The prediction of ancestral protein sequences from multiple sequence alignments is useful for many bioinformatics analyses. Predicting ancestral sequences is not a simple procedure and relies on accurate alignments and phylogenies. Several algorithms exist based on Maximum Parsimony or Maximum Likelihood methods but many current implementations are unable to process residues with gaps, which may represent insertion/deletion (indel) events or sequence fragments.Here we present a new algorithm, GASP , for predicting ancestral sequences from phylogenetic trees and the corresponding multiple sequence alignments. Alignments may be of any size and contain gaps. GASP first assigns the positions of gaps in the phylogeny before using a likelihood-based approach centred on amino acid substitution matrices to assign ancestral amino acids. Important outgroup information is used by first working down from the tips of the tree to the root, using descendant data only to assign probabilities, and then working back up from the root to the tips using descendant and outgroup data to make predictions. GASP was tested on a number of simulated datasets based on real phylogenies. Prediction accuracy for ungapped data was similar to three alternative algorithms tested, with GASP performing better in some cases and worse in others. Adding simple insertions and deletions to the simulated data did not have a detrimental effect on GASP accuracy.GASP  will predict ancestral sequences from multiple protein alignments of any size. Although not as accurate in all cases as some of the more sophisticated maximum likelihood approaches, it can process a wide range of input phylogenies and will predict ancestral sequences for gapped and ungapped residues alike. Many eve.g. ,3). In oe.g. [Nevertheless, predicting ancestral sequences is not a simple procedure. It relies on a quality alignment plus an accurate – and correctly rooted – phylogenetic tree. Strict consensus methods are quick but can suffer from over-representation of larger clades of related sequences, which contribute more sequences to the consensus than more sparsely populated clades. Maximum Parsimony (MP) methods  overcomee.g. -8) and ie.g.  and FASTe.g.  provide e.g. .GASP  is an ancestral sequence prediction algorithm that is designed to handle gapped alignments of any size using a combination of MP and likelihood methods. Although probably not as accurate as some of the more sophisticated maximum likelihood approaches, it permits the estimation of ancestral states at residues that are gapped in any sequences of the alignment with comparable accuracy to that of ungapped residues.et al. 1992 [.GASP uses input from three sources: a multiple sequence alignment (MSA); an accompanying phylogenetic tree in Newick format ; and a Pal. 1992 . SequencGASP outputs an alignment in fasta format with both input terminal sequences and predicted ancestral node sequences. Ancestral sequences can either be grouped together at the end of the file or interspersed throughout the terminal sequences to reflect the tree topology Figure . Three tpX is the likelihood for a PAM distance of X , i is the ancestral amino acid at position r,j is the descendant amino acid at position r, pijX is the substitution probability of i to j in a PAMX matrix, and N is the number of residues in the alignment. Substitutions involving gaps are ignored in this calculation.where This allows a visual comparison between the branch lengths of the input phylogeny and the predicted branch lengths given the ancestral sequence predictions.r, GASP starts at the tips and works deeper into the tree, assigning a probability of a gap at each node n, which is equal to the mean probability of a gap at the descendant nodes:If the MSA has gaps, GASP will first assign gap status to every residue at every node. Insertions and deletions are assumed to be equally likely, although a gap is assigned in the case of a tied probability (below). For each residue p is the gap probability for residue r at node n. p1 and p2 are the gap probabilities for r at the two descendant nodes.where r is fixed as a gap, otherwise r is fixed as a 'non-gap'. GASP then works back up the tree from the root, this time using the new ancestral gap probability and both descendant gap probabilities to recalculate the gap probability:Terminal branches are given a probability of 1 if a gap is present or 0 if not. Once the root is reached, the gap status is fixed for the root. If the probability of a gap is greater than or equal to 0.5, residue p0 is the gap probability for r at the ancestral node.where r is fixed as a gap if p ≥ 0.5. This continues until all nodes are assigned as 'gap' or 'non-gap'.As with the root, r is assigned a probability for each amino acid at each node n. At the tips, r has a probability of 1 for the amino acid that is present in the MSA. GASP then works down the tree assigning probabilities based on the descendant nodes, branch lengths and a substitution matrix. By default, the PAM matrix of Jones et al. 1992 [1 matrix, which represents the probability that a given amino acid will be substituted by each other amino acid when the mean substitution rate is 1/100 residues. To make a PAMX matrix, which represents a length of evolutionary time where a sequence will have undergone X substitutions per 100 residues, the PAM1 matrix is multiplied by itself X-1 times:Once gaps are assigned, ancestral sequences are predicted in a similar fashion. Each residue al. 1992  is used.i is the ancestral amino acid,j is the descendant amino acid, k is the 20 possible transitory amino acids, pijX is the substitution probability of i to j in a PAMX matrix, pik(X-1) is the substitution probability of i to k in a PAM(X-1) matrix and pkj1 is the substitution probability of j to k in a PAM1 matrix.where r, the ancestral probabilities for each amino acid are calculated for the two descendant branches individually, using a PAMX matrix, where X is 100 times the branch length as substitutions per site, i.e. a branch of 0.1 substitutions per site would use a PAM10 matrix:Unless the ancestral node has a gap  at position pi is the probability of amino acid i at residue r of node n, pijX1 and pijX2 are the probabilities of substitution from amino acid i to each amino acid j in the appropriate PAM matrix for the two descendant branches, pdj1 and pdj2 are the probabilities of amino acid j being at position r at the two descendant nodes.where Once the root is reached, the most probable amino acid is fixed as the ancestral sequence. As with gaps, GASP then works back up the tree, using the fixed ancestral node amino acid and the descendant node probabilities to give new probabilities for each amino acid. The most probable amino acid is then fixed and the process continues until all residues and all nodes have a fixed sequence.GASP is primarily designed for reasonably small trees (6–30 sequences), although there is no limit on input tree size. For larger trees, probabilities for each amino acid get very small near the root, which can lead to a heavy bias towards the fixed ancestral amino acid when GASP works back up the tree. To counter this GASP arbitrarily reduces any probabilities below a certain exclusion threshold (0.05 by default) to zero, thus reducing the slow accumulation of very unlikely amino acids.To optimise the PAM matrices used for probability calculations, GASP uses the variable branch lengths read from the input phylogeny. There is also an option to fix the PAM distance used for all branches, which would allow the use of trees without branch lengths.n and its direct ancestor n0. This ancestor itself is heavily influenced by the descendants of n but also by the 'outgroup' to n, namely those sequences that are descendant to n0 but not to n. The outgroup information contained by the ancestral node n0 can be vital in determining the correct sequence for n when the descendants of n are variable. For this reason, the GASP algorithm will, by default, fix ancestral sequences as it moves back 'up' the tree from the root, increasing the relative weighting of the outgroup to the two descendants. Because there is a chance of the wrong amino acid sweeping back up the tree , there is an option to use amino acid probabilities from the ancestral node in the last stage of GASP rather than giving the fixed amino acid an ancestral probability of 1. This option should be used with caution.Assignment of ancestral amino acids with the GASP algorithm is achieved by combining data from the descendants of a given node To test the GASP algorithm, a number of artificial phylogenies were simulated. Because there is a practically limitless number of possible tree sizes (in both numbers of sequences and branch lengths) and phylogenies, it was decided to test the algorithm on a set of simulated phylogenies based on real phylogenies that formed a subset of those for which the algorithm was originally written. This set comprised 94 neighbour-joining trees of protein families. Each tree contained at least two subfamilies of at least 3 members each, giving in total between 6 and 127 sequences. Simulations started by creating a random protein sequence 100 amino acids long. Each residue was assigned an amino acid randomly as determined by the amino acid frequencies in all the human sequences of SwissProt-TrEMBL (Release 42) . Sequencet al. 1992 [Three substitution methods were used. In the first 'PAM Equal Rates' model, the PAM1 matrix of Jones al. 1992  was usedBecause one of the main advantages of GASP is its ability to deal with gaps, a second test dataset was generated from the 'PAM Equal Rates' set of trees, this time with gaps added. The addition of gaps was kept simple so that the exact same trees could be used for the gap analysis, allowing direct comparison of the results with gaps and without.  To do this, gaps were limited to single insertion/deletion ('indel') events per column of the MSA, allowing them to overlay onto the existing simulated 'PAM Equal Rates' data. In addition, no indels occurring next to root were allowed as it is impossible to judge without an outgroup whether such an event would be an insertion or deletion.r of the simulated sequences was considered in turn and had a probability of 50% of containing an indel. Gaps were all of length 1 . Although unrealistic for testing multiple alignment or phylogeny reconstruction programs, such a simplification is not a problem for ancestral sequence prediction as each residue is treated independently. The short gaps meant that, for the same total number of gapped residues, there is a higher diversity in the phylogenetic positioning of the indels.To make the gaps, each residue T between the tip (age 0) and the oldest direct descendant node from the root. A random branch (not leading to root) is then selected for which the ancestral node is older than T and the descendant node is no older. This is the branch on which the indel occurred. The indel is randomly assigned as an insertion or deletion event with equal probability. If it is an insertion then the ancestral node plus all nodes outside the descendant clade have residue r replaced with a gap. If it is a deletion then the descendant node and all its descendants have residue r replaced with a gap.Indels were placed randomly with respect to evolutionary time. Each node in the simulated data has an 'age', which is the number of rounds of potential substitution it took to complete the simulation after that node was formed. Each indel occurs at a random age The simulated trees and alignments were run through the GASP algorithm. Because the 'real' sequence of each simulated node was known, it was then possible to determine the accuracy of GASP predictions. To test the different parts of the GASP algorithm, predictions were also made using modified GASP algorithms with parts of the model excluded.Because prediction for invariant sites is trivial for all methods, the expectation is that accuracy is inversely related to the number of variable sites. Therefore, comparisons of methods are presented as a percentage of the variable sites. In this context 'variable sites' are defined independently for each node as those sites for which not all descendant nodes (including termini) have the same sequence as the ancestral node.The simulated phylogenies are of different sizes. Considering all nodes of all trees would bias results towards the larger trees. To avoid this, each tree was arbitrarily reduced to four representative nodes:1. 'Root' = The root of the tree.2. 'Near Root' = A direct descendant node of the root.3. 'Mid Tree' = A random node approx. midway in the tree.4. 'Near Tip' = A direct ancestral node of a terminal sequence.et al. 1995 [et al. 2000 [To determine whether the GASP algorithm was useful its performance was compared to a crude consensus sequence at each node. Where two amino acids were present at equal frequencies in a column of the MSA, the most frequent amino acid in the total MSA was selected for the ancestral sequence. GASP may be considered crude compared to some existing Maximum Likelihood approaches and so its performance was also compared to that of both ML algorithms implemented by the CODEML program from the PAML package , namely al. 1995  and the al. 2000 . In addiThe GASP model marginally out-performs all methods tested for constructing the ancestral sequence at the root of the tree Figure . For allet al. 1995 and Pupko et al. 2000 performed better overall for internal nodes, this difference was not seen for every node of every tree. At each level, GASP is sometimes better and sometimes worse than all three other algorithms  using fixed PAM matrices rather than matrices derived from observed tree branch lengths.(b) fixing ancestral sequences on initial pass towards root without a second pass back up the tree.(c) no filtering of rare amino acid probabilities.(d) using ancestral probabilities when working back up the tree rather than fixed ancestral amino acids.Elements (a) and (b) were chosen for testing because they increase computational time, while (c) and (d) may not intuitively give the best results.For the phylogenies used in these simulations, all four variants performed worse than the standard GASP algorithm (data not shown). Using a fixed PAM distance for all branches rather than approximating the PAM distance using tree branch lengths (a) gives an unfair weighting to long branches and thus increases the probability of substitutions that are, in reality, unlikely. Fixing ancestral sequences on the way 'down' the tree to the root (b) does not use any outgroup information and is therefore significantly worse at distinguishing between two or more amino acids with similar ancestral probabilities. Less intuitive is the effect of reducing low amino acid probabilities to zero (c) and using fixed ancestral sequences when recalculating amino acid probabilities using all three connected nodes (d). Indeed, excluding these two elements have a much smaller effect but still reduce the overall accuracy of the algorithm (data not shown).i.e. combining (c) and (d)).Using fixed amino acids when working back up the tree increases the influence of the outgroup sequence. As was seen by the difference in accuracy between predictions at the root and nodes near the root Figure , outgroui.e. not correcting for multiple substitutions.) This is not testing the GASP algorithm per se but does provide information on the importance of using an accurate phylogeny construction algorithm. (The PAML package does not require pre-defined branch lengths and is therefore only affected by errors in supplied topology and not in branch lengths.) In many cases there was no difference. However, nearer to the root, using observed branch lengths rather than the real ones decreased prediction accuracy slightly. This decrease was correlated with total tree age (data not shown). This would imply that branch lengths corrected for multiple substitutions should be used for trees fed into the GASP algorithm, particularly with deep trees containing long branches.A final test was performed to compare the use of 'real' versus 'observed' branch lengths.  To analyse the effect of gaps on prediction accuracy, pairwise comparisons were made between the gapped datasets and the corresponding ungapped simulations Figure . As woulAlthough explicitly designed for use with protein sequence alignments and trees, it is relatively simple to convert GASP for use with nucleotide datasets. To do this, a new 'PAM matrix' should be created with substitutions probabilities for A, C, G and T only. This structure would allow the user to fit fairly complex substitution models, with different substitution probabilities for each pair of nucleotides. If the aligned sequence is coding DNA, however, it is highly recommended to use the protein sequences or a different algorithm such as those in the PAML package , as the We have presented an algorithm for predicting ancestral sequences in gapped datasets. At the root of the tree, GASP marginally outperforms three existing algorithms implemented in the PAML package. For other nodes of the tree, however, the ML algorithms of CODEML -7 generaFor real life datasets, as for all evolutionary studies, predictions are dependent on the quality of input alignments. Gapped residues are, by their nature, often located in regions of evolutionary instability and therefore the interpretations of predictions at such sites require extra care. In many scenarios, however, gaps are introduced into alignments by the missing termini of fragment sequences. In these situations, the complete sequences that form the rest of the alignment may be very well aligned and so it is highly desirable to have an algorithm that can process the gaps introduced by the truncated sequences.Project name: GASP Project home page: Operating system(s): Platform Independent. (Tested on PC (Windows 98/XP) and UNIX (Red Hat Linux 7.3))Programming language: Perl.Other requirements: None.License: None.Any restrictions to use by non-academics: Author's permission required.GASP. Gapped Ancestral Sequence Prediction.Indel. Insertion or deletion event.ML. Maximum Likelihood.MP. Maximum Parsimony.MSA. Multiple Sequence Alignment.PAM. Point Accepted Mutation.RE conceived the algorithm, coded the Perl script, designed and performed the accuracy tests and statistical analyses, designed the phylogeny simulation method, generated the simulated datasets and drafted the manuscript. DS helped in the design of test simulations and in drafting the manuscript.


Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or “noise.” Several recent studies in  Analysis of gene expression data for nearly every gene in yeast provides evidence that random variation in the production rate of proteins could significantly affect the fitness of an organism Stochasticity is a ubiquitous characteristic of life. Such apparent randomness, or “noise,” can be observed in a wide range of organisms, resulting in phenomena ranging from progressive loss of cell-cycle synchronization in an initially synchronized population of microbes to the pattern of hair coloration in female calico cats. An important source of stochasticity in biological systems is the random noise of transcription and translation, which can result in very different rates of synthesis of a specific protein in genetically identical cells in essentially identical environments .Understanding how stochasticity contributes to cellular phenotypes is important to developing a more complete picture of how cells work. Accordingly, noise in gene expression and other cellular processes has been a major focus of research for more than a decade. While several cases have been described where stochasticity is advantageous  maxTo test this prediction, we estimated protein production rates . Similar results were found when using different numbers of bins, when using halves or quartiles instead of thirds, or when separating bins by transcription rate instead of by number of translations per mRNA (data not shown). This result cannot be explained by the overall positive correlation between dispensability and rate of protein synthesis. In the first of these two methods, we binned yeast genes by their protein production rate, so that all genes in each of 15 bins had approximately equal levels of protein production see A. A Fishf, where f = 0 indicates no effect on growth when a gene is deleted, f = 1 indicates that a gene is essential, and 0 < f < 1 indicates a quantitative growth defect [f versus transcription [txn] rate | protein production rate, Spearman partial r = 0.282, n = 4,746, p = 10−87; f versus translations [tlns] per mRNA | protein production rate, Spearman partial r = −0.258, n = 4,746, p = 10−75). We also expected that the relationship between gene importance and implementation of the expression strategy that minimizes noise could additionally be seen by considering transcription rate and translation rate per mRNA together, as a ratio; a large ratio of transcription rate to translations per mRNA would indicate that transcripts are produced quickly but are translated slowly, corresponding to our expression strategy 1. Confirming this, the correlation between fitness effect and the ratio of transcription rate to translations per mRNA (controlling for protein production rate) is highly significant . Partial correlation analysis is thus in accordance with the trend illustrated in Because binning genes still allows for a small amount of variability in protein production within each bin see , we sough defect ) would cp ≤ 0.02) for all but one bin. As in In addition to essential genes, genes whose protein products participate in stable protein complexes (“complex subunits”) would also be expected to exhibit sensitivity to randomness in expression: producing too little or too much of a single protein complex subunit can compromise the proper assembly of the entire complex and waste the energy invested in the production of the other complex subunits. In support of this, it has been found that both under- and overexpression of complex subunits is more likely to result in a reduced growth rate or inviability of yeast than is misexpression of other genes, and also that complex subunits tend to be more precisely coexpressed with other genes than noncomplex subunits . Using dr = 0.203, n = 4,900, p = 10−46) and a low number of translations per mRNA . Using the ratio of transcription rate to translations per mRNA also yielded similar results . Thus, partial correlations confirm the finding illustrated in When we repeated the partial correlation analysis for complex subunits , we found similar results. When total protein synthesis was controlled for with the partial correlation, complex subunits were more likely to have a high transcription rate , suggesting that fitness effect and protein complex membership are independently associated with the expression strategy that minimizes stochastic fluctuation.  Repeating the partial correlations above with either transcription rate or translations per mRNA in place of their ratio gave significant partial correlations with both fitness effect and protein complex membership as well (data not shown).Since proteins that participate in many protein–protein interactions are more likely to be essential , it was r = −0.11, p = 10−9) between an mRNA's rate of decay and the evolutionary rate of the protein it encodes. This correlation was surprising, as it is precisely the opposite of what one would expect if the relationship between the rates of mRNA decay and protein evolution were mediated by the level of expression: slow decay would result in increased expression, which is known to be associated with slow evolution .We found that noise in protein production is minimized in genes for which it is likely to be most harmful, specifically essential genes and genes encoding protein complex subunits. This finding supports the hypothesis that noise in gene expression is generally deleterious to yeast.Yeast appear to control the noise in their gene expression at both transcriptional and translational levels preferentially for some genes; however, this noise minimization is not without a cost, as the high transcription and high mRNA decay rates that are needed to minimize noise are energetically expensive and are thus expected to be advantageous only when the benefit of reducing noise in a particular gene's expression outweighs this cost . ProteinAs is the case with many genome-wide studies, it is possible that a hidden variable could bias our results. For example, it is possible that essential genes and genes encoding protein complex subunits tend to have high transcription and low translation for reasons unrelated to noise minimization. However, until such a reason is identified, the most parsimonious interpretation of our results is that yeast adaptively minimize noise in the expression of certain genes.Escherichia coli  * A/T, where R is transcription rate, A is mRNA abundance, and T is mRNA half-life. Translation rates per mRNA in rich glucose medium were calculated from ribosome occupancy data by Transcription rates were calculated from mRNA abundances and decay rates in log-phase yeast growing in rich glucose medium  accordinFitness effect ranks were calculated from 12 replicate growth experiments for all viable homozygous yeast deletion strains in rich glucose medium; growth experiments were conducted using the method described in ADH1*) was 10-fold weaker than the other two at full induction, but all three showed very similar relationships between noise strength and percent transcriptional induction. Since we do not have genome-wide data for the percent induction for genes in rich glucose medium (or any other environment), in our analysis we make the assumption that the promoters of more highly transcribed genes tend to be at higher percent induction levels. While this certainly does not hold for all genes, we believe that it is a reasonable approximation for most genes.XYr be the correlation coefficient between variables X and Y. To control for a third variable Z,Partial correlations were calculated as described by t distribution, by the equationTo assess the significance of the partial correlation, it is transformed to be distributed according to a Student's p-value can then be calculated according to where the t-value falls with respect to its expected distribution.The two-sided Figure S1–49).Fitness effect ranks are shown on the y-axis . Protein production rate (proteins/s) is shown on the x-axis. The Spearman rank correlation coefficient is r = –0.202 (p = 10(316 KB PPT).Click here for additional data file.Table S1(37 KB DOC).Click here for additional data file.


Relatively little is known about interest in pediatric pulmonology among pediatric residents. The purpose of this study, therefore, was to determine at this institution: 1) the level of pediatric resident interest in pursuing a pulmonary fellowship, 2) potential factors involved in development of such interest, 3) whether the presence of a pulmonary fellowship program affects such interest.A questionnaire was distributed to all 52 pediatric residents at this institution in 1992 and to all 59 pediatric residents and 14 combined internal medicine/pediatrics residents in 2002, following development of a pulmonary fellowship program.Response rates were 79% in 1992 and 86% in 2002. Eight of the 43 responders in 1992 (19%) had considered doing a pulmonary fellowship compared to 7 of 63 (11%) in 2002. The highest ranked factors given by the residents who had considered a fellowship included wanting to continue one's education after residency, enjoying caring for pulmonary patients, and liking pulmonary physiology and the pulmonary faculty. Major factors listed by residents who had not considered a pulmonary fellowship included not enjoying the tracheostomy/ventilator population and chronic pulmonary patients in general, and a desire to enter general pediatrics or another fellowship. Most residents during both survey periods believed that they would be in non-academic or academic general pediatrics in 5 years. Only 1 of the 106 responding residents (~1%) anticipated becoming a pediatric pulmonologist.Although many pediatric residents consider enrolling in a pulmonary fellowship (~10–20% here), few (~1% here) will actually pursue a career in pediatric pulmonology. The presence of a pulmonary fellowship program did not significantly alter resident interest, though other confounding factors may be involved. The specialty of pediatric pulmonology is relatively new, having been recognized as a pediatric sub-specialty by the American Board of Medical Sub-specialties in 1984. In 1997, there were approximately 500 board certified pediatric pulmonologists in the United States and Canada . It has This study involved the distribution of a questionnaire to all pediatric residents. The questionnaire was initially distributed in 1992 prior to institution of a pulmonary fellowship. The questionnaire was placed in the hospital mailbox of each resident. The study was repeated (and the questionnaire redistributed) in 2002 after the fellowship, which began in 1994, had been functioning for several years. To improve the response rate, the questionnaire was distributed twice, one month apart, during each time period. The questionnaire was a three-page, 18 question form that took approximately 15 minutes to complete (copy enclosed in Appendix see ). The quThe questionnaire was distributed to 52 pediatric residents (including 2 chief residents) in 1992 and to 59 pediatric residents (including 3 chief residents) and 14 medicine/pediatric residents (including 1 chief resident) in 2002. A medicine-pediatric residency program did not exist during the initial distribution period. To avoid compromising confidentiality in the relatively small medicine/pediatric group, residents were not asked to list their residency program in 2002 and consequently the 2 resident groups during that period were combined. Forty-three of the 52 residents completed the survey in 1992 (79%) compared to 63 of 73 (86%) during 2002 (p = NS).Of the 43 respondents in 1992, 30 (70%) had considered doing a fellowship in any pediatric subspecialty and of those, 15  believed they were "very likely" to do a fellowship. Of the 63 respondents in 2002, 40 (63%) had considered doing a pediatric fellowship and 16 of those  were "very likely" to continue with fellowship training. These numbers compare with 9 of the 52 residents in 1992 (17%) who actually completed fellowship training compared to 16 of the 47 graduating residents from the 2002 survey (34%) who began fellowship training in 2003 or 2004.Eight residents 19%) in 1992 had considered a pulmonary fellowship compared to 7 residents (11%) in 2002 (p = NS). Table 9% in 199Table The last question in the survey asked residents what they thought they would be doing 5 years in the future. These responses are shown in Table This study found that a significant percentage of pediatric residents considered doing a pulmonary fellowship after their residency training, ranging from 11% in the 2002 group to 19% in the 1992 group. However, these residents also viewed themselves as less likely to enter general pediatrics perhaps suggesting that they were simply considering several pediatric subspecialties at some time during their residency training. This seems likely, as the highest scored factor by the +PF residents was the desire to continue their education after residency. Despite fairly high percentages of residents considering a pulmonary fellowship, only 1 resident in the entire group (~1%) actually believed that they would be a pediatric pulmonologist 5 years after the survey was completed. This percentage is very similar to that of first-time takers of the 1995 General Pediatrics Certifying Examination who believed they would be a pediatric pulmonologist in the future (1.1%) . If one The majority of residents who considered doing a pulmonary fellowship (8 of 15) were in their first year of residency. From personal experience, residents often consider various practice options early on in their training and frequently do not tend to narrow their choices until their second or third year of residency. This observation should be kept in mind when trying to recruit residents for pulmonary fellowship positions by seeking out those residents potentially interested in Pulmonology early on in residency rather than later.The ranking of factors that may contribute to an interest in a pulmonary fellowship were remarkably similar during the 2 time periods. The only score that approached a statistically significant difference was the statement "I enjoy the tracheostomy/ventilator population" and this score tended to increase in 2002. However, a dislike for the tracheostomy/ventilator population also received the highest score among those residents not interested in a pulmonary fellowship and was even higher than both the desire to enter general pediatrics and another fellowship program. These data may simply be a center phenomenon but might suggest a more global "disinterest" in this patient population that may need to be further studied. Two scores in the -PF resident group decreased from 1992 to 2002: not enough pulmonary patient experience to decide on a pulmonary fellowship and too few perceived pulmonary job openings. The first may be a positive reflection on the local resident experience with pulmonary patients in recent years or on the fellowship program itself though this is only speculative. The second received very low scores during both periods and may not be very relevant. However, there has been some evidence of a reversal in the prior trend of residents entering general pediatrics in recent years, suggesting greater interest among residents in fellowships . On the This study has certain shortcomings. This study asked residents to score specific factors that may or may not have been relevant to an individual resident. Although residents were given the opportunity to add personal comments, few did. In addition, certain patient populations, e.g those with respiratory infections, were not included as options in the survey and these omissions could cause study bias. Other factors that may contribute to residents' decisions regarding fellowship training were not addressed in this study. These factors include resident teaching by the faculty, resident gender, spouse occupation, mentor encouragement or other personal reasons -12. In aAlthough many pediatric residents consider enrolling in a PF (~10–20% here), few (~1% here) will actually pursue a career in pediatric pulmonology. The presence of a PF program did not significantly alter resident interest, though other confounding factors may be involved.BPD: bronchopulmonary dysplasiaPF: pulmonary fellowship+PF: those residents who considered taking a pulmonary fellowship-PF: those residents who had not considered taking a pulmonary fellowshipNone declared.The pre-publication history for this paper can be accessed here:Appendix. Resident questionnaireClick here for file


The human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional coactivator p300, a necessary step in Tat-mediated transactivation. We report here that Tat is deacetylated by human sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent class III protein deacetylase in vitro and in vivo. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV promoter. Conversely, knockdown of SIRT1 via small interfering RNAs or treatment with a novel small molecule inhibitor of the SIRT1 deacetylase activity inhibit Tat-mediated transactivation of the HIV long terminal repeat. Tat transactivation is defective in SIRT1-null mouse embryonic fibroblasts and can be rescued by expression of SIRT1. These results support a model in which cycles of Tat acetylation and deacetylation regulate HIV transcription. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional coactivator during Tat transactivation. Cycles of Tat acetylation and deacetylation, mediated by human sirtuin 1 (SIRT1), regulate HIV transcription suggesting that SIRT1 could be a therapeutic target The Tat protein of human immunodeficiency virus 1 (HIV-1) is essential for the transcriptional activation of the integrated HIV-1 provirus. Without Tat, HIV transcriptional elongation is inefficient and results in abortive transcripts that cannot support viral replication . Tat is trans-acting responsive element (TAR), an RNA stem-loop structure that forms at the 5′ end of all viral transcripts ) C. These This model was further tested in nuclear microinjection experiments using synthetic full-length Tat and AcTat. Microinjection of increasing amounts of either Tat or AcTat proteins into HeLa cells caused a marked transactivation of the HIV LTR luciferase reporter in a dose-dependent manner  of less than 5 μM  open reading frame in place of the viral nef gene  -acetylated histone H3 peptide (amino acids 1–24) [+ for 10 min at room temperature prior to the initiation of the reaction by addition of NAD+ (1 mM).The histone deacetylation assay with recombinant SIRT1  was performed as described previously for SIRT2  in 100 μds 1–24) . Splitom2PO4 (pH 7.5), 5 mM EDTA, 30 mM sodium pyrophosphate, 10 mM NaF, and protease inhibitors . Duplicates were pooled, and 1 mg of lysate was immunoprecipitated either with monoclonal α-HA  together with protein G-Sepharose  with α-FLAG M2 agarose (Sigma) or α-T7-agarose (Amersham Biosciences) for 2 h at 4 °C. Beads were washed three times in lysis buffer, boiled in SDS loading buffer, and analyzed by WB with polyclonal α-FLAG (Sigma), monoclonal α-HA (Roche), or monoclonal α-T7  antibodies. For the IP of Tat with endogenous SIRT1, HEK 293 cells were transfected only with CMV-Tat/FLAG or the CMV-empty vector using Lipofectamine reagent. Cell lysates were immunoprecipitated with rabbit α-SIRT1 antibodies (generated against amino acids 506–747) together with protein G-Sepharose (Amersham Biosciences). Immunoprecipitated material was analyzed by WB with the M2 α-FLAG antibody (Sigma) or rabbit α-SIRT1 antibodies.HEK 293 cells were cotransfected in duplicate with expression vectors for CMV-Tat/FLAG; CMV-Tat/T7 or CMV-TatK50R/FLAG; and the SIRT1/HA or SIRT1-, SIRT2-, and SIRT6-FLAG expression vectors or the respective empty vector controls using Lipofectamine reagent (Invitrogen). Cells were lysed after 24 h in 250 mM NaCl, 0.1% NP40, 20 mM NaHFor in vitro interactions, 10 U of recombinant SIRT1 (Biomol) was incubated with biotinylated synthetic Tat or acetylated Tat proteins  together with streptavidin-Sepharose (Amersham Biosciences) in lysis buffer in the presence of 5 mM nicotinamide (Sigma) for 3 h at 4 °C. Pelleted beads were washed three times in lysis buffer, resuspended in SDS loading buffer, and analyzed by WB with polyclonal α-SIRT1 antibodies, rabbit α-AcARM, or SA-HRP (Jackson Immunoresearch Laboratories).Double-stranded siRNAs directed against nucleotides 408–428 in the SIRT1 mRNA or control GL3 siRNAs  were transfected into HeLa cells plated in six-well plates with Oligofectamine reagent according to the manufacturer's guidelines (Invitrogen). The mutant SIRT1 siRNA was identical to SIRT1 siRNA except for a two-nucleotide mismatch between the target mRNA for SIRT1 and the antisense strand of siRNA at nucleotides 418 and 419. After 48 h, cells were retransfected with the HIV LTR luciferase construct (200 ng) together with increasing amounts of CMV-Tat expression vectors  and corresponding amounts of empty pcDNA3.1 vector (Invitrogen). In the control experiment, CMV-Tat was replaced by the CMV-luciferase construct, and HIV LTR luciferase was replaced by an HIV LTR promoter construct driving the expression of chloramphenicol acetyl transferase (CAT) ). Cells ΔNF-κB-luciferase reporter (200 ng) and increasing amounts of RSV-Tat  using the Lipofectamine reagent (Invitrogen). In the control experiment, RSV-Tat was replaced by RSV-luciferase (200 ng), and the HIV LTR luciferase construct was replaced by the HIV LTR CAT reporter. In transfections with HR73, HeLa cells were cotransfected with the HIV LTR luciferase reporter (200 ng) and RSV-Tat expression vectors  or the empty vector using Lipofectamine reagent. The RSV-luciferase construct was used as described above. After 4 h incubation with the DNA/Lipofectamine mix, the culture medium was changed and supplemented with indicated concentrations of HR73 dissolved in DMSO or DMSO alone. Cells were harvested 8 h later and processed for luciferase assays.In cotransfection experiments, human CMV-SIRT1 or CMV-SIRT1H363Y (600 ng) was cotransfected into HeLa cells plated in six-well plates with the HIV LTR luciferase reporter (200 ng) or the LTRSubconfluent MEFs (70%) were grown on Cellocate coverslips , and nuclear microinjections were performed at room temperature with an automated injection system (Eppendorf Micromanipulator 5171 together with Eppendorf Transjector 5246). Samples were prepared as a 20 μl injection mix containing the HIV LTR luciferase reporter or 5xUAS luciferase (each 100 ng/μl), RSV-Tat (10 ng/μl) or Gal4-VP16 (50 ng/μl), CMV-cyclinT1 (100 ng/μl), CMV-SIRT1 (100 or 300 ng/μl), together with CMV-GFP (50 ng/μl) in sterile water. At 6 h after microinjection, cells were examined under a Nikon Eclipse TE300 inverted fluorescent microscope  to determine the number of GFP-positive cells, washed in cold phosphate buffer, and stored at −70 °C for luciferase assays (Promega). In HeLa cells, synthetic Tat or AcTat proteins (each 30 or 100 ng/μl) were coinjected with the wild-type or mutant HIV LTR luciferase reporters (each 100 ng/μl) together with CMV-GFP (50 ng/μl), and harvested 4 h after injection. Cells were treated immediately after injection with DRB , TSA (400 nM), or nicotinamide (5 mM). Microinjections in siRNA-treated cells were performed 48 h after siRNA transfection.−/GFP containing the GFP open reading frame in place of the nef gene and a frameshift mutation in the env gene, as well as the method to generate pseudotyped viral particles with VSV-G, were previously described [5 Jurkat cells with different amounts of viral suspension. The titer of the viral stock was measured by flow cytometric analysis of GFP expression 48 h after infection. The pHR′-EF-1α/GFP construct is a minimal nonreplicative HIV-1 genome containing a heterologous promoter, EF-1α, driving GFP expression [−/GFP or pHR′-EF-1α/GFP viral particles at a theoretical multiplicity of infection of 0.5 in 24-well plates. Cells were repeatedly washed and resuspended in fresh medium containing HR73 (1 μM) or DMSO alone. Viral infection was monitored 36 h later by flow cytometry analysis using a Calibur FACScan .The HIV molecular clone HIV-R7/Eescribed . The numpression . Viral ppression . All vec


Iron and copper play an important role in oxidative mechanisms, producing the deleterious hydroxyl radical (*OH) that peroxides lipid membranes and damages DNA. Myeloperoxidase (MPO) and nitric oxide (NO) are known sources of free radicals and induce reduction of ferritin-Fe3+ into free Fe2+, contributing to oxidative damage.The exogenous administration of Insulin-like Growth Factor-I (IGF-I) induces hepatoprotective and antifibrogenic actions in experimental liver cirrhosis. To better understand the possible pathways behind the beneficial effect of IGF-I, the aim of this work was to investigate severe parameters involved in oxidative damage in hepatic tissue from cirrhotic animals treated with IGF-I . Fe and Cu were assessed by atomic absoption spectrometry and iron content was also evaluated by Perls' staining. MPO was measured by ELISA and transferrin and ferritin by immunoturbidimetry. iNOS expression was studied by immuno-histochemistry.Liver cirrhosis was induced by CClLiver cirrhosis was histologically proven and ascites was observed in all cirrhotic rats. Compared to controls untreated cirrhotic rats showed increased hepatic levels of iron, ferritin, transferrin (p < 0.01), copper, MPO and iNOS expression (p < 0.01). However, IGF-treatment induced a significant reduction of all these parameters (p < 0.05).the hepatoprotective and antifibrogenic effects of IGF-I in cirrhosis are associated with a diminution of the hepatic contents of several factors all of them involved in oxidative damage. Insulin-like growth factor-I (IGF-I) is an anabolic hormone produced in different tissues in response to growth hormone (GH) stimulation . Liver sIn order to give a better insight into the pathways by which IGF-I seems to exert its the hepatoprotective and antifibrogenic actions, this study was aimed at analyze several parameters involved in oxidative stress or inflammation in the liver, such as metals ions (iron and copper), iron transport and store proteins (transferrin and ferritin) and enzymes (myeloperoxidase -MPO- and inducible nitric oxide synthase -iNOS-) both in IGF-I treated and untreated cirrhotic rats.3+ into free Fe2+ contributing to oxidative damage [Metal ions, such as iron and copper, exhibit the ability to produce reactive oxygen species, resulting in lipid peroxidation, DNA damage, depletion of sulfhydryls and altered calcium homeostasis -19. Irone damage ,23.4 inhalation  twice a week for 11 weeks with a progressively increasing exposure time from 1 to 5 minutes. From that time until the 30th week rats were exposed to CCl4 once a week for 3 min. During the whole period of cirrhosis induction animals received Phenobarbital  in the drinking water (400 mg/L). Rats were housed in cages placed in a room with 12-hour light-dark cycle and constant humidity and temperature (20°C). Both food  and water were given ad libitum. Healthy, age and sex-matched control rats were maintained under the same conditions but receiving neither CCl4 nor Phenobarbital.Cirrhosis was induced as previously described ,12. BrieThe Guiding Principles for Research Involving Animals [All procedures were performed in conformity with  Animals .th -30th) of CCl4 exposure (from day 0 to day 22nd). In the morning of day 0, animals were weight and blood samples were drawn from the retroocular venous plexus from all rats with capillary tubes  and stored at -20°C until used for analytical purposes. Cirrhotic rats were randomly assigned to receive either vehicle   or recombinant human IGF-I    for three weeks. Control rats  received saline during the same period. The last dose of IGF-I was administrated the day 21st at 6 p.m.The treatment was administrated the last three weeks . The rest of liver samples were stored at -80°C.In the morning of the 22Bouin-fixed tissues were processed and sections (4-μm.) were stained with Haematoxylin and Eosin and Masson's trichrome. Liver cirrhosis was diagnosed according to the criteria previously described ,16. LiveImmunohistochemical staining of iNOS in paraffin sections (4 μm) was performed using an avidin-biotin peroxidase technique as described by Shu el al. , with soHepatic samples were homogenized in a Potter homogenizer in 7 volumes of cold buffer  containing 5 mM 2-mercaptoethanol, 0.5 μg/mL Leupeptin, 0.7 μg/mL pepstatin A and 100 μg/mL PMFS. Fibrous parts and unbroken cells debris were eliminated by centrifugion at 500 g for 5 min. Supernatans were used as the whole homogenate.® . Transferrin and ferritin were determined by immunoturbidimetry, using a Hitachi 710 autoanalyzer  and kits for clinical human, from the same laboratory. MDA was assessed after heating samples at 45°C for 60 minutes in acid medium. It was quantitated by a colorimetric assay using LPO-586 , which after reacting with MDA, generating a stable chromophore that can be measured at 586 nm . Total proteins were assessed by Bradford's method [MPO was measured by ELISA, using a commercial kit from BIOXYTECHs method .Representative samples (approximately 1 g. of each rat liver) were collected, weighed and later dried in stove (70°C) to constant weight. Iron and copper concentrations were determined by flame atomic absorption spectrophotometry  .P value < 0.05 was considered to be statistically significant. Calculations were performed with SPSS program version 6.0 .Data were expressed as mean ± SEM. To analyse the homogeneity among groups, Kruskall-Wallis test was used, followed by multiple post-hoc comparisons using Mann-Whitney U tests with Bonferroni adjustment. Any 4.Liver cirrhosis was histologically proven and ascites was observed in all rats treated with CClTable As shown in Figure Figure In order to find a relationship between the studied parameters and oxidative liver damage, MDA levels, an index of lipid peroxidation, were evaluated . Hepatic4-induced cirrhosis associated with iron and copper overload and an increase of myeloperoxidase and iNOS expression.These results show that the treatment with low doses of IGF-I induces a reduction of all studied parameters involved in oxidative damage mechanisms in this model of cirrhosis. These findings support the hepatoprotective and antifibrogenic effects previously reported ,16. ThisIt is well known that iron and copper promote oxidant forces ,18,21,302*- radicals [Free iron (or low molecular iron or chelatable iron pool) facilitates the decomposition of lipid hydroperoxides resulting in lipid peroxidation and induces the generation of OH radicals and also accelerates the nonenzymatic oxidation of glutathione to form Oradicals ,19,30,323+ per mole of protein [2*-, etc. [Most of the body's iron is tightly bound to transferrin, entering cells via receptor-mediated endocytosis. Transferrin avidly binds 2 moles of Fe protein . Normall protein ,30,31. I*-, etc. . EnhanceIn the present study, we have found that hepatic transferrin and ferritin levels increased in cirrhotic rats with a parallel rise in iron deposition, whereas in cirrhotic rats treated with IGF-I all the above-mentioned parameters appeared diminished  and secrete myeloperoxidase (MPO) [2*- formed during this respiratory burst is converted to the bactericidal oxidant hypochlorous acid (HOCl) via a series of reactions catalyzed by superoxide dismutase and MPO [After hepatic injury, several kinds of cells  are activated in the subsequent inflammatory response . Free rase (MPO) . The maj and MPO ,29. Nume and MPO . MPO has and MPO . The sam and MPO . In our Another result which deserves particular mention is that iNOS expression was significantly lower in cirrhotic rats treated with IGF-I compared to untreated cirrhotic animals. This finding is in accordance with those reported by other groups -53. Howe4-induced cirrhosis. The hepatic levels of both metals diminished in cirrhotic animals treated with IGF-I. MPO content, iNOS immunohistological expression and hepatic ferritin and transferrin levels were increased in untreated animals and returned to normal in cirrhotic animals treated with IGF-I.In conclusion, these results show that the hepatoprotective and antifibrogenic effect of IGF-I in rats with liver cirrhosis is associated with a significant reduction of the hepatic levels of several parameters such as Fe, Cu, MPO, iNOS, ferritin and transferring, all of them involved in oxidative damage. In this work, iron and copper overload have been demonstrated in the liver from rats with CClThe IGF-I effects described in the present study suggest that a therapeutical approach targeted at lowering oxidative stress marker levels could be effective in the chronic liver disease.2*-, superoxide radicals; MPO, myeloperoxidase; iNOS, inducible nitric oxide synthase; AU, arbitrary units.IGF-I, insulin-like growth factor-I; Fe, iron; Cu, cooper; MDA, malondialdehyde; CO, control healthy group; CI, untreated cirrhotic rats; CI + IGF, IGF-treated cirrhotic rats; OThe author(s) declare that they have no competing interests.MG: Analytical studies, hypothesis and paper elaboration.ICC: Experimental design and treatment (induction of liver cirrhosis and IGF-I administration), hypothesis, histopathological study and scores.MDS: Analytical studies and in vivo assay.IN: Atomic absorption spectrometry assay.JEP: In vivo assay.AC: Hypothesis and experimental design and revision.ADC: Experimental treatment and documentation.EC: Histopathological study and measurements.SGB: Revision.The pre-publication history for this paper can be accessed here:


The character of upper limb disorder in computer operators remains obscure and their treatment and prevention have had limited success. Symptoms tend to be mostly perceived as relating to pathology in muscles, tendons or insertions. However, the conception of a neuropathic disorder would be supported by objective findings reflecting the common complaints of pain, subjective weakness, and numbness/tingling. By examining characteristics in terms of symptoms, signs, and course, this study aimed at forming a hypothesis concerning the nature and consequences of the disorder.I have studied a consecutive series of 21 heavily exposed and severely handicapped computer-aided designers. Their history was recorded and questionnaire information was collected, encompassing their status 1/2 – 1 1/2 years after the initial clinical contact. The physical examination included an assessment of the following items: Isometric strength in ten upper limb muscles; sensibility in five homonymously innervated territories; and the presence of abnormal tenderness along nerve trunks at 14 locations.Rather uniform physical findings in all patients suggested a brachial plexus neuropathy combined with median and posterior interosseous neuropathy at elbow level. In spite of reduced symptoms at follow-up, the prognosis was serious in terms of work-status and persisting pain.This small-scale study of a clinical case series suggests the association of symptoms to focal neuropathy with specific locations. The inclusion of a detailed neurological examination would appear to be advantageous with upper limb symptoms in computer operators. Upper limb pain and dysfunction are frequent complaints associated with computer work. However, the responsible pathology and the pathophysiological mechanisms are insufficiently understood. In addition, there is no consensus with regard to physical findings that may reflect symptoms.The involvement of the nerves in "non-specific" upper limb disorder, e.g. in computer operators, is suggested by various observations: The demonstration of an elevated threshold to vibratory stimulation -3; abnorUpper limb pain in computer operators shares the features of a neuropathic pain: Common analgesics tend to be ineffective. Pain may be evoked spontaneously or may appear to constitute an abnormal response to stimuli with frequent occurrence of allodynia. In addition, there are often non-painful abnormal spontaneous or evoked sensory phenomena such as numbness/tingling. The common experience of weakness which may further deteriorate on use would also be compatible with an upper limb nerve affliction.A precise and accurate diagnosis is crucial for effective management and rehabilitation, and also for epidemiological studies concerning causation. In order to get a better understanding of the pathophysiological mechanisms, the injured tissue should be precisely located. This might not necessarily be where symptoms predominate.I have aimed at studying a clinical series of computer operators with upper limb complaints and dysfunction in terms of• exposure characteristics;• symptoms and past treatment;• physical findings which may reflect an affliction of the peripheral nerves;• prognosis with regard to symptoms and work-status.This study comprises a consecutive series of 21 computer-aided designers with pain and functional limitations in the dominant upper limb. All patients were referred to a department of occupational medicine for diagnostic and aetiological assessment and management. Three patients were males of median age 27 years (range 25–41) and 19 were females of median age 35 years (range 25–55).Patients were interviewed about the character, distribution, initial presentation and development of their symptoms. Special attention was given to the presence of upper limb pain, subjective weakness and numbness/tingling, and to other symptoms included in a standard protocol for work-related upper limb disorders .A subsequent physical examination included extracts of diagnostic criteria for selected clinical disorders  .Upper limb nerve afflictions were defined from an additional neurological examination consisting of the following components:• Manual assessment of the isometric strength in a selection of ten upper limb muscles Figure . Any red• The sensibility  was ass• The axillary nerve (the deltoid area);                                       • The musculocutaneous nerve ;                                       • The radial nerve ;                                       • The median nerve (the tip of the second finger);                                       • The ulnar nerves (the tip of the fifth finger).                                       • The perception of vibration .rk 256 Hz) was addAny sensory deviation from normal was registered as abnormal.• Assessment of tenderness with slight pressure at 14 locations along the course of nerves . Any mec• The brachial plexus ;                                       • The suprascapular nerve (suprascapular notch);                                       • The axillary nerve ;                                       • The musculocutaneous nerve ;                                       • The median nerve ;                                       • The radial nerve ;                                       • The posterior interosseous nerve at the arcade of Frohse (supinator tunnel);                                       • The ulnar nerve .                                       Assessments in patients with unilateral disorder were based on comparison to contra-lateral findings defined as normal. In patients with bilateral disorder, test-results were related to other findings in the same limb assumed to be normal, e.g., strength in adjacent muscles or sensibility in adjacent innervation territories .The definition and location of a nerve affliction ("neuropathy") was based on a traditional approach with a focus on the topography and innervation patterns of the upper limb nerves. Special consideration was given to the presence of normal strength in certain muscles and of reduced strength in others ,19, and I have operated with two sets of criteria for the definition of focal neuropathy assuming the second criterion to be more convincing:Criterion 1: The presence of a pattern of muscle-weakness suggesting a focal neuropathy at a defined location, at which mechanical allodynia with slight pressure at the nerve is present.• Criterion 2: Criterion 1 plus sensory deviations from normal in one or several sensory territories located peripherally to focal neuropathy.• In addition, double crush  at the aBrachial plexus neuropathy at chord level was defined with reduced strength in the deltoid, biceps, and radial flexor of the wrist muscles, when weaknesses were accompanied by brachial plexus tenderness at its passage behind the pectoral muscle. Depending on the extent of brachial plexus involvement, additional muscles may be weak and mechanical allodynia may extend in the proximal or medial direction.Median neuropathy at elbow level was defined with reduced strength in the radial flexor of the wrist muscle along with mechanical allodynia involving the median nerve at elbow level . With an isolated median neuropathy, the deltoid, biceps, and ulnar extensor of the wrist muscles must be intact.and the median nerve was defined in the following situations:Double crush involving the brachial plexus • Strength in the radial flexor of the wrist muscle was reduced as much as / more than it was in the deltoid or biceps muscles.• Mechanical allodynia was either the same or more conspicuous at the median nerve at elbow level than it was at plexus level.Posterior interosseous neuropathy was defined with reduced strength in the ulnar extensor of the wrist muscle along with tenderness at the nerve-passage below the arcade of Frohse in the dorsal proximal forearm. With an isolated posterior interosseous neuropathy, the deltoid, biceps, short radial extensor of the wrist, and radial flexor of the wrist muscles must be intact.and the posterior interosseous nerve was defined in the following situations:Double crush involving the brachial plexus • Strength in the ulnar extensor of the wrist muscle was reduced as much as / more than in the deltoid, biceps, or radial flexor of wrist muscles.• Mechanical allodynia was either the same or more conspicuous at the arcade of Frohse than it was at plexus level.Other potential focal neuropathy was defined according to similar criteria, e.g., an isolated carpal tunnel syndrome would require reduced strength in the short abductor of the wrist muscle but preserved strength in the radial flexor of the wrist muscle. An isolated ulnar neuropathy at elbow or wrist level would require reduced strength in the abductor of the fifth digit and intact proximal muscles. In addition, mechanical allodynia should be present at the appropriate locations along nerve trunks.Patients were recommended to freely move and use the symptomatic upper limb within the limits of immediate and subsequent pain aggravation. All patients were offered physiotherapy based on the concept of adverse neural tension ,24 and e1/2 – 1 1/2 years after the initial examination the patients responded to a questionnaire: The exposure characteristics; symptoms ; past treatment; pain intensity at the first encounter and at follow-up to be quantified on a VAS-scale from 0 (no pain) to 10 (extreme pain); and the present status with regard to functional limitations and work.The change of the level of reported pain between the first consultation at the department and at follow-up was assessed by Friedman's test.All 21 patients returned the questionnaire. The mean duration of work with computer-aided design was 95 months (16–260 months). The self-reported daily mean of time spent with computer work constituted 81% (50–100%) of the total working time. 86% of the respondents reported aggravating factors during the months prior to the onset of symptoms, including high work intensity, overwork or other work conditions causing an unusual strain.Pain in the dominant upper limb was common to all patients. It had a mean duration of 24 months (1–60 months) and was the main symptom in 13 patients. All but one patient had a subjective feeling of weakness/fatiguability. Five patients reported this to be the most disturbing symptom. 19 patients experienced numbness/tingling which constituted the main symptom in three of them. Five patients had bilateral symptoms.All patients had received treatment prior to admission: A limited and transitory effect of past physiotherapy was reported in four out of 17, of pain killers in one out of 10, and of local steroid injections in two out of three patients. For the remaining patients the past treatment had no effect.According to the defined criteria for work-related upper limb disorders , non-neuIn all 21 patients reduced strength was demonstrated in the following muscles: Deltoid, biceps, triceps, and the radial flexor, short radial extensor and ulnar extensor of the wrist. In a smaller number of patients there were additional strength-reductions in the pectoral, infraspinatus, latissimus and abductor of the fifth digit muscles  or unemployed (eight patients).2 = 8.0 and 9.0, p < 0.005 and 0.003, respectively). However, the pain persisted on a disturbing level in the majority of patients  were invariably involved. In a few limbs there was an additional involvement of the pectoral, small abductor of the fifth digit, latissimus dorsi, and infraspinatus muscles Figure . This isCaution should be exercised when drawing a comparison between the outcome of this study of patients, referred with a serious disorder, and studies of "healthy" computer operators in occupation. Still, it would be relevant to compare with upper limb findings in computer operators described by others. A study of 533 visual display terminal workers has suggested an array of upper limb disorders in 22%, dominated by tendon related conditions in 15% and probable nerve entrapment in 4% . In a reHowever, the diagnoses depend on the choice and validity of the clinical tests employed and on the diagnostic criteria applied. The "somatic shoulder/neck syndrome"  is charaMy findings are more in accordance with those of Pascarelli, who studied 485 upper limb patients out of which 70% were computer operators. A detailed and comprehensive physical examination demonstrated protracted shoulders in 78% and head forward position in 71%. This was also frequent in my study-patients but not systematically registered. A neurogenic thoracic outlet syndrome in 70% was suggested by tests stressing the brachial plexus and by the demonstration of mechanical allodynia . In a foThe limited success of the prevention and management of computer-related upper limb disorders demands new approaches to practice and research in the field. The inclusion in future studies of the presented systematic examination of the upper limb nerves may provide additional diagnostic information. This may lead to future improvement of the prevention and management of computer-related upper limb disorders.None declared.JRJ designed the study and conducted all the clinical examinations, and wrote the manuscript.The pre-publication history for this paper can be accessed here:


What were the critical steps in the development of ATRA and arsenic as treatments for APL? Researchers in Shanghai tell the story and look to the future Acute promyelocytic leukemia (APL) was first identified as a distinct subtype of acute myeloid leukemia in 1957 by Leif Hillestad. It is called M3 in the French–American–British classification, with a variant type referred to as microgranular (M3v in the French–American–British nomenclature) Apoptosis: A genetically determined process of cell death in which the cell uses specialized cellular machinery to kill itself and is then eliminated by phagocytosis or by shedding.Caspase: A family of cysteine proteases with aspartate specificity that are essential intracellular death effectors.Disseminated intravascular coagulation: A hemorrhagic disorder that occurs following the uncontrolled activation of clotting factors and fibrinolytic enzymes throughout small blood vessels, resulting in depletion of clotting factors and tissue necrosis and bleeding.Fibrinogenopenia: A decrease in concentration of fibrinogen in the blood.Granulocyte: Terminally differentiated myelocyte or polymorphonuclear white blood cell  with granule-containing cytoplasm.Ligand-inducible transcription factors: Transcription factors that structurally have domains associated with DNA binding and ligand (hormone) recognition. When binding to its specific ligand, the transcription factor initiates a series of conformational changes and interacts efficiently with its specific DNA response element to recruit components of the transcriptional machinery.Nuclear receptor superfamily: One of the most abundant classes of transcriptional regulators including receptors for steroid hormones , RAs, thyroid hormones, and so on. These transcription factors regulate diverse functions such as homeostasis, reproduction, development, and metabolism in animals.Promyelocyte: Granule-containing cell in bone marrow that is in an intermediate stage of development between myeloblasts and myelocytes and that gives rise to a granulocyte.Proteasome: Proteolytic complex that degrades cytosolic and nuclear proteins.Sumoylation: Post-translational modification of proteins by the small ubiquitin-like modifier SUMO.Ubiquitin: A chiefly eukaryotic protein that when covalently bound to other cellular proteins marks them for proteolytic degradation.APL accounts for 10%–15% of all cases of acute myeloid leukemia, with several thousand new cases diagnosed worldwide each year. Before the advent of differentiation therapy, APL was treated with anthracycline-based chemotherapy with a complete remission rate of 60%–76% and a 5-year event-free survival rate of 23%–35% [Failure to differentiate terminally characterizes most, if not all, cancer cells of every origin. Whether the induction of differentiation could be a treatment strategy for cancers was hotly debated for decades before the advent of differentiation therapy.cis RA in people with refractory or relapsed APL [cis RA was ineffective in treating APL An important discovery of the early 1970s was that myeloid leukemic cells could be reprogrammed to resume normal differentiation and to become non-dividing mature granulocytes or macrophages as a result of stimulation by various cytokines ,4. Basedpsed APL ,9,10, bu2/day). The result was dramatic: 23 patients (95.8%) went into complete remission (CR) without developing bone marrow hypoplasia or abnormalities of clotting. The remaining one patient achieved CR when chemotherapy was added Beginning in the early 1980s, the Shanghai Institute of Hematology conducted a series of experiments on differentiation therapy for APL. These experiments showed that all-trans RA (ATRA) could induce terminal differentiation of HL-60, a cell line with promyelocytic features, and fresh leukemic cells from patients with APL. These intriguing results were the impetus for a clinical trial. Twenty-four patients with APL were treated with ATRA , yellow arsenic , and white arsenic, or arsenic trioxide (As2O3) Arsenic is a common, naturally occurring substance that exists in organic and inorganic forms in nature. The organic arsenicals consist of an arsenic atom in its trivalent or pentavalent state linked covalently to a carbon atom. There are three inorganic forms of arsenic: red arsenic  and induces partial differentiation at low concentrations (0.1 × 10−6 to 0.5 × 10−6 M). The rapid modulation and degradation of the PML-RARa oncoprotein by arsenic trioxide could contribute to these two effects Studies have shown that arsenic trioxide exerts dose-dependent dual effects on APL cells—it induces apoptosis (programmed cell death) preferentially at relatively high concentrations  of retinoids  and retinoid X receptors (RXRs). These belong to the steroid/thyroid/retinoid nuclear receptor superfamily of ligand-inducible transcription factors. Both RAR and RXR families consist of three subtypes: α, β, and γ etinoids .More than 95% of patients with APL have the tq22;q21) translocation. This results in the fusion of the RARα gene on 17q21 and the promyelocytic leukemia (PML) gene on 15q22, which generates a PML-RARaα fusion transcript [ translocPML-RARa may affect transcription in other pathways including those in which the transcription factor AP1 and interferon-responsive factors are involved. PML-RARα also binds to promyelocytic leukemia zinc finger (PLZF) protein and potentially affects its functions  −7–10−6 M) exert their effects through targeting the PML-RARα oncoprotein, triggering both a change in configuration and degradation of the oncoprotein and the activation of transcription, leading to differentiation. Cleavage of the PML-RARα fusion protein by caspases at residue D522 has been shown in APL cells induced to differentiate by ATRA ATRA and arsenic trioxide degrade and cleave the PML-RARα oncoprotein. Although we now have a good understanding of the molecular mechanisms underlying ATRA in differentiation therapy for APL, these mechanisms were shown long after the identification of the efficacy of this drug in treating the disease. Now it is well established that pharmacological concentrations of ATRA , and this difference persisted after consolidation therapy (p < 0.05). Notably, all 20 patients in the combination group remained in CR whereas seven of 37 cases treated with monotherapy relapsed (p < 0.05) after a follow-up of 8–30 months .Since ATRA and arsenic trioxide degrade the PML-RARa oncoprotein via different pathways, and since studies in animal models have shown synergic effects of both drugs in prolonging survival or even eliminating the disease ,35, the It seems that a combination of ATRA and arsenic trioxide for remission and maintenance treatment of APL produces better results than either of the two drugs used alone, in terms of the time required to achieve CR and the length of disease-free survival. We hope that the use of three treatments—ATRA, arsenic trioxide, and chemotherapy—will ultimately make APL a curable human acute myeloid leukemia The story of ATRA in the treatment of APL shows that by targeting the molecules critical to the pathogenesis of certain diseases, cells can be induced to return to normal. Differentiation therapy is therefore a practical method of treating human cancer that has shown consistent effectiveness in trials. The clarification of the underlying molecular abnormalities of APL is an example of the benefits of a close collaboration between bench and bedside, and is necessary for our understanding of the mechanisms of ATRA in differentiation therapy. It is clearly important to elucidate the molecular and cellular basis of a particular cancer if we are to further develop mechanism-based target therapies.The sequencing of the human genome and ongoing functional genomic research are now accelerating the dissection of disease mechanisms and identification of therapeutic targets. This in turn may facilitate the screening of promising treatments. What we learn from developing curative treatment approaches to APL may help to conquer other types of leukemias and cancers.


Increasing prevalence of obesity and disorders associated with sedentary living constitute a major global public health problem. While previous evaluations of interventions to increase physical activity have involved communities or individuals with established disease, less attention has been given to interventions for individuals at risk of disease.ProActive aims to evaluate the efficacy of a theoretical, evidence- and family-based intervention programme to increase physical activity in a sedentary population, defined as being at-risk through having a parental family history of diabetes. Primary care diabetes or family history registers were used to recruit 365 individuals aged 30–50 years, screened for activity level. Participants were assigned by central randomisation to three intervention programmes: brief written advice (comparison group), or a psychologically based behavioural change programme, delivered either by telephone (distance group) or face-to-face in the family home over one year. The protocol-driven intervention programme is delivered by trained facilitators, and aims to support increases in physical activity through the introduction and facilitation of a range of self-regulatory skills . The primary outcome is daytime energy expenditure and its ratio to resting energy expenditure, measured at baseline and one year using individually calibrated heart rate monitoring. Secondary measures include self-report of individual and family activity, psychological mediators of behaviour change, physiological and biochemical correlates, acceptability, and costs, measured at baseline, six months and one year. The primary intention to treat analysis will compare groups at one-year post randomisation. Estimation of the impact on diabetes incidence will be modelled using data from a parallel ten-year cohort study using similar measures.ProActive is the first efficacy trial of an intervention programme to promote physical activity in a defined high-risk group accessible through primary care. The intervention programme is based on psychological theory and evidence; it introduces and facilitates the use of self-regulatory skills to support behaviour change and maintenance. The trial addresses a range of methodological weaknesses in the field by careful specification and quality assurance of the intervention programme, precise characterisation of participants, year-long follow-up and objective measurement of physical activity. Due to report in 2005, ProActive will provide estimates of the extent to which this approach could assist at-risk groups who could benefit from changes in behaviours affecting health, and inform future pragmatic trials. This trial addresses the rise in the burden of disease associated with sedentary living: a major public health problem. Physical inactivity accounts for up to 11.7% of all deaths in developed countries [Reversal of this trend will require not only public health programmes to increase activity at societal level, but also interventions to help high-risk individuals increase physical activity and maintain beneficial activity patterns . This is2 [The study targets people with a parental family history of Type 2 diabetes and a sedentary lifestyle, who constitute a clearly identifiable high-risk population . A consi2 , and pro2 ,6.Most trials have evaluated increasing physical activity in the context of established disease. The few published trials of primary prevention in high-risk groups have methodological limitations. They have mainly evaluated brief interventions to increase exercise in the general population delivered through primary care practitioners -9, oftenThree trials among individuals with impaired glucose tolerance in China , FinlandProActive intervention programme included a review of psychological theories and evidence, through systematic reviews [Many of the available trials evaluated interventions that were not explicitly based on psychological theory and evidence, and did not specify clearly which behaviour change techniques were applied by the providers -20. In a reviews ,20 and e reviews  was seleet al., 2004. A causal modelling approach to the development of theory-based behaviour change programmes for trial evaluation. Submitted).A range of behaviour change techniques with evidence for their effectiveness was used to bridge the gap between intention and action: goal setting and review, action planning, use of prompts, self-monitoring, and reinforcement ,18,23,25ProActive.Major challenges in promoting physical activity are maintenance of behaviour change, and the avoidance of drop out rates that can approach 50% . ReviewsProActive is to determine the effects of a theoretical- and evidence-based intervention programme on objectively measured physical activity after one year, in sedentary individuals at risk of diabetes and related metabolic abnormalities due to their family history. Three questions are posed:The primary objective of 1. Behaviour change: Can an innovative approach to increasing physical activity achieve clinically important change in this behaviour when offered to a group at increased risk of diabetes?2. Disease impact: If so, what is the potential for the changes in behaviour achieved in mid-life to reduce the incidence of diabetes in later life?3. Dose finding: How does delivery of the approach, at two levels of intensity, affect acceptability, efficacy and costs?The trial will estimate the extent to which physical activity and its key psychological mediators are altered by the intervention programme, and assess its acceptability to this high-risk group. It will document the extent to which behaviour change is associated with reduction in weight gain and improvement in physiological and biochemical correlates, and will model the potential impact of the intervention on future risk of diabetes.Intensive, face-to-face interventions may not be a feasible health service model, and there is some evidence that less intensive, continuous support may be as effective ,23. The ProActive is a four-year study with a complex randomised trial design [l design , with ceThe study design and patient flows  are shown in Figure The study is explanatory in design, and the quality-assured intervention programmes are delivered by carefully trained and supervised family health facilitators with experience of working in primary care or the community, and backgrounds in health promotion, dietetics and nursing.The study is set in urban, suburban and rural Cambridgeshire, Essex and West Suffolk, England, in the homes of participants and their families. The study population consists of offspring of people with Type 2 diabetes, aged 30–50 years, without a diagnosis of diabetes, and not considered very active based on self-report at the start of the study (see below). This age range defines a group at risk of weight gain ,32. Any Once the relevant ethical and PCT approval had been obtained, 53 practice teams in the locality were approached by letter, inviting them to take part in the study, and highlighting the reimbursement of all costs involved. Personalised letters were sent to the practice manager (who we asked to collate responses and reply using a reply slip and Freepost envelope), all partners and nursing staff. Included with each letter was a brief summary of the study and a Research Information Sheet for Practices (RISP) form . If no rInitially participants were recruited through their parents; patients with Type 2 diabetes on the diabetes registers of 20 practices ('recruitment method 1'). Patients were written to by their general practitioner, with a description of the study, and asked to provide contact information for any offspring aged 30–50 years, living locally. Consent was also sought for the practice to pass the contact details of the offspring to the research team so that they could invite the offspring directly into the study. Piloting demonstrated feasibility and acceptability of the method, and one reminder was sent after three weeks if no reply was received.From 20 practices, 2631 patients were approached and 2025 (77%) replied, yielding 1238 potentially eligible offspring who were invited to take part in the study. The ratio of approximately one potentially eligible offspring to two patients with diabetes was half our pilot projections, so to increase recruitment we developed a second recruitment approach ('recruitment method 2'). This approach recruited potential participants with a recorded family history of diabetes directly from practices with family history registers, and was feasible in seven of the 20 practices. General practitioners wrote to all patients aged 30–50 years with a recorded family history of diabetes, enclosing a study information sheet, and asking those willing to complete and return to the practice a questionnaire to determine which family member(s) had diabetes, and of which type. Consent was sought for this information and contact details to be passed on to the research team. Using this method, with again one reminder letter, 1340 patients were written to, and 896 (67%) responses were received, with 283 patients interested and eligible. Both recruitment approaches provided 1521 potential trial participants. Practitioners used their discretion in applying both approaches to the exclusion of patients who were physically or mentally unwell.Potential participants recruited by both methods were next written to by the research team with full information about the study and a screening activity questionnaire, describing occupational and leisure activity, based on published questionnaires ,36, to eTo fulfil measurement requirements participants had to be able to walk briskly, without help, on the flat for 15 minutes. Participants also had to live within reach of the measurement centre and the Family Health Facilitators; defined as a 30-minute average travel time from the study co-ordination centre. Other exclusion criteria included individuals with serious physical or psychiatric illness limiting programme involvement; people with life issues interfering with the study; those known to be pregnant or have diabetes before baseline measurement; and those planning to move away. As shown in Figure ProActive. Brief details of the trial were sent, together with a request for feedback if the practitioner had any concerns about the offspring's participation, or about the safety of the facilitators making home visits.Eligible offspring were registered with general practitioners in the Eastern Region of the UK. Prior to both baseline measures and randomisation, these doctors were individually informed about their registered patients' intention to participate in Randomisation was carried out centrally by the trial statistician, using a partial minimisation procedure that dynamically adjusted the randomisation probabilities in order to balance important covariates; body mass index, sex, age, physical activity , family size, and behavioural intentions. Randomisation thus used baseline measures. Thirty-two pairs of siblings and two sibling-triples were cluster randomised to the same study group to avoid contamination, and the remaining 295 participants (81%) were individually randomised. Overall, 365/465 (78%) of those eligible went forward to randomisation.At baseline and the end of the study, all participants attend the study centre at either Ely or Cambridge for questionnaires, physiological and anthropometrical measures, and venesection. At six months, psychological and self-reported physical activity data are collected by postal questionnaires. Measures relating to the intervention programme evaluation are collected by the facilitators during the intervention, and we assess reported use of self-regulatory strategies by participants to increase their activity levels at six and twelve months.In similar primary care based trials we have achieved attrition rates of 30% or less ,39, and Maximising retention is an important issue, particularly as the comparison group do not benefit from regular contact with a facilitator. At recruitment, the introductory leaflets for all three arms emphasised the importance of follow-up, irrespective of treatment group. Participants who drop out of the intervention programme are contacted by a principal investigator, and offered an opportunity to give feedback and to confirm drop out from the intervention programme only, or from trial measurement as well.The distribution of measures across baseline, six-month and one-year follow-up are shown in Table 2/kg/body weight) is measured by indirect calorimetry during a submaximal graded treadmill exercise test, and maximal cardiorespiratory fitness (VO2max) is estimated using predicted maximal heart rate (i.e. 220 minus age) [Oxygen uptake (ml Onus age) ,42.Self-report measures of well-being and quality of life include subjective health and energy (SF-36) , anxietyThe frequency and severity of physical activity related injury is assessed by study questionnaire at one year.Psychological mediators of physical activity include intention to increase activity over the next year, and its predictors . These key measures have been developed for the study following the recommendations of Ajzen . PhysiolProActive intervention programme to have an impact on health service costs in the short term, we are monitoring health service utilisation  in the last 20% of participants recruited to the study.The economic analysis will explore the impact of a physical activity intervention programme on NHS costs. As the study is explanatory in design, we will not conduct a full cost-effectiveness analysis, but aim to provide a cost-description of the delivery of the intervention programmes. We are measuring the costs of delivering the 'face-to-face' and 'distance' intervention programmes via family health facilitators. These costs primarily comprise the training of facilitators, educational materials, travel, and the time that facilitators spend contacting and visiting families (including cancelled visits). Travel costs and contact time are recorded by the facilitators for every trial participant. The cost of facilitator time will be based on national average salaries, employment costs, qualifications, overheads and indirect costs . AlthougProActive are cardiovascular and musculoskeletal events associated with the laboratory procedures of treadmill exercise testing and injuries sustained as a consequence of increasing physical activity in everyday life. The cardiorespiratory fitness test used in this study is submaximal, and only undertaken following extensive screening procedures. If a participant exhibits a positive Rose angina questionnaire [The primary safety concerns for participants in ionnaire , a positionnaire  or an abRanges for acceptable results are set for all clinical measures. If these are exceeded, the information is sent to the general practitioner, and the participant informed and advised to consult.As the intervention programme is based on participants' own preferred activities, and emphasises small achievable goals set by the participants, the risk of excess injury is small. Group information about injury will be reported.Participants previously unaware of their familial risk of diabetes may experience anxiety related to awareness of their increased risk status. This is considered in facilitator training, and measures of anxiety, worry about diabetes and perceived vulnerability are included (see above).Physiological and anthropometric measures are made in two centres by observers unaware of individuals' group allocation. Biochemical measures are made in one laboratory with established quality assurance systems. Randomisation was undertaken by the trial statistician, independently of the trial co-ordination team, and the data entry team are unaware of study group.The administrative database (participant information), dayPAR values and blood test results are managed in-house, with the latter being double entered. Numeric fields have limiters set so that values outside a defined range cannot be entered. Additionally, any blood results outside the 'normal range' are flagged for confirmation of value. Random checks on administrative data are performed regularly, checking the data on the database against paper records and correcting any errors found.Double data entry of all anthropometric and questionnaire measures is undertaken by an experienced, independent agency, blind to study group . In addition, random checks are applied as described above.The family health facilitator contacts participants randomised to the 'face-to-face' and 'distance' interventions, and arranges a home interview including family members. At this introductory interview, personal reasons for increasing physical activity are elicited and reinforced, family participation is encouraged, and the relationships between physical activity, weight gain and prevention of Type 2 diabetes are explained and discussed.In the 'face-to-face' arm this is followed by four visits and two brief support telephone calls over five months. During these interactions the participant and willing family members learn strategies to increase physical activity, for instance selecting activities that they enjoy doing, setting achievable goals, defining action plans, self-monitoring, self-reinforcement and relapse prevention. Pedometers are available for self-monitoring among participants who have chosen walking as their goal. A key difference between this intervention and others currently under evaluation (e.g. ACT) is that there is no absolute target for physical activity defined at the outset. Family members are encouraged to make gradual and continuous increases in their activity, as much as they feel able to, on the understanding that all increases, if maintained, are beneficial. Follow-up continues by monthly telephone calls up to one year, to discuss any difficulties in applying the strategies, and to encourage family members to increase activity further.In the 'distance' arm, following the introductory meeting the intervention programme is delivered by six telephone calls over five months, and then monthly by post up to one year, with content similar to the 'face-to-face' arm. During the phone calls the facilitators encourage the participants to involve family members. Visits and telephone calls take approximately one hour and 45 minutes, respectively.An arm-specific introductory leaflet is used, but otherwise materials are the same for the face-to-face and distance arms. All introductory leaflets include text to encourage retention in the trial. In the comparison arm the leaflets offer brief advice on the benefits of physical activity. Participants in the intervention programme arms are given an educational manual describing the strategies that participants are encouraged to use to increase their habitual activity in a step by step fashion.Various mechanisms are used to promote the fidelity of delivery of the intervention programme to the underlying psychological theories and intervention programme protocols. A detailed training manual and protocols for each contact were developed, and a Training Officer appointed. Facilitators attended a five day phased course in psychological theories, behaviour change techniques and experiential training in techniques, with six half-days initially, followed by refresher sessions at six months and continuing supervised practice by a clinical psychologist and through peer-appraisal. Facilitators complete a checklist for the introduction of and mastery of self-regulatory strategies by the participant after each contact, and monitor intervention programme attendance and drop-out for each participant.An assessment of adherence by facilitators to the behaviour change techniques specified in the protocols was conducted among a random sample of 27 participants, using reliable coding frames and transcripts of the sessions. The intervention programme evaluation includes: an assessment of the frequency of meetings and telephone calls, proportion of progress reports and postcards sent and progress reports returned, satisfaction with the intervention programme, reported use of self-regulatory strategies by participants at six months and one year, and drop-outs at one year.The sample size calculation was initially based on physical activity level (PAL), the ratio of total energy expenditure to estimated basal metabolic rate ,41, and Main analyses will be at one year, comparing combined 'face-to-face' and 'distance' versions of the intervention programme with 'brief advice', comparing 'face-to-face' with 'distance' modes of the intervention programme, and estimating the difference between each intervention programme group and 'brief advice' to inform a larger pragmatic trial. Analysis by intention-to-treat will retain individuals within their randomised group regardless of participation. Comparisons will involve an adjustment for baseline physical activity and other variables used in the randomisation. We will undertake sensitivity analyses, assuming a range of potential outcomes for non-completers, informed by available baseline and interim data on non-completers. Non-completers will have multiple data imputed with a 'missing at random' assumption and with sensitivity analyses to represent optimistic and pessimistic scenarios for drop out. Clustering effects by family will be estimated for the primary outcome.A secondary 'dose-response' analysis will use all three randomised groups, over baseline, six months and one year. A 'per protocol' analysis will also be undertaken among those completing the intervention programme.The incremental cost of delivering the 'face-to-face' intervention programme will be compared to the 'distance' and 'brief advice' groups.Stage 1) The trial will provide evidence on the relationship between observed behaviour change, weight change, and biochemical and physiological correlates. Modelling is facilitated by reference to the Ely Cohort; a prospective population cohort study that began in 1990 and involved 1122 people without known diabetes [ProActive.diabetes . MeasureStage 2) Using models based on past cohort data, the influence of behaviour change on future diabetes incidence [ncidence ,41,51,52Stage 3) We will undertake sensitivity analyses on the projections at Stage 2, using a range of plausible assumptions about how behaviour change might affect other risk factors and hence indirectly influence future diabetes risk.ProActive is the first efficacy trial of physical activity promotion in a defined high-risk group accessible through primary care, evaluating an intervention programme based on theory and evidence. It supports increases in informal activity, through the introduction and facilitation of self-regulatory strategies with regular reinforcement by the facilitator. Due to report in 2005, ProActive has the potential to make substantial contributions to understanding the extent to which such approaches could assist the wide range of at risk groups who could benefit most from increasing their physical activity.The trial team brings together expertise in the epidemiology of diabetes  with intet al., 2004. A causal modelling approach to the development of theory-based behaviour change programmes for trial evaluation. Submitted). This will enable replication and further strengthening of effective intervention steps, as well as development of theory.In terms of the intervention itself, careful measurement along the hypothesised causal path from cognition, through self-reported behaviours to energy expenditure, will enable testing of the application of the Theory of Planned Behaviour in this setting, and of the relationship between the everyday activities that the programme has as its focus and the objectively measured physical activity VOThe authors declare that they have no competing interests.ALK, NW, SG, SS, WH, DS, – Principal InvestigatorsTP – Trial StatisticianKW – Trial Co-ordinatorWill H – Trial EconomistUE – Physical activity measurementAll authors read and approved the final manuscript. ALK is the paper guarantor.The pre-publication history for this paper can be accessed here: