Three prenylation motif predictors are presented that allow discrimination between proteins that are unique substrates of farnesyltransferase (FT) and those that can be alternatively processed by geranylgeranyltransferase I (GGT1). ) which also allows evaluation of evolutionary motif conservation. PrePS can predict partially overlapping substrate specificities, which is of medical importance in the case of understanding cellular action of FT inhibitors as anticancer and anti-parasite agents.We refined the motifs for carboxy-terminal protein prenylation by analysis of known substrates for farnesyltransferase (FT), geranylgeranyltransferase I (GGT1) and geranylgeranyltransferase II (GGT2). In addition to the CaaX box for the first two enzymes, we identify a preceding linker region that appears constrained in physicochemical properties, requiring small or flexible, preferably hydrophilic, amino acids. Predictors were constructed on the basis of sequence and physical property profiles, including interpositional correlations, and are available as the Prenylation Prediction Suite (PrePS,  Prenylation refers to the posttranslational modification of proteins with isoprenyl anchors -3. TheseThe CaaX box was initially understood to consist of a cysteine (C), followed by two aliphatic residues (aa) and a terminal residue (X) that would direct modification by either FT or GGT1, but newly found substrates and kinetic studies of mutated substrate peptides and enzyme inhibitors have shown that the motif recognized by the enzymes appears to be more flexible . FurtherAn unsolved problem so far is accounting for the complexity of the prenylation substrate recognition motifs in theoretical models in order to identify substrate proteins from their amino-acid sequence. No available method has been able to selectively assign the correct modifying enzyme, which determines the types and number of lipid anchors. The high probability of motifs similar to the small CaaX box occurring by chance is a general problem that has so far prohibited large-scale proteome analyses . We descThe first task consists of collecting sequences that are known substrates for the respective enzymes. Typically, a good starting point is the Swiss-Prot database . HoweverUnfortunately, such justified concerns dramatically lower the amount of data in the learning set. However, because of earlier interest in developing peptide-based inhibitors of FT and GGT1 as anticancer treatments, the kinetics of the enzymes with various tetrapeptide substrates already modified with lipid anchors by the enzymes have been measured . Hence, However, possession of a valid CaaX box might not be a sufficient selection criterion. Typically, short terminal sequence motifs are connected to the rest of the protein by a linker region that experiences only limited constraints on specific amino acids per position but often has a compositional bias towards small or hydrophilic amino acids in connecting sequence stretches . This prTaking account of the considerations above, the following procedure has been applied to obtain conservative and reliable learning sets of FT and GGT1 substrates. First, a literature search for known prenylated proteins and valid tetrapeptides (see ). SecondThis resulted in learning sets of 692 FT and 486 GGT1 substrates, respectively (see ). Among Compositional analysis of residue frequencies at single motif positions reveals that major restrictions to specific amino acid types exist only for positions within the CaaX box . Whereas a broad variety of residues are allowed in motifs recognized by FT , mainly leucine and methionine appear to be preferred by GGT1 in agreement with experimental evidence . InteresUsing the Fisher criterion , interpositional correlations of residue sizes within positions +1, +2 and +3  from both FT and GGT1 substrates have been identified. Often, when a very large residue occurs at specific positions, neighboring residues compensate to obey the overall physicochemical constraints  in the binding pocket. Similarly, compensatory effects appear to exist regarding hydrophobicity between positions +1 and +3 in FT and between +1, +2 and +3 in GGT1 substrates , -13 (PDB 1MH1), -15 (PDB 1AM4), -12 (PDB 1A4R)). In the structure of a G protein gamma subunit, the linker region also appears to be extended and wrapped around the beta subunit in the heterotrimeric G protein signaling complex (PDB 1GG2). It needs to be emphasized that the linker region must not necessarily be in an unstructured conformation after the anchor has been attached , as folrotein 1 , myristorotein 1  motifs. S consists of a term Sprofile scoring a query sequence against the redundancy-corrected profile of the learning-set sequences and another term Sppt that penalizes deviation from the physicochemical motif requirements.Following the approach already applied to the prediction of GPI and myristoyl anchors and PTS1-mediated targeting -10, a scS = Sprofile + SpptSprofile distinguishes the three positions +1, +2 and +3 of the CaaX box as well as the linker region (-1 to -11). Sppt comprises a sum of terms that are constructed from the physical property requirements for FT and GGT1 substrates that were outlined in the section describing the motif refinement (and listed in Tables Sppt).The term The threshold for a query protein to be a predicted farnesylation or geranylgeranylation target by FT or GGT1, respectively, is set to include all sequences in the learning set. Hence, the self-consistencies or upper bounds of sensitivities of the FT and GGT1 predictors are 100%. Additionally, the robustness of the method has been cross-validated in jackknife tests . In the cross-validation over the complete scoring function, the rates of finding known substrates after excluding them and their close homologs from the learning procedure  were 92.6% for FT and 98.6% for GGT1, respectively.As required for a good predictor , the scoin vitro studies have shown that motifs like CVIL, CVLL, CAIL and CCIL (single-letter amino-acid code) are valid for FT as well [in vivo [in vivo [Previously, the assignment of CaaX box substrate proteins to either FT or GGT1 has been based mainly on the identity of the final residue in the motif (position +3) where FT allows several amino-acid types and GGT1 clearly prefers leucine ,27. This as well . Mutatio[in vivo . Also, R[in vivo . Similar[in vivo .in vivo when farnesyltransferase is inhibited [In the same way that FT can accept CaaX box motifs ending in leucine and phenylalanine, GGT1 appears to tolerate methionine at this position, which was previously thought to direct farnesylation. This has important consequences in the case of the oncoprotein K-Ras (in variants with CVIM and CIIM motifs) which becomes geranylgeranylated nhibited .in vivo activity of GGT1 compared to FT [As we have experienced with our earlier predictors for myristoylation and PTS1 targeting, we find even some correlations of the prediction scores with experimentally measured substrate-enzyme affinities. Interestingly, the scores of the GGT1 predictor give better agreement with the experimental data when divided by 3, in agreement with a threefold lower ed to FT . To estied to FT  and we tUnlike FT and GGT1, substrate recognition by GGT2 is less dependent on strictly defined carboxy-terminal motifs, but on the complex formation of the substrate with an escort protein . As illuRab proteins are small GTPases (around 60 different have been identified in humans)  that shaTwo alignments (F3F4 region and full length) were therefore constructed and after removal of entries with a maximal redundancy of 90% identity over the whole sequence length (117 of 179 entries annotated in Swiss-Prot remaining), hidden Markov models (HMMs) were created and calibrated. The choice of this methodology for the GGT2 prediction was strongly influenced by the fact that the HMMer  algorithThe three tools to predict lipid modification by FT, GGT1 and GGT2 are available as Prenylation Prediction Suite (PrePS), which is accessible online . Users cUntil now, the only available tool to predict protein prenylation has been the Prosite  search wThe inadequacy of the Beese, Casey and colleagues  motif inFarnesyltransferase inhibitors (FTIs) have been developed to prevent prenylation of oncogenic Ras proteins and are currently undergoing phase II and III clinical trials . While FNP_004528)) has a CKQQ farnesylation motif that is further retained in mouse, rat, frog, fish, fungi and plants, as predicted by PrePS. This taxonomically widespread evolutionary conservation would rather indicate a relevance of the lipid anchor for the function of this protein, which is part of a family involved in transcriptional activation and chromatin formation, including histone binding [We would like to mention here one example prediction of PrePS for a protein that would be a candidate for a previously unknown FTI target. The human nucleosome assembly protein I-like protein  .P07355) terminating in a CGGDD motif, which is not at all predicted by PrePS as it is mechanistically unlikely to be processed by farnesyltransferase. Another rather surprising prediction resulting from the tagging experiment is the farnesylation of Rab21 (Q9UL25), which has a double cysteine motif followed by three additional residues (CCSSG) which, at least formally, resembles a CaaX box. Rab proteins with CaaX boxes such as Rab5 (CCSN), Rab8 (CVLL/CSLL), Rab11 (CQNI) and Rab13 (CSLG) are usually modified by GGT2 in vivo [in vitro [Xenopus (GenBank AAH60498.1) and Drosophila (AAH60498.1) share the double cysteines but their motif is different and shorter by one residue, pointing to a higher importance of the conservation of the cysteine doublet than the rest of the motif. The evOluation, furthermore, shows that Rab5 is the most closely related prenylated Rab-family member. Interestingly, both cysteines in the CCSN CaaX box motif of Rab5 were shown not only to be geranylgeranylated by GGT2 in vivo but are also required for proper localization and function of the GTPase [While this paper was in preparation, farnesylation of the NAP1-like protein has been suggested experimentally through a special tagging and purification technique , giving  in vivo ,53,54 buin vitro ,55. PrePA complete analysis of large-scale predictions of prenylated proteins ranked by functional importance as estimated by evolutionary motif conservation and medical implications will be published in a follow-up work.We identified physicochemical requirements for each motif position by correlating 20-dimensional vectors filled with the positional frequencies of occurrence of the 20 amino-acid types in the carboxy-terminally aligned learning set with a library of over 650 amino-acid physical properties . This haF of the sum of variances of single positions with the variance over multiple positions for pairs and triplets of positions is calculated, allowing gaps of up to two residues between pairs.The Fisher ratio F values for probabilities p = 0.05 are taken as thresholds for evaluating significance of interdependence of physical properties between motif positions. These are 1.288 for the FT and 1.355 for the GGT1 nonredundant learning set, respectively.The To identify regions in the FT and GGT1 binding pockets that are characteristic for the respective enzyme, we analyzed the pattern of residue conservation in vicinity to the CaaX substrate peptide and the prenylpyrophosphate Figures  and 5. AConstruction and calculation of the scoring function essentially follows the methodologies ,16,60 apThe composite score function consists of a profile and physical property term.S = Sprofile + Sppt     (2)f of an amino acid type a at a given alignment position i is down-weighted proportionally to the number of other positions that are identical in sequences sharing a at i, resulting in subscores SPSIC representing the natural logarithms of these redundancy-corrected frequencies. These are summed over the respective regions The profile matrix is calculated using the PSIC algorithm to account for redundancy in the learning set. The frequency of occurrence Sprofile:before they enter αprofile is a normalization factor to compensate for differing lengths of the regions and is derived from:Cregion is a relative weighting of motif regions. We propose a rational basis for its approximated computation. Cregion is derived from the average Shannon entropies of the regions in the alignment of the nonredundant learning set sequences. Shannon's information theoretic entropy has already previously been investigated as measurement of sequence variability in alignment positions and residue conservation [ervation . We calcna,i is the number of occurrences of amino-acid type a at the investigated position i of the region and N the total number of sequences in the alignment. Only amino-acid types that occur at least once  are included in the sum. In order to avoid overly precise values, the final values are rounded up to the last decimal that showed variation in the conservation measure when comparing the small learning set based on Swiss-Prot annotation with the larger learning set described in this work. The position of the absolutely conserved cysteine in the CaaX box would have the maximum conservation value of 1. Table Cregion for the regions +1, +2, +3 and -11 to -1.where Sppt) and the biophysical meaning discussed earlier.The primary role of the physical property term is to exclude query sequences that do not fit into the determined physical property profile of the motif  can be defined as follows:As an example, a penalty for a physical property Sppt as a sum with a term-specific weighting factor αj that emphasizes varying importance of the single terms.The natural logarithms (to be comparable to the profile score) of these single terms enter Sppt, the FT and GGT1 score functions also include a penalty for query proteins when the carboxy terminus appears inaccessible as a result of structural constraints. For example, helices with strongly hydrophobic sides are either buried in the structure or fold back to the protein core and reduce the flexibility and accessibility of the carboxy terminus. We recognize these if hydrophobic residues (LIVMFYW) appear in patterns like i,i+3,i+6 (hXXhXXh) or i,i+3,i+7 (hXXhXXXh) or i,i+4,i+7 (hXXXhXXh) within 20 residues preceding the cysteine of the CaaX box.As part of Sprofile + Sppt recalculated after removal of the entry to be validated from the learning set) resulted in sensitivities of 99.4% for FT and 99.8% for GGT1, respectively. This generally indicates that the learning sets are large enough for the method not to suffer from removal of individual entries and, hence it should also be able to find valid motifs not yet included in the learning set. At the same time, we acknowledge that the similarity of sequences within homologous groups in the learning set influence the above estimated sensitivity. However, we emphasize that the values calculated for the profile matrix vary even when only one sequence from a group of homologs is left out, since the method used for profile extraction [We have performed three jackknife tests to validate the robustness of our predictors. First, cross-validation over the whole scoring function . We find that in the case of the predictor for GGT1, the sensitivity of 98.6% is very close to the first jackknife test. For the FT predictor, a sensitivity of 92.6% is obtained in this test, which is only slightly lower than in the other jackknife procedures. The decrease can be almost exclusively attributed to the large group of highly similar sequences of hepatitis delta antigen that all share the uncommon motif CRPQ with both arginine at position +1 and proline at +2 being rather exceptional amino acids. Hence, the CRPQ sequences remain below the prediction threshold in the jackknife test if no example of this motif is present in the learning set. When leaving the CRPQ group out of the cross-validation, the FT predictor is calculated to have a sensitivity of 97.9%.Sppt are overfitting the learning data. To exclude bias through similar sequences in homologous groups, the test was executed only over the learning set after removing redundancy . Sppt alone is recalculated after removal of the entry to be validated from the parameter calculation procedure. The obtained sensitivities of 100% for FT and 99.8% for GGT1 indicate that the parameterizations of physical property terms in Sppt are not overfitting the learning data.A third cross-validation test aims to elucidate whether the 39 parameters  introduced through the physical property terms in S greater than a threshold score Sth can be formulated as follows:To estimate probabilities of false-positive prediction, a set of sequences had to be defined whose carboxy-terminal amino-acid pattern should not be subject to selection for a valid CaaX prenylation motif. This is fulfilled for sequences in the NCBI nonredundant database lacking a cysteine at the fourth position from the carboxy terminus. Hence, any compliance with motif restrictions of such sequences apart from the fourth last position could only be attributed to random or at least prenylation-independent appearance. Following earlier experience with GPI- and myristoyl-anchor prediction ,9, a polA polynom of the sixth degree was used to improve the residual fit. Polynoms with degrees higher than six would not result in an increase of the relative improvement of the residual. The probabilities of false-positive prediction for scores at the prediction threshold are extrapolated to 6.3% for FT and 1.2% for GGT1 for sequences that contain a cysteine at the fourth-last position . Given that appearance of a cysteine at this position is rare in databases (1.7%), the independent general probabilities of false-positive prediction by FT and GGT1 for all protein sequences are as low as 0.11% and 0.02%, respectively. This corresponds to specificities of 99.89% and 99.98%.in vivo prenylation when a corresponding motif has been predicted, we also tried to estimate probabilities of false-positive prediction for differently localized subsets of proteins. These subsets were retrieved from the Swiss-Prot database [Since the subcellular context of a protein can be relevant to judging the likelihood of database  and assi


Walking speed, which is often reduced after stroke, can be influenced by the perception of optic flow (OF) speed. The present study aims to: 1) compare the modulation of walking speed in response to OF speed changes between persons with stroke and healthy controls and 2) investigate whether virtual environments (VE) manipulating OF speed can be used to promote volitional changes in walking speed post stroke.Twelve persons with stroke and 12 healthy individuals walked on a self-paced treadmill while viewing a virtual corridor in a helmet-mounted display. Two experiments were carried out on the same day. In experiment 1, the speed of an expanding OF was varied sinusoidally at 0.017 Hz (sine duration = 60 s), from 0 to 2 times the subject's comfortable walking speed, for a total duration of 5 minutes. In experiment 2, subjects were exposed to expanding OFs at discrete speeds that ranged from 0.25 to 2 times their comfortable speed. Each test trial was paired with a control trial performed at comfortable speed with matching OF. For each of the test trials, subjects were instructed to walk the distance within the same time as during the immediately preceding control trial. VEs were controlled by the CAREN-2 system (Motek). Instantaneous changes in gait speed (experiment 1) and the ratio of speed changes in the test trial over the control trial (experiment 2) were contrasted between the two groups of subjects.When OF speed was changing continuously (experiment 1), an out-of-phase modulation was observed in the gait speed of healthy subjects, such that slower OFs induced faster walking speeds, and vice versa. Persons with stroke displayed weaker  correlation coefficients between gait speed and OF speed, due to less pronounced changes and an altered phasing of gait speed modulation. When OF speed was manipulated discretely (experiment 2), a negative linear relationship was generally observed between the test-control ratio of gait speed and OF speed in healthy and stroke individuals. The slope of this relationship was similar between the stroke and healthy groups .Stroke affects the modulation of gait speed in response to changes in the perception of movement through different OF speeds. Nevertheless, the preservation of even a modest modulation enabled the persons with stroke to increase walking speed when presented with slower OFs. Manipulation of OF speed using virtual reality technology could be implemented in a gait rehabilitation intervention to promote faster walking speeds after stroke. Regulation of walking involves the integration of visual, proprioceptive and vestibular information. Optic flow (OF) is a typical pattern of visual motion generated at the eye as the person moves through the environment . OF is aSlow speed, a feature that characterizes locomotion after stroke , can impThe specific aims of this study were: first, to compare the non-volitional modulation of walking speed in response to an OF of variable speed between persons with stroke and healthy controls and second, to investigate whether a virtual reality-based paradigm that manipulates OF speed could be used to promote volitional changes in walking speed in persons with stroke. We hypothesized that persons with stroke, although presenting with an altered modulation of walking speed in response to OFs of changing speed, would still present sufficient modulation such that a paradigm based on OF speed manipulation could be used to promote faster walking speeds.Twelve subjects with a hemiparesis  due to a first-time stroke in the middle cerebral artery region and twelve healthy controls  participated in this study Table . The locSubjects were evaluated while walking on a self-paced treadmill and watching, in a head mounted display (HMD), virtual environments representing corridors Figure . The HMDSubjects were first habituated to walk on the self-paced treadmill without the HMD, and then with the HMD while watching a virtual corridor scene expanding at a speed matching their comfortable walking speed. Comfortable walking speed was determined after a minimum of 4 to 5 minutes of habituation while walking with the HMD. Subjects listened to white and pink noises through earphones during testing, to avoid any speed feedback from auditory cues. White noise has equal energy at all frequencies, while pink noise has more power in the lower frequencies that the higher frequencies. The latter provides additional masking power for low frequency noises.Experiment 1, the speed of an expanding OF was varied sinusoidally , from 0 to 2 times the individual's comfortable walking speed, for a total duration of 5 minutes. The sine wave was preceded and ended by 30 seconds of OF matching comfortable speed. In Experiment 2, subjects were instructed to walk at a comfortable pace  on the self-paced treadmill through a 13 m-long virtual corridor comprising of 2 doorways located 10 m apart. Subjects started walking 1.5 m before the first door and ended at 1.5 m after the second door when a stop sign appeared. Every control trial was followed by a test speed trial, during which expanding OFs corresponding to 0.25, 0.5, 0.75, 1.00, 1.25, 1.50, 1.75 and 2 times the individual's comfortable speed were randomly presented. For each test speed trial, subjects were instructed to cover the corridor distance within the same time span as the previous control trial.Two series of experiments were conducted on the same day, with experiment 2 following experiment 1. Because of fatigue or time constraint, one healthy control and 3 subjects with stroke did not complete experiment 2. In the first experiment, the instantaneous and non-volitional changes in walking speed in response to an OF of continually changing speed (Experiment 1) were examined. In the second, the voluntary or intentional changes in walking speed in response to OFs at discrete constant speeds (Experiment 2) were examined. In ® system (Motek) that also manipulated the OF speeds. In addition, the treadmill speed signal was recorded at 360 Hz using the Vicon-512 A-D system. Speed signals were low-pass filtered at 0.02 Hz .Distance travelled and walking speed were provided by the treadmill tachometer and recorded at 100 Hz using the Caren-2® and the level of significance was set p < 0.05.For Experiment 1, the strength of the relationship and phase lag between the OF speed and walking speed signals were analyzed by means of cross-correlations. For Experiment 2, the mean walking speed over the 10 m separating the 2 doorways was calculated for every trial. Changes in walking speed for the speed trials were expressed as a ratio of the mean walking speed in the test trial over the control trial (gait speed ratio). The slopes between gait and OF speed ratios were obtained using linear regressions. All outcome variables, including cross-correlation coefficients, phase lags and slopes were compared between subjects with stroke and healthy controls using Student t-tests for independent samples. The relationship of these variables with the subjects' comfortable gait speeds was also quantified using Pearson correlation coefficients. Statistical analyses were carried out in Statistica 7.0 2 = 0.08, P = 0.4) nor the phase lags  were associated with the comfortable overground walking speed post stroke.Examples of modulation of walking speed in response to an OF of continually changing speed are illustrated for a healthy subject and subjects with stroke in Figure 2 = 0.53, p < 0.05) and higher ratios of speed increment  than those who walked at faster initial speeds.Figure The purpose of this study was to investigate the ability of persons with stroke to modulate their speed of walking in response to changing OF speeds. The main findings are that subjects with stroke present with alterations in their speed modulation response to changing OF speeds. However, when instructed to use OF speed information to scale their walking speed, they display patterns of modulation that are similar to those observed in age-matched healthy subjects.Results of the present study demonstrate that walking speed is modulated out-of-phase with OF speed in healthy subjects, which is consistent with previous studies -8, despiResults from Experiment 2, in which subjects voluntarily attempted to walk a fixed distance within the same time span while exposed to OFs of different speeds, yielded larger walking speed changes than when exposed to the sinusoidally changing OF speeds in Experiment 1. This difference was observed despite the fact that the range of OF speed was smaller in Experiment 2  than during Experiment 1 (0 to 2 times). The larger modulation observed in Experiment 2 may be attributed to the fact that information from constant OF speeds is easier to perceive and integrate than continually changing speeds, while a cognitive intentional process was also involved. The fact that the walking distance was shorter and that perception of time  may have been a useful source of information to complete the task may also have contributed to the larger gait speed modulation responses observed in the second experiment. Gait speed adaptation did not fully compensate the mismatch with OF speed, even by our healthy aged-matched subjects, as indicated by slope values that did not approach -1.0. This partial compensation suggests the involvement of other sensorimotor transformations based on an internal representation and the central nervous system's integration of other sensory cues, such as leg proprioceptive information.Present results also showed that subjects with stroke displayed abnormalities in their non-volitional or unintentional modulation of walking speed. Both animals -22 and hIt is also noteworthy that the down-regulation response observed in the subjects with stroke differs from the up-regulation response observed in subjects with Parkinson's disease, which manifests as an abnormally large modulation in walking speed in response to changing OF speed . This fiResults from Experiment 2 also reveal that subjects with stroke, once cognitively though intentionally involved in the task, produce speed modulation responses that are similar to that observed in the age-matched healthy subjects. The fact that the stroke group performed within the norms in the second experiment, but not during the first experiment, suggests that the two tasks involved different stimuli  and that incorporating a volitional component yields more accurate, yet not fully adapted, speed modulation responses. Most importantly, the adaptability of subjects with stroke in the second experiment provide a strong rationale to incorporate the manipulation of OF speed in a virtual-reality training paradigm that aims at promoting faster walking speeds. Present results indicate that subjects with stroke could instantaneously increase their walking speed by 1.44 times, which represents an increase close to 50%. To our knowledge, it is the first evidence that a virtual environment simulating OF could induce such large changes in gait speed in subjects with stroke. Before such an approach can be implemented in a virtual reality-based gait rehabilitation program, further work is needed to determine whether habituation occurs in the intentional speed modulation paradigm, as for the unintentional paradigm , and theThe author(s) declare that they have no competing interests.Lamontagne conceived and carried out the experiment and analyses presented in this paper. She also drafted the manuscript. Fung and McFadyen contributed to the design of the study, data analyses and reduction, as well as revision of the manuscript. Faubert contributed to the design and psychophysical aspects related to optic flow perception. All authors read and approved the final manuscript.


We do not yet know how to use blood glucose self-monitoring (BGSM) most effectively in the self-management of type 2 diabetes treated with oral medication. Training in monitoring may be most effective in improving glycaemic control and well being when results are linked to behavioural change.DiGEM is a three arm randomised parallel group trial set in UK general practices. A total of 450 patients with type 2 diabetes managed with lifestyle or oral glucose lowering medication are included. The trial compares effectiveness of three strategies for monitoring glycaemic control over 12 months (1) a control group with three monthly HbA1c measurements; interpreted with nurse-practitioner; (2) A self-testing of blood glucose group; interpreted with nurse- practitioner to inform adjustment of medication in addition to 1; (3) A self-monitoring of blood glucose group with personal use of results to interpret results in relation to lifestyle changes in addition to 1 and 2.The trial has an 80% power at a 5% level of significance to detect a difference in change in the primary outcome, HbA1c of 0.5% between groups, allowing for an attrition rate of 10%. Secondary outcome measures include health service costs, well-being, and the intervention effect in sub-groups defined by duration of diabetes, current management, health status at baseline and co-morbidity. A mediation analysis will explore the extent to which changes in beliefs about self-management of diabetes between experimental groups leads to changes in outcomes in accordance with the Common Sense Model of illness. The study is open and has recruited more than half the target sample. The trial is expected to report in 2007.DiGEM intervention and trial design address weaknesses of previous research by use of a sample size with power to detect a clinically significant change in HbA1c, recruitment from a well-characterised primary care population, definition of feasible monitoring and behaviour change strategies based on psychological theory and evidence, and measures along the hypothesised causal path from cognitions to behaviours and disease and well being related outcomes. The trial will provide evidence to support, focus or discourage use of specific BGSM strategies.The  Diabetes is now a major public health problem. The number of people with diabetes is estimated to reach 330 million by 2030 . There iThere is now strong evidence for the effectiveness of tight glycaemic control in reducing complications among people with diabetes . HoweverBGSM was used to underpin insulin dose adjustment in the Diabetes Control and Complications Trial among people with type 1 diabetes, which clearly demonstrated the efficacy of glycaemic control in reducing diabetic complications. However, neither the rationale for BGSM nor its efficacy or effectiveness among people with type 2 diabetes is clear. Yet BGSM is now widely accepted as part of management of people with type 2 diabetes ,6, and tPeople with type 2 diabetes are at risk from a range of macrovascular and microvascular diabetic complications. Large trials have confirmed the effectiveness of intensive glycaemic control at reducing these complications [A recent qualitative study has suggested that self-monitoring may be an important factor in helping people achieve a better understanding of their condition . HoweverThree systematic reviews have provided no evidence that self monitoring is effective in improving glycaemic control for people with type 2 diabetes when compared to urine testing and measurement of glycosylated haemoglobin (HbA1c) -14. The There are a small number of studies that offer some insight into how BGSM might lead to improved blood glucose control among people with type 2 diabetes. BGSM may be helpful in the titration of therapy by either patients or by practitioners or both, but whether regular monitoring is more effective than periodic measurement of HbA1c is unknown. Evidence from qualitative studies with patients suggests that awareness of fluctuations in blood glucose levels may promote adherence to self-care behaviours, including medication taking, diet and physical activity in selected patients ,17.There is increasing work in the area of diabetes self-management that uses psychological theory to guide intervention and measurement of the processes of behaviour change. One approach, the Common Sense Model (CSM) , proposeThe majority of previous trials have used reflectance meters rather than biosensor technology. The older meters required larger quantities of blood and took longer to produce a reading than current systems. Although when used correctly the older meters provided reliable information, in practice their accuracy, usability and so potential impact was limited and may have formed a barrier to their effectiveness without high levels of motivation.Only one identified randomised trial specified the approach used with patients to support their interpretation of test results . PatientPrevious trials have not specified the efforts made to monitor delivery of interventions. These include both the process of attempting to ensure that participants understand the techniques being used, and that efforts are made to ensure that the intervention is delivered as per protocol, and that there is adequate separation from the comparison groups in the intervention delivery.Our primary objective is to determine whether HbA1c is significantly lower in patients with type 2 diabetes allocated to each of two intervention groups  compared to patients allocated to a control group .Secondary objectives are:(i) To compare well-being, satisfaction, health service use and economic cost between allocated groups.(ii) To conduct an exploratory analysis of changes in mean HbA1c between regimens among sub-groups defined by duration of diabetes, current management, self-reported health status and co-morbidity.(iii) To test how self-monitoring influences beliefs and behaviour using measures chosen on the basis of theoretical models of behaviour change.DiGEM is a four-year study with an open, randomised controlled pragmatic parallel group trial design Figure  with seqThe study design is shown in Figure (i) a control group receiving standardised usual care and three monthly measurement of HbA1c,(ii) a self-testing group who, in addition to the above, are carrying out blood glucose self testing with the results interpreted by the study nurse,(iii) a self monitoring group who, in addition to both of the above, are given support in interpreting and applying the results of blood testing to enhance motivation and maintain adherence to diet physical activity and medication regimens.Following randomisation, patients receive the allocated education and training appropriate for their group, with follow up to maintain the interventions at four, 13, 26, and 39 weeks with a final assessment at 52 weeks.Non-insulin using patients with type 2 diabetes have been recruited from general practices in Oxfordshire and are being recruited from South Yorkshire to take part in the study. The 48 recruited practices represent a geographical spread of rural/suburban centres and cover a wide socio-economic range of patients. The mean number of patients recruited in the 24 practices in Oxfordshire was 10.2, standard deviation (SD) 5.6.Patients suitable for trial inclusion are identified from practice generated computer lists. Eligible patients are sent an invitation to participate signed by their general practitioner accompanied by an information sheet and a reply paid envelope to facilitate response. One further letter is sent if no response is received in one month.Inclusion criteria are type 2 diabetes, aged 25 years or more at diagnosis, managed with lifestyle or oral hypoglycaemic agents, independent for activities of daily living. Exclusion criteria are use of blood glucose monitor twice a week or more often over the previous three months, current use of insulin, co-morbidity or limited life expectancy that would make intensive glycaemic control inappropriate, last clinic HbA1c or HbA1c at the assessment visit less than 6.2%, or unable to follow trial procedures.).Participants are randomly allocated to one of the three groups using a partial minimisation procedure to adjust the randomisation probabilities between groups to balance important covariates including duration of diabetes, HbA1c, and prior medication using a computer programme  , medicatBeliefs about diabetes and its management are assessed using the Illness perceptions Questionnaire , BeliefsBlood glucose monitoring resources are measured by counting recorded entries in diaries. Medication use, episodes of hospitalisation and their duration will be noted and non-hospital health care resource use will be recorded by a questionnaire administered to all patients at each visit.Participant eligibility for the study and willingness to be randomised to a group in which they might be required to test their own blood glucose six times a week or more is confirmed at the assessment visit. Following consent, beliefs about diabetes, the role of eating, physical activity and medication are discussed with all participants. A goal setting approach to lifestyle change is introduced and continued in subsequent visits. Baseline blood tests and clinical measurements are taken and questionnaires completed at this visit.Following the assessment visit and confirmation of eligibility on the basis of HbA1c measurement, patients are randomised to one of three groups: control group, self-testing and self-monitoring.At a visit two weeks after the assessment visit participants receive training and education appropriate to their allocated study group. The control group receives 3-monthly HbA1c measurements and identifies behavioural goals to improve glycaemic control. The self-testing group, in addition, is asked to use a blood glucose meter to record three fasting, pre-meal or 2-hour post meal readings on two days during the week. Treatment targets of fasting and pre-meal levels of 4–6 mmol/l and post meal levels of 6 to 8 mmol/l and advice about using these readings to identify high (>15 mmol/l) and low (<4 mmol/l) blood glucose readings are given. The self-monitoring group, in addition, is provided with training and support to encourage interpretation of readings and application to goals for lifestyle change based on the CSM in order to reach treatment targets.Subsequent follow up includes a telephone call 2 weeks after randomisation (after the post-randomisation visit rather than randomisation), and further visits at 4, 13, 26 and 39 weeks. The follow up visits differ according to the allocated group. Those allocated to the control group have a HbA1c measure two weeks before their scheduled visit in order for their glycaemic control to be discussed. The two groups using a meter are managed on the results of their blood glucose self-monitoring. The GP is notified of all HbA1c results and asked to consider changes in medication in line with the National Institute for Clinical Excellence diabetes guidelines. The GP is also notified if blood glucose readings are consistently above 15 mmol/l.Baseline self-report measures and measures of belief are completed at the assessment visit. Baseline biochemical measures and clinical measurements are also made. Repeated measurements are made at the 52-week follow up visit. Data on adverse reactions or complications are collected at each study visit along with information about use of medication and health services.Patients are supplied with a blood glucose meter calibrated to provide plasma equivalent results. Meters are checked at the beginning of the study and at 26 weeks by study nurses with a test aliquot.A script outlining the topics to be covered, and explanation of the theoretical basis of the intervention are used to support the nurses in their adherence to study protocol. Study nurses attended a six day phased course in psychological theory, behaviour change techniques and skills training in the intervention. Additional measures to ensure fidelity include self-review of taped interventions by the study nurses and external review by a researcher using a checklist to ensure delivery of the intervention according to protocol. Prompts built into the patient diaries help participants adhere to their allocated group intervention.In the absence of data relating to change in HbA1c we have calculated sample size conservatively based on the absolute level of HbA1c at follow up. We set out to detect a difference in HbA1c of 0.5% between any two groups. At the outset we estimated the SD of HbA1c as 1.5, based on data from the UKPDS. In practice, the SD of baseline HbA1c among the 245 patients recruited in Oxford was 0.9, but we assumed that the SD among additional patients recruited elsewhere could be as high as 1.5. With 205 further patients, the overall SD would be 1.2, and there would be 150 patients in each group . These numbers would give 93% power to detect a difference of 0.5% in HbA1c between any two trial groups . Figure We propose to conduct a single analysis of main trial endpoints at the end of the study. The proposed intention to treat analysis will compare mean levels of HbA1c at follow up between the three study groups, with baseline HbA1c as a covariate, using analysis of covariance. Post-hoc t-tests between groups will be conducted in the event of a statistically significant result. Subsequent analysis will include comparing the two intervention groups against the control group.We will estimate the intervention effect in sub-groups defined by duration of diabetes (above or below median duration), current management , health status at baseline (above or below median EQ-5D score) and co-morbidity (presence or absence of diabetes related complications). We will also explore the extent to which the measures of beliefs included in the study can explain changes in behaviour; firstly by comparing mean levels of beliefs e.g. about controllability of diabetes between experimental groups and secondly by a more formal mediation analysis . Within The economic evaluation will be on an intention-to-treat basis. A cost-effectiveness/cost-utility analysis will be performed in which the difference in effectiveness will be compared to the difference in total costs between each study intervention group, and the results will be expressed as incremental cost-effectiveness ratios. Effectiveness will be measured in terms of change in HbA1c, and modelled for life years gained and quality adjusted life years gained. Unit costs will be attached to the resource items collected from the healthcare resource utilisation assessment using published national average costs and tariff averages for procedures to calculate costs. Mean values and 95% confidence intervals will be reported for each component of resource use and cost and for total costs and effectiveness. Sensitivity analyses will be performed on all aspects of the economic evaluation that are subject to uncertainty.This study will make an important contribution to the evidence-base for the use of blood glucose self-monitoring in non-insulin using patients with type 2 diabetes. It will provide a robust estimate of overall effect of use of the meters for self-testing alone and with a more intensive programme to support people in using their test results actively in self management of health related behaviours.This trial will address two main problems associated with previous trials. Firstly, the study is adequately powered to identify a reduction in HbA1c of 0.5%, which is associated with clinically important reductions in diabetic complications. Secondly, study participants allocated to the most intensive group are explicitly supported in interpretation of their study results in relation to lifestyle changes. This is the first study in this field to use standardised delivery of these interventions together with the other measures supporting fidelity to protocol.A further strength of the trial design is the inclusion of measures that explore the extent to which the interventions being used influences beliefs and behaviour. It will be possible to test whether the interventions, especially that based on the CSM, are successful in altering beliefs about diabetes, and whether the interventions were delivered with fidelity to their theoretical basis. It will also be possible to test whether differences in behaviour and HbA1c between experimental groups are due to the interventions bringing about changes in beliefs, as proposed by the CSM. Depending on the pattern of findings from the mediation analysis, it will be possible to conclude there is no mediation , total mediation  or partial mediation (part of the effect is transmitted by beliefs and part is direct). This will provide information about the extent to which the CSM can usefully inform intervention content to change beliefs and behaviours.The trial is mainly generalisable to that group of patients willing to be randomised to no self-testing. It will be limited in its ability to inform management of people who are enthusiastic about regular meter use. Not only does this group include people who have already been recommended to use a meter by their doctor or nurse, but also includes people who have obtained a meter in the absence of medical advice. In health service terms, these are people who are likely to obtain a meter whether or not they are prescribed or offered one. A trial to address management in this group would be more difficult because of difficulties in identifying individuals who are both enthusiasts, yet willing not to be exposed to the use of self-monitoring. However, the detailed information about beliefs and the pre-specified sub-group analyses will provide information to inform design of future studies in this area by allowing refinement of interventions and accurate estimates to inform sample size calculations The detailed information from blood glucose diaries and relation to outcomes will also inform the refinement of training programmes and data interpretation.This trial has features of both a pragmatic and an explanatory trial . The useThe results of this trial will be available in 2007.The authors hold no financial or non-financial competing interests. The views expressed in this paper do not necessarily reflect those of the NHS.AF, A-LK and AN had the original idea for the study and wrote the study protocol. AF, AW, DF and ALK developed study measures and intervention. AF, AW and LG have set up the study and made it run. AF is the guarantor of this paper.Members of the writing committee for this paper were A. Farmer, A. Wade, D French E Goyder HAW Neil, A-L Kinmonth.The study investigators are Oxford: A Farmer, D Mant, R Holman, S Ziebland, R. Holman, A Gray, and P Yudkin ; Cambridge A-L Kinmonth; Birmingham D FrenchMembers of the trial steering committee are N Stott (Chair), A Farmer, HAW Neil, S Sutton, H Tewson, D Chapman, H Hearnshaw, E Goyder, and M Jiwa.Members of the data monitoring committee are C. Baigent (Chair), J Levy and K Wheatley.Coordinating Centres: (Oxford) A Wade , A Craven , J Simon  and A Fuller (data manager); (Sheffield) Vivienne WalkerStudy Nurses (Oxford) M Selwood, H Kirlow, M Chapman, and S Turner; (Sheffield) A Casbolt, K Dobson, and A Willert.Health Technology Assessment Programme and NHS R&D NHS Support Funding. AF is supported by an NHS R&D Career Development AwardThe pre-publication history for this paper can be accessed here:


Objective: The purpose of this report is to present the results of a preliminary treatment regimen for hypertrophic scars combining topical 2% salicylic acid cream (Avosil) with an overlay of hydrogel dressing (Avogel). Methods: The study group consisted of 3 patients with symptomatic hypertrophic scars: 2 presternal and 1 on the inner thigh. Scars were divided into 3 equal-size areas: (1) untreated control, (2) hydrogel alone, and (3) 2% salicylic acid with hydrogel cover. Treatments were applied every 8 to 12 hours and a Velcro appliance was employed to cover the area during treatment. The total length of treatment was 60 days. Results: At the end of the 60-day treatment protocol, the area treated with 2% salicylic acid and hydrogel was asymptomatic. In contrast, the hydrogel-treated and untreated control areas remained erythematous and symptomatic for burning pain and pruritis. Conclusion: This small study suggests the efficacy of combined salicylic acid and hydrogel therapy in the treatment of hypertrophic scars. More extensive studies of scar treatment with salicylic acid and hydrogel are needed. These studies must be larger in scope to carefully document the spectrum of patient responses and should include methods for evaluating alterations in the levels of different inflammatory mediators. Thirty years ago, Larson et al observed that elastic pressure wrap dressings were effective in reducing scar hypertrophy in healing burn scars.In the past decade, another topical device, silicone gel sheeting, has been found to be useful clinically in controlling scar formation.8Inflammation is critically important for healing and scarring. Dang et alThe first 2 cases were part of an ongoing pilot study at St. Mary's Hospital designed to document the effect of topically applied salicylic acid on symptomatic hypertrophic scars. Both patients had tender and pruritic hypertrophic scars following median sternotomy. For study inclusion, the length of the scar needed to be sufficient to accommodate a control area and 2 experimental areas . The control area was left untreated while one experimental area was covered with Avogel hydrogel  and a second area of equal length was treated with 2% salicylic acid in Eucerin  and covered with Avogel hydrogel. The study regimen called for treatments to be performed for 8 to 12 hours per day employing a Velcro appliance specially constructed to cover the scar during treatment and to prevent salicylic acid from spreading into the control areas. Exclusion criteria included the use of anti-inflammatory agents during the previous 2 weeks or known allergies to anti-inflammatory agents. The scars were assessed at 2-week intervals for 60 days. Direct measurements, histories, and digital photographs were obtained. An end stage was achieved when the experimental region of the scar was flat and asymptomatic and when erythema had receded.The third patient was treated in a similar fashion by a Chicago area plastic surgeon for an 11-month-old hypertrophic scar from a burn injury on the inner thigh. This case is included to demonstrate that similar effects have been observed independently. The protocol used to treat this patient is described below.The Institutional Review Board of St. Mary's Hospital approved this study, and all participants gave informed consent. The procedures followed were in accordance with the ethical standards as set forth by the institution and with the Helsinki Declaration of 1975, as revised in 1983.All patients experienced significant symptomatic relief from tenderness and pain within days in the area of the scar treated with 2% salicylic acid and hydrogel. Some relief in symptoms was recorded in the area treated with hydrogel alone, but no symptomatic relief was experienced in the untreated area. Noticeable decreases in scar width and height were noted in all patients over the 60-day study period in the area treated with salicylic acid and hydrogel. Comparatively small changes in scar dimensions were noted with hydrogel alone, and no change was noted in the control region. Toward the end of the study period, scar erythema was greatly decreased in the area treated with hydrogel and salicylic acid in all patients.This patient is a 49-year-old man Fig  who undeThis patient is a 65-year-old woman Fig  who undeThis patient is a 42-year-old firefighter who suffered a flame burn injury to the skin on the left inner thigh 11 months before presentation. He was treated as an outpatient at one of the burn centers in Chicago where he received topical anti-microbials and dressing changes. The wound healed by epithelialization and hypertrophic scarring. The patient was referred to the University of Chicago Scar Clinic because the burn scar had become quite hypertrophic and was unresponsive to compression stocking therapy. A photograph of the scar taken at that time is shown in Figure Therapy was initiated with Avogel hydrogel with 3% salicylic acid in a cream base applied to the scar each night and left for 8 to 10 hours. The use of compression garment therapy was discontinued. One month later, treatment regimen was adjusted such that 2% acetylsalicylic acid in Eucerin ointment  with hydrogel was used to achieve better symptomatic relief. On 7-week follow-up  regulation, inhibiting histamine binding to its receptors, and inhibiting the NF-κB signal that upregulates inflammation. The most widely used anti-inflammatory agents belong to the broad category of nonsteroidal anti-inflammatory drugs. They inhibit either prostaglandin production or NF-κB generation or both. It is noteworthy that steroids also exhibit anti-inflammatory effects by inhibiting inflammatory response gene promoters NF-κB, AP-1 (activator protein-1), and NF-AT (nuclear factor of activated T lymphocytes).13The most common mode of prostaglandin synthesis blockade is inhibition of cell membrane–bound COX. There are 2 isoforms of COX expressed in human cells, COX-1 and COX-2.1 blockers, inhibit the inflammatory response, resulting in reduced scar formation and reduced discomfort. Patients scratch the inflamed scar less frequently, which probably reduces scar growth rate. Finally, antihistamines are well known to inhibit collagen synthesis.20Antihistamines are commonly used only to control the symptoms of scar pruritus. However, they have other important effects that may function to reduce scarring. Antihistamines, particularly HFrom the above, it is clear that more extensive studies of scar treatment with salicylic acid and hydrogel are needed. These studies must be larger in scope to carefully document the spectrum of patient responses and should include methods for evaluating alterations in the levels of different inflammatory mediators.


In order to evaluate metabolic pathways associated with obesity, global gene-expression data were integrated with phenotypic and genetic segregation analyses, identifying 13 metabolic pathways the genes of which are coordinately regulated in association with obesity. Four genomic regions were found to control the coordinated expression of these pathways and novel genes potentially associated with the identified pathways were identified. A segregating population of (C57BL/6J × DBA/2J)F2 intercross mice was studied for obesity-related traits and for global gene expression in liver. Quantitative trait locus analyses were applied to the subcutaneous fat-mass trait and all gene-expression data. These data were then used to identify gene sets that are differentially perturbed in lean and obese mice.We integrated global gene-expression data with phenotypic and genetic segregation analyses to evaluate metabolic pathways associated with obesity. Using two approaches we identified 13 metabolic pathways whose genes are coordinately regulated in association with obesity. Four genomic regions on chromosomes 3, 6, 16, and 19 were found to control the coordinated expression of these pathways. Using criteria that included trait correlation, differential gene expression, and linkage to genomic regions, we identified novel genes potentially associated with the identified pathways.This study demonstrates that genetic and gene-expression data can be integrated to identify pathways associated with clinical traits and their underlying genetic determinants. Comprehensive high-throughput analytical techniques such as expression microarrays have brought about a rethinking of the possibility of understanding biology and disease at a global ('systems biology') level ,2. TheseTraditional quantitative trait locus (QTL) analyses of complex traits in model organisms have focused on the identification of specific causative genes that differ between the originating strains and that are directly responsible for the variation in trait expression . The avaet al. [We have been interested in using these approaches in mice to understand the pathogenesis of obesity and vascular disease, among other related conditions ,13. Metaet al. . This seTo identify functionally related gene sets that are differentially perturbed in lean and obese mice of the BXD cross, two methods were applied: gene set enrichment analysis (GSEA)  and overAs a second approach, we used an analysis of over-representation of gene set members by applying the Fisher's exact test, as implemented by the Expression Analysis Systematic Explorer (EASE) software . The resp < 0.05 (not corrected for multiple comparisons). A large majority of these genes (61/68) showed a significant positive correlation , with relatively few being negatively correlated (10%). Within individual pathway sets, between 42% and 63% of the genes were significantly correlated. The majority of correlated genes in a given set were always positively correlated. The magnitude of the correlations was comparable across the 13 pathways.The gene sets identified as being differentially regulated between obese and lean mice represented metabolic pathways involved in energy and lipid metabolism. Note that genes can contribute to multiple pathways, as different pathways can be biologically interrelated. The majority of pathways feed into the tricarboxylic acid (TCA) cycle Figure . The aggWe hypothesized that the composite set of pathways represented a functionally interrelated gene set responding to common genetic controls (referred to subsequently as the obesity-associated pathway set). Two approaches were taken to test for such common genetic controls. The first was to test for the enrichment of pathway gene-expression quantitative trait loci (eQTLs) along genomic intervals, and the second was principal component analysis (PCA) of the gene set, followed by QTL mapping of the derived principal components.10 of the odds (LOD) score greater than 2.0. Of these eQTLs, 278 corresponded to 77 genes represented in the 13 differentially regulated pathways identified above by the GSEA procedure. To examine whether there was an enrichment of pathway gene eQTLs among the eQTLs of the 4,670 most differentially expressed genes along the genomic intervals, we first divided the genome into sequential overlapping 20 centimorgan (cM) intervals, beginning a new interval every 10 cM. We then counted how many of the eQTLs overall for the 4,670 differentially expressed genes and how many of the eQTLs specific for the 77 pathway genes defined above mapped to each interval. In general, assuming under the null hypothesis that the genomic location of genes influencing the transcript levels of other genes in common pathways is independent, the expected number of pathway gene eQTLs mapping to each interval is (M/B) × K where B is 4,670, M is the subset from the 4,670 genes that have eQTLs mapping to the interval, and K is 77. To compute the statistics we applied Fisher's exact test. The p-values obtained for the statistics are shown in Table p-value', which reflects the p-values after applying the Bonferroni correction criteria .When eQTLs for the 4,670 most differentially expressed genes were mapped we found that there were 15,262 eQTLs with logAfter correcting for multiple comparisons, significant over-representation of pathway gene eQTLs was obtained for four loci located on chromosomes 3 (80-100 cM), 6 (30-50 cM), 16 1-20 cM), and 19 (20-50 cM)  and Cyp2c39 (LOD = 12.4). To see whether these two genes were the major contributors to mapping of the PCs to chromosome 19, we carried out the analysis without these two genes for each of the pathways separately. The resulting components showed no evidence of significant linkage genome-wide.An alternative approach that has been proposed to test for coordinated regulation of the genes in individual pathways is PCA ,18. PCA The loci identified above are genomic regions with apparent regulatory control of pathway genes that are differentially perturbed between obese and lean mice. The locus allele effects on the subcutaneous fat-mass trait, using the nearest markers at each locus, are shown in Figure cis-eQTL [Cis-eQTLs were defined as any gene with eQTLs mapping to 20 Mb (approximately 10 cM) on either side of the gene's physical location. This definition reflects the relative accuracy of the 13 cM dense BxD QTL map. The decision to choose such a definition for cis-eQTLs is supported by the work of Doss et al. [cis nature of such defined eQTLs and by showing the extreme bias in the mapping of these cis eQTLs to regions which are not identical by descent between the B6 and DBA strains of mice.The chromosome 19 locus had the greatest impact on the expression of pathway set gene expression, and it was of interest to identify the candidate responsible genes for further study. For candidate genes regulated at the level of expression , an eQTL will be present proximal to the physical location of the gene; we have referred to such an eQTL as a cis-eQTL . Cis-eQTcis-acting eQTL (LOD score > 4.3) that map to chromosome 19 between 20 and 50 cM, and determined the genomic location of each gene using the University of California Santa Cruz (UCSC) Genome Browser . Of 249 eQTLs mapping to the chromosome 19 locus, 19 overlapped the physical location of their respective gene  and subsequently for the presence of eQTLs falling within the identified regulatory regions for both chromosome 6  and 19 . A total of 117 genes were identified that were not members of one of the 13 obesity-associated pathways. These include 20 genes with defined relationships to the starting pathways (based on published literature), but also 28 expressed sequence tags (ESTs) with no functional annotation. A complete list is provided in Additional data file 1.The above analyses were restricted to the genes that had been assigned to gene sets in the starting databases such as KEGG. To identify novel genes that may belong to the same aggregate group of obesity-associated pathway genes, we filtered the 4,670 differentially regulated genes first for correlation with the fat-mass trait at This study shows that the incorporation of genome-wide gene-expression data with traditional phenotypic trait data in a mouse intercross population enables the identification of regulated metabolic pathways and the genomic regions that control the expression of the constituent pathway genes. In contrast to studies with genetically altered mice, we have studied a heterogeneous population where the genetic variations leading to the phenotypes and the changes in gene expression are all naturally occurring. In this work, we identified metabolic pathway gene sets with altered gene expression in association with the subcutaneous fat-pad mass trait, but the concept is applicable to other phenotypic traits where the target organ examined would be expected to show changes in relevant pathway gene expression. While gene-expression changes are not sufficient to imply actual metabolite flux through a pathway , changesTwo approaches were used to identify gene sets whose transcripts were altered in association with the fat-mass trait: the GSEA method and Fisher's exact test. These yielded comparable results, although the GSEA appeared to be more sensitive. Central to both is the availability or construction of predefined gene sets to test. We used two primary sources, the KEGG database, composed primarily of traditional metabolic pathways, and the Biocarta database, composed of gene sets of varying type. The total number of genes represented in these was relatively low . There are certain to be biologically important gene sets as yet unrecognized, as well as currently uncategorized genes that belong in the existing sets. Our approach allows tentative functional assignment to such uncategorized genes when they share properties of correlation of expression levels with the phenotypic trait and co-localization of eQTL with the trait QTL.Aoc3) (12.5-fold change in expression level). This observation is consistent with the study in which overexpression of human AOC3 in mice resulted in an increase in weight and subcutaneous white adipose tissue when the animals were fed an atherogenic diet for 15 weeks [Aldh1a1, Aldh2, and Aldh3a2)). These enzymes are involved in the generation of acetate, which can be converted to acetyl CoA. The family is also involved in ascorbate/aldarate metabolism, valine, leucine, and isoleucine degradation, propanoate metabolism, fatty-acid metabolism, glycerolipid metabolism, β-alanine metabolism, bile acid biosynthesis, and pyruvate metabolism pathways (eight out of the 13 pathways). The increased expression of the aldehyde dehydrogenases such as Aldh3a2 in mice with higher subcutaneous fat-pad mass is consistent with recent studies that correlate hyperinsulinemia with increased hepatic expression of Aldh3a2 [Consistent with an obesity trait, the gene sets identified were of pathways directly or indirectly related to energy metabolism. Within any given pathway, only about half of the genes showed a correlation between transcript levels and fat mass, consistent with the concept that regulation of a metabolic pathway requires the control of only selected elements rather than all constituents. It is of interest that the pathways are at least loosely interconnected, and several of the genes examined have been shown to influence adipose tissue mass in other studies. For example, of the total of 150 pathway genes represented on the microarrays, the gene with the highest differential expression between obese and lean mice was amine oxidase copper-containing 3 , just as for a traditional physiologic trait -6,26. Loet al. [The loci on chromosomes 6 and 19 had the greatest number of eQTLs, and were the two loci identified in previous studies to be significantly linked with the subcutaneous fat-mass trait. It is of interest that the most significant locus controlling this fat-mass trait is located on distal chromosome 2 [H-2 locus, are also known. Yet another possible explanation is that the genes with eQTL mapping to a common hotspot are in linkage disequilibrium (LD). Two or more genes that differ genetically between the two strains and that are in LD will show correlated expression levels [trans-eQTL hotspot for these genes which is more difficult to interpret [cis-eQTLs for which there is a minimal clustering of physical location (data not shown). On this basis we conclude that LD is unlikely to explain the hotspots identified in this study.We show that there are hotspots of eQTL activity for pathway gene members along the genome. One explanation for the presence of these hotspots is the presence of a major regulator gene within each locus that coordinately regulates obesity-related pathways. Alternatively, these loci may contain several such regulators that are closely linked. It is well known that clusters of duplicated genes with related functions occur commonly in the genome. Other clusters with diverse genes but related functions, such as the n levels . This spnterpret . We detecis-acting regulation can be identified by the coincidence of an eQTL for a gene and its physical location. We have experimentally verified that the majority of such putative cis-eQTLs are real [An approach to prioritizing candidate genes using eQTL information is presented for the chromosome 19 locus. This uses the concept that for genes regulated at the level of transcription, are real . This apIn conclusion, this study presents one approach for using gene-expression microarray data in conjunction with genetic segregation to identify metabolic pathways that are altered in association with obesity, and to identify specific genomic regions that exert regulatory control over these pathways. These findings contribute, along with other approaches developing genetic regulatory networks , to the et al. [et al. [This study, along with others previously reported ,19,28, w [et al. , and an All F2 progeny were fed a chow diet until 12 months of age and then put on a high-fat high-cholesterol atherogenic diet containing 0.5% cholic acid for 4 months . At 16 months of age these mice were euthanized and liver and other organs were removed and frozen at -70°C for subsequent RNA and DNA extraction. Body weight was determined before organ removal and adipose tissue depots were removed and weighed for a subset of 96 animals in the phase 1 group.RNA extraction and expression microarray profiling were carried out as described using a custom ink-jet microarray (Agilent Technologies) containing 23,574 60mer oligonucleotide non-control probes for profiling gene expression in liver of the F2 mice  of the N10 expression ratio values (averaged over fluor-reverse pairs) as quantitative traits for the 111 mice that were part of the phase 1 group described above. The linkage map was constructed using microsatellite markers at an average density of 13 cM using MapManager QTX version 0.30 and QTL Cartographer version 2.0 [QTL analysis was performed using mean logsion 2.0 . For eacA total of 378 gene sets were developed for gene set enrichment analysis (GSEA). Gene sets (118) representing metabolic pathways were obtained from KEGG ,33,34. Tp-value for differential expression (compared to the control pool) for each gene were calculated as described [p-values of 0.05 or less in 10 or more F2 mice were defined as differentially expressed, which yielded a set of 4,670 genes. To study gene subsets  that are associated with obesity, the mice comprising the upper and lower 15th percentiles of the subcutaneous fat-pad mass trait were selected. The average and standard deviation of the mean log ratio of expression of each differentially expressed gene for these two groups of mice were calculated and used for the GSEA and the Fisher's exact test analyses described below.A subset of genes represented on the microarray were selected on the basis of differential expression across all the 155 mice arrayed, irrespective of phenotype. This was done to ensure a starting gene set that was active and differentially expressed in liver among mice. To filter for these genes, the expression ratio and the corresponding escribed ,39. Genep-value of 0.05.Gene sets that are differentially regulated in relation to the subcutaneous fat-pad mass trait were identified using the GSEA method as described in . The groThe test subset was composed of transcripts identified as being the 20% most differentially regulated between obese and lean groups of mice. Fisher's exact test was applied as implemented in the Expression Analysis Systemic Explorer (EASE) program , downloacis-eQTL definition as described in [cis-eQTL when the eQTL mapped within a 20 megabase (Mb) window on either side of the physical location of the gene. This corresponds to an approximately 20 cM segment of the genetic map, which is the approximate level of accuracy for a QTL given a genetic map with an average inter-marker distance of 13 cM. The following approach was taken to equate cM locations on the genetic map (constructed by the QTL software for this specific cross) with the Mb locations of the physical map, in order to be able to define eQTL as cis or trans. The physical location of the gene was obtained from the UCSC Genome Browser . To map the genetic map location of the eQTL to the approximate equivalent location on the physical map, the physical map locations of the microsatellite markers immediately adjacent  to the eQTL were identified from the UCSC genome browser . Then the Mb 'location' of the eQTL was determined by interpolation based on the relative location of the eQTL between the markers on the genetic map.We adopted the same ribed in . An eQTLPCA was performed as implemented in the R statistical software version 1.8.1  using thThe following additional data are available with the online version of this paper. Additional data file A table listing genes that were not included in any tested gene set but are significantly correlated with subcutaneous fat mass and have eQTLs on chromosomes 6 and 19 .Click here for fileA table listing genotype and fat-mass trait data for the mice used in this study.Click here for file


PPARA gene have been found to associate with ischaemic heart disease in non diabetic men. The L162V variant was found to be protective while the C2528G variant increased risk. L162V has also been associated with altered lipid measures. We therefore sought to determine the effect of PPARA gene variation on susceptibility to myocardial infarction in patients with type 2 diabetes. 1810 subjects with type 2 diabetes from the prospective Go-DARTS study were genotyped for the L162V and C2528G variants in the PPARA gene and the association of the variants with incident non-fatal myocardial infarction was examined. Cox's proportional hazards was used to interrogate time to event from recruitment, and linear regression for analysing association of genotype with quantitative clinical traits.Common variants of the The V162 allele was associated with decreased risk of non-fatal myocardial infarction  whereas the C2528 allele was associated with increased risk (HR = 2.77 95%CI 1.34–5.75 p = 0.006). Similarly V162 was associated with a later mean age of diagnosis with type 2 diabetes and C2582 an earlier age of diagnosis. C2528 was also associated with increased total cholesterol and LDL cholesterol, which did not account for the observed increased risk. Haplotype analysis demonstrated that when both rare variants occurred on the same haplotype the effect of each was abrogated.PPARA locus is important in determining cardiovascular risk in both male and female patients with diabetes. This genotype associated risk appears to be independent of the effect of these genotypes on lipid profiles and age of diagnosis with diabetes.Genetic variation at the  PPARA gene is a strong candidate for a genetic determinant of CVD risk in people with type 2 diabetes [Dysregulation of fatty acid metabolism plays a pivotal role in the aetiology of type 2 diabetes , and expdiabetes .PPARA gene has been screened for common variation [in vitro [The ariation -8. The min vitro ,9, and tin vitro ,8,10, dein vitro . A seconin vitro . Recentlin vitro .PPARA gene variation with risk of CVD and diabetes related traits in Caucasian subjects with type 2 diabetes participating in the prospective population-based Genetics of Diabetes Audit and Research in Tayside Scotland (Go-DARTS) study [PPARα activators improve the dyslipidemia associated with type 2 diabetes and may be particularly beneficial in lowering risk of CVD in subjects with type 2 diabetes or metabolic syndrome . We therS) study -16.The clinical characteristics of the genotyped cohort are shown in table During a median follow up time 49.6 months there were 108 non-fatal myocardial infarction events and 355 deaths from all causes. In a fully adjusted Cox's proportional hazards model  and even in this high risk population did not account for the increased cardiovascular risk associated with C2582. This was not unexpected, as in keeping with the previous studies in non-diabetic men, inclusion of total cholesterol or LDL cholesterol in the model did not affect the association with cardiovascular outcomes, indicating that the increased risk associated with the C2528 variant is not likely to be through its influence on lipid levels.PPARA was used to construct haplotypes with L162V and G2825C [Few studies have considered cardiovascular disease or considered variants other than the L162V. One recent study also demonstrated a non significant trend towards a protective effect of V162 in individuals with diabetes . This std G2825C .PPARA variation is associated with CVD risk regardless of diabetic status.In this study, as in ours, the C2825 was associated with an earlier age of diagnosis, with the V162 allele being associated with protection from early diagnosis, in a manner consistent with the modulation of CVD risk. This is the first study to present the association with age-of-diagnosis of type 2 diabetes in the same population with the association with cardiovascular risk, and we can state that inclusion of age of diagnosis does not modulate the observed associations with cardiovascular risk and vice-versa, demonstrating that these are independent observations. This is not surprising, as PPARA gene.The G2825C variant is in a non-coding region (intron 7) and therefore unlikely to be a directly causal variant, however several other studies have indicated its potential biological importance ,11,12 anPPARG locus in which the biological effect of a variant with probable functional consequences is consistently modified in an opposing manner by the presence of a variant for which a function is difficult to ascribe [We have previously demonstrated a similar situation in the  ascribe ,15. Give ascribe .PPARA locus in modulating susceptibility to cardiovascular disease, and have shown that this association is relevant to individuals with type 2 diabetes.We have confirmed the potential importance of genetic variation at the In the Tayside region of Scotland detailed clinical information on all individuals with diabetes mellitus is recorded on a continuously updated electronic clinical information system known as DARTS (Diabetes Audit and Research in Tayside Scotland) . ValidatFollowing written informed consent from individuals registered on DARTS, blood samples for genetic studies have been collected, thereby creating a genetic sub-study known as Go-DARTS. Rigorous compliance with NHS data protection and encryption standards is maintained and the study was approved by the local research ethics committee.CTTTCTGTCGGGATGT, V162 probe (TET labelled) – TTCACAAGTGCGTTTCTGTCGGGATGT.The PPARα L162V and G2528C genotypes were determined in 1,810 individuals all of whom were Caucasian with type 2 diabetes diagnosed between the age of 35 and 70 years. Taqman (Applied Biosystems) allelic discrimination assays were used. The primers and probes used for the allelic discrimination assays were as follows: L162V Forward primer-CAGAAACAAATGCCAGTATTGTCG, Reverse primer-GGCCACCTTACCTACCGTTGTG, L162 probe (FAM labelled) – TTCACAAGTGCGGATTCAAAAGCTTCATTTCCCA, C2528 probe (TET labelled) – ATATCTAGTTTCGATTCAAAAGCTTCATTTCCCA.G2528C Forward primer-TCCTTAAATATGGTGGAACACTTGAAG, Reverse primer-TCACAACCACCAGTTTTGCAT, G2528 probe (FAM labelled) – ATATCTAGTTTFor each clinical measure the mean was determined from multiple measures obtained up to a maximum period of three years . LDL cholesterol was estimated through the use of the Freidwald equation. Linear regression was used to determine the association of genotype with each measure corrected for age at genotyping. For determining the association of genotype with age of diagnosis, this was corrected for gender and presence of a history of smoking by determining residuals and adding these to the overall mean age of diagnosis. Cox's proportional hazards was used to model time to first event. All individuals were followed from the point of genotyping until a non-fatal myocardial infarction occurred, or a composite of non-fatal myocardial infarct or all cause death. Censoring occurred either at the end of the study or death from any cause. Both variants were included in the model and it was found that a co-dominant model for each variant produced the best fit. The following variables were also included in the model; age at genotyping, gender, history of smoking, treatment with insulin, a previous history of a myocardial infarction, prevalent angina and prevalent cerebrovascular disease. Haplotype frequency estimates together with haplotype effects were determined using the THESIAS Program ,29. In tDARTS Diabetes Audit and Research in Tayside StudyGo-DARTS Genetics of DARTSPPARα Peroxisome Proliferator Activated Receptor alphaPPARA Gene for PPARαPPARD Gene for PPARδCVD Cardiovascular diseaseLOCAT Lopoid Coronary Angiography TrialNPHS2 Second Northwick Park Heart StudyHR Hazard RatioCI Confidence IntervalThe author(s) declare that they have no competing interests.ASFD and CNAP wrote the manuscript and performed the analysis, CNAP and ADM conceived the study and participated in its design and coordination. GL helped draft the manuscript and contributed to the data analysis. BF and SL performed the genotyping. All authors read and approved the final manuscript.


The number of women working in general practice internationally has been steadily rising. In Scotland there have been concerns that such a change may lead to increased part-time working and subsequently to a fall in available general practice manpower despite an apparently rising overall number of general practitioners. However, there is very little information on the actual hours worked by men and women general practitioners or on the types of work they are undertaking.Anonymous workload questionnaires of all Scottish general practitioner principals and non-principalsResponse rates for general practice principals and non-principals were 67.2% and 65.2% respectively. Male principals spent on average 18% more time on general medical services (GMS) and 50% more time on non-GMS activities  than women (both p <0.01). This difference was similar for non-principals. In no age group did the hours worked by women doctors approach that of male doctors.Women doctors in primary care in Scotland work fewer hours in all age groups than their male counterparts. The rapidly increasing proportion of women in general practice may lead to an increasing shortfall of medical availability in the future if current work patterns are maintained. Further longitudinal research is required to establish this and man-power planning is required now to address this. More worryingly auxiliary activities such as teaching and administrative duties are not being taken up by women. This may have serious implications for the future development of the specialty in Scotland. Faced with an aging population and the falling popularity of general practice as a career there have been concerns that the future Scottish general practitioner (GP) work force may be insufficient to manage a rising clinical workload . As in oGeneral practice in the UK is largely managed in partnerships of GPs with a few working single-handedly . Up untiAnother group of doctors known as non-principals did not usually have a direct contract with a primary care organisation but worked as locums, assistants or on the doctors retainer scheme (a scheme to maintain doctors with domestic commitments in practice) and were employed usually on a sessional basis by GP principals . These doctors generally spend less time on the managerial and strategic planning aspects of general practice and they were largely made up of relatively young women. They too were free to take up other types of employment. In April 2004 these titles were re-designated with all doctors providing primary care services now called performers and those with contracts to provide services with the local primary care organisations additionally called providers. However despite the change in name their day-time roles remained largely the same and work patterns were maintained. Out-of-hours responsibilities passed away from GP principals to primary care organisations in April 2004.Current information on workload relies largely on contractual information which formerly divided principals into those working in to part-time (two categories), job share or full time which was defined as more than 26 hours per week. Information on non-principals was largely restricted to those who worked on the retainer scheme and a small group of practice assistants. Little at all is known about the workload contribution of the increasingly large number of doctors who work as locums in primary care.While it was largely assumed that doctors contracted to work more than 26 hours were working considerably more than this, it is not known to what extent doctors were working either above or below their contracted hours or if some of the contracted hours were being taken up either with National Health Service (NHS) related activity , education, research or private medical work. The last re-organisation of the health service (1991) saw many doctors take roles in NHS management and in specialist clinics and there has been a shift of medical undergraduate education to the community.Although the number of full-time equivalent GP principals in Scotland has been steadily rising, this increase is largely due to women doctors . It has Information on workload is very sensitive as it may reveal a situation where some doctors who may be paid as 'full-time' were in reality spending only half of this time on NHS related work. Potentially, previous non-anonymous surveys may have resulted in a perception of workload that was inflated because of concerns on the part of some respondents about revealing contractual non-adherence.In addition there is very little information on future work intentions. While there have been studies on retirement intentions -10, thesThe aim of this study was to discover how male and female general practice performers  and non-principals) divided their time between general medical service (GMS) activity and other activities such as teaching and administrative tasks. In order to obtain an honest picture of current GMS and non-GMS workload we decided to use an anonymous survey method. As out-of hours work was shortly to be contracted out we decided to restrict our survey to in-hours work.We realised that busy professionals would be unlikely to be able to devote a large amount of time to completing a questionnaire on workload. We aimed therefore for a questionnaire which took no more than five minutes to complete and which covered only two sides of A4.The main components of the principal questionnaire see  were: inThe non-principal questionnaire see  also conDuring construction both questionnaires were piloted on 4 occasions until we were satisfied that the questionnaires were both easy to understand and complete.All general practice principals in Scotland were identified from databases held by Information and Statistics Division of NHS Scotland (ISD) and non principals from accrued Supplementary Medical Lists derived from Scottish Primary Care Trusts. Because of the way it was assembled, and the highly mobile nature of the doctors on it, this non-principal database was known to be imperfect. It contained doctors who while trained in general practice no longer practiced , some doctors who had registered for work in more than one trust appeared twice, some had gone abroad and some had become principals since the list had been created. In both databases some doctors had retired or were on long term sick leave.In May-June 2004 all GP principals and non-principals in Scotland from the 'cleaned' databases were sent the questionnaire along with a covering letter which outlined the purpose of the study and reassured the participants of complete confidentiality. Enclosed with the questionnaire was a reply paid postcard which participants were asked to return by separate post to inform us that they had completed the survey. Four weeks later those who had not returned a card were sent a reminder.Data was entered into SPSS . CompariThe response rate for the GP principal questionnaire was 2541/3783 (67.2%) and for the non-principal questionnaire 749/1149 (65.2%). While the GP principal response rate is reasonably accurate great caution must be used in interpreting the non-principal response rate. This group of doctors is highly mobile with many retiring from practice, leaving the country or giving up general practice to work in other parts of the NHS (over 200 indicated they had plans to behave similarly in the next 5 years in this sample group). It is very likely that the non-principal response rate is a higher proportion of those non-principals currently working in general practice in Scotland than the figures here reported suggest. We were able to compare the demography of most of our respondent samples with figures from ISD. Age and sex data were not available for the whole of the non-principal sample at the time of the study from ISD, but sex data was available for those doctors who have contracts (i.e. associates/assistants and GP retainees). Where they could be compared the demographic pattern was almost identical for the respondents and the whole Scottish database.Figure However, although 66.7% women are described as being 'full-time' in terms of the old GMS contract, in fact only 37.3% were paid a full share of partnership profits compared with 75.7% of males. "Full-time" males >26 hours) provided 8.1 sessions of GMS while their female equivalents provided 7.5 sessions (p < 0.001). Table 6 hours pTable Table Of those GP principals who were not planning to retire in the following 2 years, 73 (3.1%) planned to increase their sessions by an average of 1.5 sessions per week and 288 (13.3%) planned to decrease their sessions by an average of 2.0 sessions a week. Women were more likely than men to be planning an increase in sessions (p < 0.05). 178 (7%) planned to retire in the next two years. Doctors of both sexes planned to retire on or before 60. The mean planned age of retirement for women was 58.8 and for men 59.9.Responses from non-principals reflected the type of post they held. When appropriate, data is presented grouped then broken down by the larger groups of non-principal type.The age-sex distribution of the respondents is shown in table Table Respondents contributed an average of 5.15 (SD2.49) sessions per week to clinical general practice. Exclusive GP retainers contributed on average 3.5 (SD 0.8) and exclusive locums 5.7 (SD 2.6) sessions per week. Men provided on average 5.8 (SD 2.8) sessions and women 4.9 (SD 2.3) sessions per week. Doctors over the age of 35 infrequently worked more than 5 sessions.36 (14.8%) men and 66 (13.3%) women undertook other NHS work outside of general practice mainly hospital sessions and teaching. Those that took part in other NHS work spent on average 2.8 sessions per week on it.More men (66 (28.6%)) than women (72 (15.1%)) undertook non-NHS sessions (p < 0.001). These were very wide ranging, the commonest being occupational medicine (32), DSS medical work (26), prison medical work (4) and sports medicine (4). Those that took part in non-NHS work spent on average 10.5 sessions per month on it.Respondents were asked what their likely career intentions were in the next five years. There were significant differences in career intention between men and women, while the majority of both sexes had decided against a traditional partnership career path a greater proportion of women were intending to become partners (196 (39.6%) v 72 (29.5% p <0.01). Relatively more men opted to remain as locums or leave the NHS. However decisions to leave GP, NHS and Medicine were heavily weighted by those intending to retire (22 respondents) who were mainly male. Twelve respondents gave reasons for leaving general practice related to the stress of the job, the remainder intending career changes.Respondents were asked if they thought their commitment to general practice was likely to change in the next 5 years. Most thought it would remain the same, however, women were more likely than men to think it would increase (125/439 (28.5%) v 38/198 (19.2%), p < 0.01).Clinical commitment to non-GP NHS and non-NHS roles was considered likely to remain stable overall.Clearly these figures rely exclusively on self report of work-load. Doctors like most people are unlikely to minimise their workload, but unlike other surveys which have been carried out specifically with calculation of remuneration in mind this was an anonymous survey which sought to inform the need for future GP training. Previous work in this area in Scotland has been dogged with a poor response . ResponsOur data seem to suggest that the previous assumption that full-time doctors worked nine GMS sessions is an over-estimate and this is particularly true for women doctors. Overall doctors provide 7.4 clinical GMS sessions and an additional 0.9 session of NHS, educational or research related work. In comparison with previous surveys the perception of time actually spent on GMS appears to have recently increased [Over recent years the rise in non-principals  has been considered to be boosting the work force, however, their average GMS commitment is 5.15 sessions per week. This data includes accurate data on the contribution of GP locums for the first time. Perhaps of some concern is the fact that most of these largely female doctors do not see themselves following the partnership route in general practice, but intend to stay on as locums or assistants of some kind. This clearly will have implications for the way general practice is managed with the current structure of equal partnerships giving way to the models seen in the legal and accountancy sectors of a small number of executive partners aided by salaried assistants. It appears that this latter group are likely to be dominated by female doctors. This has also been found in other European countries ,13.Additionally and perhaps more worryingly, women are contributing only about 60% of the activity of men in aspects of general practice involved with its development such as training, teaching and research. It is not clear why women are not more involved in non-GMS activities. This may be down to a prioritisation between home and work life, active discrimination or unintentional "glass-ceiling" effects. It is a phenomenon described by other researchers  and poseGiven that most of the current GPs in Scotland intending to retire within the next 10 years  are likely to be replaced by a mainly female and less than full time work force there are concerns about the numbers of doctors currently being trained and retained in general practice in Scotland . With the likely medical shortfall expected to start to become apparent about five years from now, hard choices on what constitutes medical primary care may have to be made. It is likely that the generic family practitioner of today is going to become a luxury. Such concerns are also being voiced internationally where women are becoming an increasing part of the general practice workforce -15.It is not clear why women opt for part-time working. There is evidence that women are more likely to change their preferred career to general practice than men . A desirThe Kerr report has emphWomen doctors in primary care in Scotland work fewer hours in all age groups than their male counterparts. The rapidly increasing proportion of women in general practice may lead to an increasing shortfall of medical availability in the future if current work patterns are maintained. Further longitudinal research is required to establish this and man-power planning is required now to address this. More worryingly auxiliary activities such as teaching and administrative duties are not being taken up by women. This may have serious implications for the future development of the specialty in Scotland.GP General PractitionerGMS General Medical ServicesNHS National Health ServiceAll the authors are currently or have been employed by NHS Education for Scotland, the organisation responsible for GP training in Scotland.BM conceived and planned the research, helped analyse the data and wrote the paper. IC, entered and analysed the data and helped write the paper, KE distributed and helped design the questionnaires, entered the data, and proof-read the paper. Colin Hunter helped with recruitment and helped to plan the research and design the questionnaires. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Questionnaire for principalsClick here for fileQuestionnaire for non-principalsClick here for file