , this modization  predictsdization B.The effects of non-idealities can also be explored in this model. For example, mcG ≈ 2seG, which corresponds to the melting temperature of the crystals, such untemplated nucleation is inhibited by a kinetic barrier—the existence of a critical nucleus size  in principle can be controlled by slowing down the growth processes, making experimental investigations the appropriate next step.Abstract Wang tiles are implemented as DNA tiles according to the scheme described earlier : each moSince untemplated crystals were not expected to produce recognizable Sierpinski triangles, it was necessary to create a proper nucleating structure to provide the initial input for the algorithmic self-assembly. Previous work using DNA tiles to self-assemble an initial boundary had proven to be difficult , so in tThe DAO-E Sierpinski tile set B consistIn order for the nucleating structure for the DAO-E lattice to assemble onto a long PCR-generated nucleating strand, the tiles on the input row must be of the DAE-O variant. Further, we simplified the construction so that all nucleating strands contain the same repetitive sequence, but the input tile strands are doped with a fraction of strands containing a ‘1' sticky-end, and again the nucleating structure contains a few randomly located sites from which a Sierpinski triangle should grow.In principle, two approaches can be taken for initiating algorithmic self-assembly of DNA tiles. In the preformed tile approach, each tile is prepared separately by mixing a stoichiometric amount of each component strand in the hybridization buffer and then annealing from 90 °C to room temperature over the course of several hours. The nucleating structure is similarly prepared by annealing the nucleating strand with input tile and capping strands. Then the rule tiles and nucleating structure are mixed together at a temperature appropriate for crystal growth. In the bulk annealing approach, the nucleating strand, the capping and input tile strands, and the strands for all rule tiles are initially mixed together and then annealed. Since, at the concentrations we use, the tiles themselves have melting temperatures between roughly 60 °C and 70 °C while the crystals have a melting temperature within a few degrees of 40 °C , during Results for the DAE-E tile set are shown in Shown in S of possible states for the memory cells and an update function f : S × S → S, one can create a set of |S|2tiles according to the scheme of S|) to about 20 for the DAO-E and DAE-E tile designs used here, but this is already sufficient to implement several known universal Turing machines and cellular automata  A rigid or quickly straightening nucleating structure was simulated by setting iS = 4, so that near the crystal melting temperature where mcG ≈ 2seG, the border growth is strongly favorable. This was used for iS = 0.25 for the border tiles; in this case, near the melting temperature border growth requires stabilization by growth of rule tiles, resulting in faceted crystals. In combination with doubled concentrations of T-00 and T-11 (iS = 2), this case was used for The xgrow program simulates the kTAM for a set of square Wang tiles see , beginniThe strong effect of these variations may be seen in not seen for the T-11 tile, despite its increased concentration. This is because, regardless of what information is presented on the facet, there is no way to create a layer containing more than 50% T-11 tiles and no mismatches; T-01 or T-10 tiles must intervene. Thus the nucleation rate is substantially reduced, relative to T-00 nucleation on all-zero facets. This can be assessed in simulations by measuring the probability, p(L), that a T-00 tile will be found after L layers of growth from a facet. Simulations with parameters similar to p(L) ≈ 0.66−L/27e + 0.34 for all-zero facets, indicating strongly preferential nucleation, but for all other facets p(L) relaxes quickly to the asymptotic distribution. Simulations with parameters similar to p(L) relaxes to the asymptotic distribution immediately for every facet type investigated.Simulations confirm the preferential nucleation of T-00 tiles on all-zero facets when T-00 and T-11 concentrations are doubled . In contDesign of DNA Wang tiles occurs in three steps. First, the tile and lattice geometry must be determined. Here, the sizes (number of base-pairs) of each double-helical domain and sticky end, and other structural adornments such as contrast hairpins, are decided. These decisions impact the stability of each tile molecule, as the natural geometry of the DNA double-helix (10.5 bp for a full turn of B-form DNA)  constraiAt the second level, specific sequences must be chosen. The issue here is that we wish to prevent undesired associations between strands that might inhibit formation of the correct molecular structure. We used the heuristic principle of sequence symmetry minimization , 1990 toThe third level of design concerns variations. We conceptualize DNA Wang tiles as consisting of three modules: the sticky ends, the core helical regions, and adornments such as the hairpin structures that provide contrast for AFM imaging. A given double-crossover core can be given different sticky ends (reprogrammed) by replacing just one or two strands, thus allowing reuse of core designs to implement different tile sets. In our experience, the structural and thermodynamic stability of a given core is not significantly affected by changes in the sticky-end sequences. Similarly, using additional strands, a given core can be used with or without the hairpin adornments, which can be inserted at various locations. Although the hairpin adornments can affect the integrity of a DNA tile , we have seldom found the undesired products to exceed 20% of the material.The core sequences for R-00 and S-00 are identical to the A and B tiles from a previous study . We usua2O  based on extinction coefficients estimated using a nearest-neighbor model , PAGE purified, and quantitated by UV absorption at 260 nm in Hor model . DNA tilThe single-stranded nucleating strands were synthesized using a procedure based on Stemmer's assembly PCR . In asse+)*. The fraction of NRE subsequences is controlled by the amount of SplintNREUE2 and SplintNUERE2, which mediate the transitions into and out of the NRE sequence.  The NRE input tile outputs ‘0' and ‘1,' while the NUE input tile outputs ‘0' and ‘0'. To generate a different language, or a different distribution of sequences in the same language, a new assembly PCR must be run. Assembly reactions for DAO-E and DAE-E nucleating strands were designed using slightly different principles. The improved design used for DAO-E nucleating strands is simpler: A single periodic sequence is generated. The fraction of ‘1' sites is determined by the stoichiometry of input tile strands used in subsequent self-assembly reactions—strands A4SV and A4-S00 both assemble in the input tile in the same place, but one carries a ‘1' sticky end while the other carries a ‘0' sticky end. The approach used for DAE-E nucleating strands is more complex but more powerful for generating non-trivial input patterns. By having multiple splints that can overlap a given sequence, the assembly can be directed to non-deterministically choose one of several ways to extend a sequence. Thus, assembly PCR can be used to generate any regular language . In thisN types) is prepared without polymerase . To avoid mispriming events, the splints are annealed in the reaction mixture at 37 °C for 5 min. The polymerase (0.4 μl) is added and the reaction is subjected to an initial 72 °C extension step, followed by 40 cycles . In stage 2, 40 μl of new PCR Mix B , is added to the first reaction volume and the reaction cycled for an additional 25 cycles . In stage 3, the 60 μl reaction volume is split into three 20 μl volumes, an additional 40 μl of Mix B is added to each and an additional 20 cycles  are performed. At this point, long double-stranded product should be formed. (We have observed that such products remain in the well of an agarose gel long after a 20 kb marker has entered the gel.) Also the dNTPs in the mixture are presumably nearly exhausted—specifically there is little dGTP left. (Any remaining dGTP will be used up early in stage 4.) In stage 4, to create single-stranded nucleating strands, 5 μl of the stage 3 product are mixed with 55 μl of fresh PCR mixture  for additional 60 cycles of the stage 3 program . While addition of asymmetric primers at this stage might yield more single-stranded product, a satisfactory yield of single-stranded product results without doing so. After the final PCR, the reaction mixture is extracted with phenol:chloroform:isoamyl alcohol , ethanol precipitated, and resuspended in purified water; the yield was estimated by UV absorbance. Typically, three 60 μl tubes of stage 4 product were pooled in a single recovery step and DNA was resuspended in 200 μl of water. Absorbance measurements of freshly resuspended material appeared unstable, perhaps because clumps of nucleating material scatter light. Long single-stranded DNAs may be prone to hydrolysis in water or strand-breakage upon freeze–thaw. However, after storage in water at 4 °C for a year, the nucleating structure still works well  MX 4000 real-time PCR instrument using a Perkin-Elmer  GeneAMP XL kit that uses rTth polymerase. In stage 1, a 20 μl reaction mixture containing 1 pmol total of splints , and began with preannealed samples at 15 °C, increasing to 80 °C over the course of several hours. Single-tile melts were superimposable with the reanneal from 80 °C back to 15 °C, indicating that equilibrium values were measured. Raw absorbance values were normalized. Whereas S-00 has a sharp melting transition  near 65 °C, the R-00-23J tile has a somewhat more gradual transition, which we attribute to the presence of the hairpin. Above 40 °C, the absorbance of the mixture equals the average absorbance of the individual tiles, indicating that crystals have completely melted by that point. Prior to the crystal melting transition between 36 °C and 40 °C, there is significant noise in the measurement, presumably due to light scattering.Melting temperatures for tiles and crystals were estimated based on UVth tiles . These t260 melts of all tiles; however, several other DAO-E and DAE-E tiles have similar transitions between 50 °C and 70 °C. Therefore we assume that the templated and untemplated Sierpinski crystals also melt at approximately 40 °C and that at that temperature, the DNA tiles are reasonably well formed.We have not performed UV2+ buffer , annealing from 90 °C to 20 °C at a rate of 1 °C/min (taking about 1 h). Longer annealing schedules  did not seem to decrease the error rate or the number of untemplated tubes or crystals.Self-assembly was performed by bulk annealing of all relevant rule tile, input tile, capping, and nucleating strands in a  50 μl volume of 1× TAE/MgDAO-E reactions contained nucleating strands sufficient to bind 0.004 μM of input tile (as estimated from binding capacity gels), 0.2 μM of each capping and input tile strand , and 0.2 μM of each rule tile strand (for each of the five or six tiles used). An excess of input tile strands was used to ensure complete coverage of the nucleating strand. The excess partial input tiles appeared not to significantly interfere with the self-assembly of algorithmic crystals.DAE-E reactions contained nucleating strands sufficient to bind 0.002–0.008 μM of input tile (as inferred from the estimated yield of the PCR), 0.2 μM of each capping and input tile strand , and 0.2 μM of each tile strand (for each of the four tiles used). Again, an excess of input tile strands was used to ensure complete coverage of the nucleating strand.2+ buffer on a Digital Instruments Nanoscope III  equipped with a nano-Analytics Q-control III  and a vertical engage J-scanner, using the roughly 9.4 kHz resonance of the narrow 100 μM, 0.38 N/m force constant cantilever of an NP-S oxide-sharpened silicon nitride tip (Veeco Metrology). After self-assembly is complete, samples were prepared for AFM imaging by deposition of 5 μl onto a freshly cleaved mica surface (Ted Pella) attached by hot melt glue to a 15 mm metal puck; an additional 30 μl of buffer was added to both sample and cantilever (mounted in the standard tapping mode fluid cell) before the sample and fluid cell were positioned in the AFM head. The tapping amplitude setpoint, after engage, was typically 0.2–0.4 V, the drive amplitude was typically 100–150 mV, scan rates ranged from 2 to 5 Hz. Individual tiles are most clearly resolved for low amplitude setpoint and high drive amplitude values. However, under such conditions, the greatest damage is done to the sample and the hairpin labels are less distinct, sometimes disappearing entirely. Thus, to prevent damage to samples, amplitude setpoint was maximized and/or drive amplitude minimized subject to the constraint that tiles and their hairpin labels be visible.AFM imaging was performed in tapping mode under TAE/MgAfter acquisition, most images were flattened by subtracting a low-order polynomial from each scan line, or by adjusting each scan line to match intensity histograms. For some images see , bottom,Figure S1(57 KB PDF).Click here for additional data file.Figure S2(160 KB PDF).Click here for additional data file.Figure S3(126 KB PDF).Click here for additional data file.Figure S4(16 KB PDF).Click here for additional data file.Figure S5(21 KB PDF).Click here for additional data file.Figure S6(16 KB PDF).Click here for additional data file.Figure S7(22 KB PDF).Click here for additional data file.Figure S8(244 KB PDF).Click here for additional data file.Figure S9(22 KB PDF).Click here for additional data file.Figure S10(25 KB PDF).Click here for additional data file.Figure S11(53 KB PDF).Click here for additional data file.Figure S12(33 KB PDF).Click here for additional data file.Figure S13(234 KB PDF).Click here for additional data file.Figure S14(203 KB PDF).Click here for additional data file.Figure S15(226 KB PDF).Click here for additional data file.Figure S16(256 KB PDF).Click here for additional data file.Figure S17(234 KB PDF).Click here for additional data file.Figure S18(146 KB PDF).Click here for additional data file.Figure S19. Compiled Figures S1–S18(1.7 MB PDF).Click here for additional data file.Video S1Each frame is an average of three raw images. At the center is an amalgamation of many individual algorithmic crystals, each with its own characteristic pattern of tiles . While no large undamaged Sierpinski triangles were seen in this series of images, in some frames it is possible to see both double-helices within the tiles, as well as the major and minor grooves within the helices.(17.8 MB MPG).Click here for additional data file.