There is a growing body of evidence linking health and well-being to key business issues. Despite this, corporate uptake of workplace health promotion programmes has been slow outside the USA. One possible reason for this is the lack of a generally available health risk measure that is quick and easy to administer and produces data that is rich enough to inform and direct subsequent employee health promotional interventions.We report on the development and validation of the health and well-being (HWB) assessment, a free to use health risk appraisal questionnaire that has been specifically developed for use in the corporate setting. The HWB assessment focuses upon modifiable health issues that directly impact upon business drivers. Development involved interviews with business leaders to ascertain their key areas of focus, scientific and general literature review to find evidence for health status having an impact upon these areas, and end user testing.Three UK-based organisations  participated in the research. A total of 2224 employees completed the HWB assessment, the short-form 36 (SF-36) and the World Health Organisation Health and Work Performance questionnaire (WHO-HPQ) as part of the validation process.th percentile were more likely to achieve workplace productivity standards than those with scores below the 25th percentile .The HWB assessment is a twenty item questionnaire covering ten areas of health and well-being. Completion of the HWB assessment generates a global health risk score and ten sub-scores corresponding to the ten areas covered. It is easy to use and quick to complete (average completion time was eight minutes) and showed good internal consistency and test-retest reliability. Statistically significant correlations with similar SF-36 variables were observed. A significant negative correlation between HWB score and productivity decrement, as measured by the WHO-HPQ, was observed (r = -0.4). Individuals with HWB scores above the 25The HWB assessment generates reliable business focused health risk data that can be used to direct and target appropriate interventions within corporate populations. It may also be useful in quantifying the financial impact health status issues have upon organisations. The last decade has seen increasing interest in the health and well-being of the workforce. This has been driven partly by the increasing burden of direct healthcare costs, but also from a recognition that the economy within the developed world has appreciably changed,2. The rThe evidence for the impact of many lifestyle factors upon long-term health is overwhelming. Smoking, excess alcohol intake, poor nutritional status, a sedentary lifestyle and psychological distress have all been associated with numerous diseases -10. IndeWith these issues gaining greater ascendancy in the corporate world, we saw a need for a short, easy to administer questionnaire that could capture this business critical health status information. By conducting a confidential survey of all employees, aggregated data can be used to provide a first step by which organisations can target and monitor appropriate population-based health interventions within their workforce. A key issue in conducting such surveys is maintaining individual privacy and ensuring confidentiality of information. The majority of US organisations who already conduct annual health surveys of their employee populations do so either via their occupational health departments or external third parties.Although there are a number of general and specific health risk appraisal measures available for corporate use, they are either not well validated, suffer from being too long and cumbersome to administer, or cost an appreciable amount to use. In the case of health related quality of life measures, such as the SF-36, or specific stress indicators such as the general health questionnaire (GHQ), the data that is generated is not specific enough to direct health and well-being interventions within the corporate setting.We report on the development and validation of the health and well-being (HWB) assessment, a free to use twenty item questionnaire. We also describe its use in assessing the impact employee health has upon productivity and performance.Our principal aim was to develop a questionnaire that focused upon business pertinent health and well-being issues. A secondary aim was that it should be quick and easy to administer with the amalgamated results serving as a baseline from which employers can start to implement appropriate health promotion interventions within their employee populations.We initially surveyed a sample of twelve business managers and executives to ascertain the key issues currently facing their organisations. Interviewees came from four different business sectors, namely (i) Technology (ii) Engineering (iii) Banking and Insurance and (iv) Public sector / Health. Interviews lasted no more than 30 minutes and were semi-structured, asking each interviewee to describe the key issues they faced in their day-to-day operations. We then searched the general and scientific literature for evidence of the effect health parameters have upon the issues identified. The key business issues facing our sample of corporate leaders could be categorised into four separate areas. Table Initial questionnaire development involved formulating items that corresponded with the health and well-being areas that impact upon the four key business issues. An initial set of 40 questions was tested and discussed in one to one interviews and focus group discussions. Thirty-five employees and managers, from companies in the four industry sectors described previously, participated in these sessions. Question changes and selection were an iterative process, with the final questionnaire formulated after three rounds of small group testing and one round of initial data collection from 100 volunteers. This background research and subsequent refinement led us to construct a 20-item questionnaire covering ten areas of health and well-being  were invited to complete the questionnaire via the internet. All data transmission utilised 128-bit encryption and all data storage was fully compliant with the UK Data Protection Act (1998). All participants were required to electronically sign an agreement for their anonymised data to be used in amalgamated format for purposes of research. A draw with a prize of a weekend break was offered as an incentive to participate for each company group. Thirty employees re-took the questionnaire four weeks after the initial completion date in order to provide test re-test data.As well as completing the newly developed questionnaire, participants were also asked to concurrently complete the Short Form 36 (SF-36) and part B of the World Health Organisation's Health and Work Performance (WHO-HPQ) questionnaire in order to assess criterion validity,25. The For each participant in the study details on age, gender, sickness absence in the preceding three months, company position, marital status and weekly working hours were also collected.Statistica, a statistical software package distributed by Statsoft Inc. All data analysis was carried out using Of the 3000 employees invited to participate in the study, 2224 completed the questionnaires (74% response rate). Online completion ensured that there were no missing data points in completed questionnaires. The mean age was 38.1 years (standard deviation 10.7). Fifty-nine per cent of respondents were female , mental health vs. symptoms of stress (r = 0.70) and mental component summary measure (MCS) vs. stress (r = 0.71). Additionally, there was a clear association between the overall HWB score and the General Health and Vitality scores of the SF-36 (r = 0.59 and 0.49 respectively). All SF-36 multi-item scales were significantly correlated with the overall HWB score (p ≤ 0.01).th percentile was 10% and 75th percentile was 33.5%)The 2224 individuals who completed the HWB assessment and the SF-36 also completed part B of the WHO-HPQ. The output from the WHO-HPQ is a calculated productivity decrement for each respondent, i.e. the proportion of the week that the individual is not working optimally, either because they are absent or because they are not working effectively [A negative correlation between the HWB score and calculated productivity decrement was observed , i.e. better health status, as measured by the HWB assessment, was associated with less weekly productivity decrement.General linear model analysis indicated that age and the overall HWB score were the only two variables that remained as significant predictors of weekly productivity decrement (p < 0.0001 for both).th percentile figure of 33.5% productivity decrement per week was taken as the cut-off for achieving the productivity standard within the current population.The 75Similarly, the lower quartile HWB score of 52.1 was used as the cut-off to define poor health. 2 × 2 table analysis using these cut-offs demonstrates an odds ratio of 3.62  for making the productivity standard if HWB score is above the lower quartile value, Chi squares 158.82 (Yates Corrected), p < 0.0001.There was a significant correlation between the single question on effectiveness contained in the HWB assessment and the productivity decrement, as calculated by the WHO-HPQ, .Thirty individuals re-took the HWB assessment four weeks after their original completion date. During this time no information or intervention with regard to health and well-being was delivered to them. The correlation between HWB scores at both time points was excellent (r = 0.90), with no significant differences between mean scores or variance of the data sets.Table The association between employee health status and costs incurred by employers is incontrovertible. Numerous studies have clearly shown how health risk factors directly impact upon medical care costs, short- and long-term absence and workers' compensation,27-29. AAs already mentioned, although well-established questionnaires have been extensively validated in many different populations the data that is generated is often of limited value in specifically directing health and well-being interventions. We have presented the first steps of the development and validation of a health risk appraisal measure that has been specifically designed for use in the corporate setting. As well as having good content, criterion and construct validity, the generated data can help health promotion specialists develop appropriate and targeted interventions for the respondent population. The questionnaire provides information on areas such as nutritional choices, levels of habitual physical activity, sleep difficulties and stress symptoms. Amalgamated answers can be used to ensure the correct and most appropriate health interventions are delivered to the population being assessed. In addition, the single question on work effectiveness can be used to confirm the link between the health of the population being studied and their performance, prior to more in-depth evaluations of productivity such as can be made with a specific productivity measure.One would naturally expect those individuals who have taken more time off due to illness to have worse health than those who have been absent for less time. We have demonstrated that sickness absence in the preceding three months is a significant predictor of HWB score and remains so when other variables are controlled for. This is an indication that the HWB assessment is indeed measuring the health and well-being issues that are critical to businesses as a whole. Further confirmation of the discriminant validity of the HWB assessment is needed, however a suggestion that it can detect real differences in health status between groups is also seen in the significantly better scores observed between those with more senior positions as compared with those in junior positions. This difference possibly reflects the better financial rewards, the better access to healthy alternatives and the superior levels of job control associated with more senior corporate positions.The fact that the HWB score and sub-indices were significantly correlated with the broadly similar SF-36 multi-tem scales is an indication that the majority of the constructs assessed by the SF-36 are at least partially reflected in the HWB.Productivity whilst at work can be influenced by a multitude of different factors, however as demonstrated by Burton and colleagues, health is a major contributor. Our stuAlthough these initial results appear promising, data collection from a larger employee sample, from different sectors and incorporating a wider age range, is necessary in order to confirm that our observations still hold true. Normalising the scoring (as is often performed with SF-36 data) would also make interpretation easier and more user friendly. Additionally, longitudinal data on whether the HWB assessment can be used as a predictive tool for populations, and hence provide businesses with visibility on how their employee health status issues are likely to affect their bottom line, is the logical next step. This process is already underway in four multinational organisations with populations in both the USA and the UK and is being overseen by the Institute for Health and Productivity Management (IHPM).In summary we present a new health risk measure, the Health and Well-being Assessment (HWB), which has the following key features:(i) has been specifically designed for the corporate environment addressing the health and well-being issues that affect key business drivers(ii) is quick, easy and free to use(iii) the generated data is useful for guiding future interventionsBy combining medical health issues with other more "lifestyle" and well-being focused areas within a short, easy to use questionnaire we believe that we have created a useful corporate tool.GHQ – General Health QuestionnaireHWB – Health and well-beingROI – Return on InvestmentSF-36 – Short Form 36 questionnaireWHO-HPQ – World Health Organisation Health and Work Productivity QuestionnairePM has a part-time salaried role with health and well-being business consultants Vielife.No other financial competing interestsNo non-financial competing interests.PM performed all of the work that is contained within this paper.Health & Well-being Questionnaire Questionnaire and scoring algorithms.Click here for file


Allermatch™ A query amino acid sequence is compared with all known allergenic proteins retrieved from the protein databases using a sliding window approach. This identifies stretches of 80 amino acids with more than 35% similarity or small identical stretches of at least six amino acids. The outcome of the analysis is presented in a concise format. The predictive performance of the FAO/WHO criteria is evaluated by screening sets of allergens and non-allergens against the Allermatch databases. Besides correct predictions, both methods are shown to generate false positive and false negative hits and the outcomes should therefore be combined with other methods of allergenicity assessment, as advised by the FAO/WHO.Allermatch™ provides an accessible, efficient, and useful webtool for analysis of potential allergenicity of proteins introduced in genetically modified food prior to market release that complies with current FAO/WHO guidelines. A step-by-step procedure to assess allergenicity is described by the Codex alimentarius and the FAO/WHO consultation group [The safety of genetically engineered foods must be assessed before authorities in most nations will consider granting market approval. An important issue in current food safety assessment is the evaluation of the potential allergenicity of food derived from biotechnology. Since many food allergens are proteins, introduction of a new ("foreign") protein in food by genetic engineering can in theory cause allergic reactions. Therefore the allergenicity of novel proteins needs to be assessed. Potential allergenicity of a protein is a complex issue and various tests can be used for prediction, including bioinformatics, on group ,2. An imon group  to estab(1) Obtain the amino acids sequences of known allergens in protein databases in FASTA format .(2) Prepare the complete set of 80-amino acid length sequences derived from the query protein .3) Compare each of the sequences of (2) with all sequences of (1), using the program FASTA [ Compare According to the Codex alimentarius , potenti(a) More than 35 % similarity over a window of 80 amino acids of the query protein with a known allergen.(b) A stretch of identity of 6 to 8 contiguous amino acids.This procedure is described in more detail by the expert consultation and the Codex Alimentarius. Potential allergenicity requires further testing of the protein with panels of patient sera and possibly animal exposure tests ,2.. Leader sequences were, if annotated, trimmed from the sequence. The SwissProt allergen list contains 334 mature protein sequences, while the WHO-IUIS allergen list  contains 632 sequences . These two databases contain 236 duplicate entries. The non-redundant combined database contains 730 sequences  windows using a sliding window with steps of a single residue. Each of these windows is compared with all sequences in the allergen database of choice. All database entries showing a similarity higher than a configurable threshold percentage (default is 35%) to any of the 80 aa query sequence windows are flagged. Upon completion of the analysis, a table is shown with all flagged database entries. Per entry, the highest similarity score is given, as well as the number of windows having a similarity above the cut-off percentage. For each allergen database entry identified, more detailed information on the similarity between the allergen and query sequence can be retrieved, such as those areas of both proteins within all 80 aa windows scoring above the cut-off percentage. The similarity score calculated by FASTA can apply to stretches smaller than 80 aa, Allermatch™ converts such a similarity score to an 80 aa window. For example, 40% similarity on a stretch of 40 aa converts to 20% similarity on an 80 aa window.This method looks for short sub-sequences (words), which have a perfect identity with a database entry. The wordsize is configurable (default is 6 aa). The output given is similar to the output given by Mode 1. All database entries with at least one hit are listed and for each of these, more detailed information can be retrieved upon request.The Allermatch™ webtool also offers a full alignment of the query sequence with either of the allergen databases using FASTA. Although this full alignment is currently not required by the FAO/WHO guidelines, the full alignment of protein sequences helps positioning of regions of potential allergenicity in the whole primary structure of the protein. The FASTA output is parsed and information from the allergen database is added and presented.To examine the predictive performance of the FAO/WHO criteria for potential allergenicity, we have performed two tests. The first test determines the percentage of false negative and the second test assesses the amount of false positives. Both tests are performed with standard settings; for the sliding window approach an 80 amino acid window with a 35% similarity cutoff is used and for the wordmatch approach 6, 7 and 8 aa word sizes are tested.The false negative error-rate is estimated by a leave-one-out method, testing all sequences in each Allermatch™ database against that database with the tested sequence excluded. Each sequence not resulting in a hit is considered a false negative. The results of each method/database combination are summarized in Table i.e. T1 (related to Bet v 1), human serum albumin , and human heat shock protein 70 . A selection of unrelated, non-allergenic proteins is therefore likely to give a lower false positive rate. Caution should be taken in interpreting these false hit rates. The used methods might perform differently with other sets of proteins. For example, a member of a completely novel group of valid allergens is likely to generate a false negative result.In the second test, we assess the odds of a false positive by testing 12 protein sequences known to be non allergenic. This is based on non-reactivity of these proteins towards IgE-sera of allergy patients or on the inability to cause IgE-responses in experimental animals  and confirm results, before arriving at conclusions. To prevent false positives as much as possible, one should choose for the well-annotated SwissProt database. To prevent false negatives, the combination of the larger WHO-IUIS database with that of SwissProt is more appropriate. Updates to the SwissProt and WHO-IUIS allergen lists will be incorporated in the Allermatch™ databases on a regular basis.The prediction of potential allergenicity by primary sequence comparison depends on the quality of the data used for comparison. Addition of a non-allergenic or poorly annotated protein to any of the Allermatch™ allergen databases would obviously result in undesired false positives and should be prevented. A workable strategy could be to use multiple databases, Several other websites in the public domain offer sequence alignment facilities that support the prediction of potential allergenicity, such as SDAP ,11, AlleAllermatch™ is an efficient and comprehensive webtool that combines all bioinformatics approaches required to assess the allergenicity of protein sequences according to the current guidelines in the Codex. The application will be kept up to date with the FAO/WHO criteria and the SwissProt and WHO-IUIS allergen lists. It will be extended with other, supplementary methods to support and refine the prediction of allergenicity..Allermatch™ is platform independent and accessible using any Netscape 4+ compatible webbrowser at MF developed and implemented the Allermatch™ webtool. HN provided the domain name registration and advised in the web site development. GK and AP provided the scientific background and constructed the sequence databases. JPN and RvH provided time, resources and ample discussion. All authors have read and approved the final manuscript.


The protein structure prediction problem is one of the most challenging problems in biological sciences. Many approaches have been proposed using database information and/or simplified protein models. The protein structure prediction problem can be cast in the form of an optimization problem. Notwithstanding its importance, the problem has very seldom been tackled by Constraint Logic Programming, a declarative programming paradigm suitable for solving combinatorial optimization problems.Constraint Logic Programming techniques have been applied to the protein structure prediction problem on the face-centered cube lattice model. Molecular dynamics techniques, endowed with the notion of constraint, have been also exploited. Even using a very simplified model, Constraint Logic Programming on the face-centered cube lattice model allowed us to obtain acceptable results for a few small proteins. As a test implementation their (known) secondary structure and the presence of disulfide bridges are used as constraints. Simplified structures obtained in this way have been converted to all atom models with plausible structure. Results have been compared with a similar approach using a well-established technique as molecular dynamics.The results obtained on small proteins show that Constraint Logic Programming techniques can be employed for studying protein simplified models, which can be converted into realistic all atom models. The advantage of Constraint Logic Programming over other, much more explored, methodologies, resides in the rapid software prototyping, in the easy way of encoding heuristics, and in exploiting all the advances made in this research area, e.g. in constraint propagation and its use for pruning the huge search space. Notwithstanding the continuous improvement in predictive methods, witnessed every two years by the world wide CASP experiment ,2, predi1. assemblying the structure of a protein using structural fragments of similar sequences, available in the protein structure repository (the Protein Databank ), and la2. representing the protein chain by a highly simplified model which is, hopefully, treatable.) lattice studied by Toma and Toma , 1, 10)domain domain {1, 2, ..., 10}. Suppose we wish to state that the weight of each item of X is 3, of Y is 4, and of Z is 5 and the total weight of selected items must be less than or equal to 40. Moreover, we wish to state that the number of items of X plus those of Y must be less than those of Z. This can be simply stated as:is a constraint that states that the three variables X + 4 * Y + 5 * Z ≤ 40, X + Y <Z3 * knapsack problem using . In general, in the modeling stage we can use constraints as well as declarative programs involving them.We have modeled a sort of constraint solver that is available in the language. The constraint solver uses constraints for sensibly pruning the search tree. One of the main capabilities is called constraint propagation. Constraint propagation reduces the domains of the variables eliminating those values that cannot lead to constraint solutions. For instance, in the considered example, constraint propagation reduces the domains of the variables X, Y, and Z to {1, ..., 4}, {1, ..., 4}, and {3, ..., 6}, respectively. For finding a possible solution, a further built-in capability – the labeling predicate – can be used. We can look for a generic solution as well as for a solution minimizing some function. In the example above, we could ask for minimizing the function -2X2 + Y + 4Z. This can be done by adding a constraint of the form:Solution's search is performed by a F = -2 * X * X + Y + 4 * Z, labeling([minimize (F)], ).The constraint solver then exploits the solution's search using constraint propagation and branch-and-bound techniques returning the answer:F = 3, X = 3, Y = 1, Z = 5F be a linear function.The library clpfd of SlCStus Prolog  allows tConstrain & Generate technique introduced as opposed to the Generate & Test technique of the classical Logic Programming approach . In theWe have followed the Constrain & Generate programming style for encoding the protein structure prediction problem. As a matter of fact, the main predicate of our solution is of the form reported in Figure Methods Section), writetime and print_results are output predicates. The constraint predicate is defined using several predicates each of them modeling one of the properties of the problem. For instance, the predicate next_constraints sets the distance between consecutive aminoacids .Briefly, next_constraints recursively calls the predicate next for each pair of consecutive aminoacids. Assume that < code described in the Methods Section. All tests are done using SICStus PROLOG 3.11.1 [In Table G 3.11.1  and a PCN of aminoacids, the execution time, the energy of the best model found and its RMSD from the native structure for all the residues and for the core residues only. When there is not explicitly written "limit" it means that the program successfully terminated in the time reported; otherwise the program terminated due to time limit. We wish to observe that the results with time limit 10 h/24 h are typically computed in few hours. The rest of the time is used to further explore the solutions' space.From left to right, the meaning of each column is as follows: the protein PDB identification code, the number η is reported a further constraint on the compactness ratio η is added before the search. CF = η bounds the linear distances |Xi - Xj|, |Yi - Yj|, and |Zi - Zj| between all pair of residues i and j to ηN where N is the length of the primary list. If η is low (e.g. 0.2), this constraint imposes a compact form to the protein and strongly reduces the running time.When a CF = ss = γ is reported.One of the structural constraints considered is the presence of disulfide bonded residues (ssbonds). The rigid structure of the lattice is such that a low value of Euclidean distance  between ssbonds often precludes all possible solutions. For this reason the default is chosen as 6. However, in some cases we tried computations with lower value. In these cases in the table the text GOR IV secondary structure prediction method [The secondary structure, as computed from the deposited structure in PDB, has been input as constraint. As a unique exception, in the case of 1VII(*) we have instead predicted it using the n method .Detailed models from lattice models Section. There is some improvement in general on RMSD from native structure. This is especially significant when the starting structure is already close to the native one, being not merely due to increasing compactness of the structure. It is moreover reassuring that the procedure we are discussing is able to recover realistic models starting from the very simplified lattice models. The RMSDs of the resulting detailed models from the corresponding native structures are reported in Table The predicted structures have been also transformed into all atoms models as described in the 3. For the sake of comparison, we have used it as an ab-initio predictor . The comparison is obviously not fair because in our case secondary structure is known and not predicted. Times are obtained from the result files, but it is not clear to which machine/CPU occupation they refer. Results are reported in Table We conclude the section comparing some results of our prediction with those returned by the well-known HMMSTR/Rosetta Prediction System . This pr on the  lattice. Secondary structure elements have been imposed through a constraining potential as described in the Methods Section. In order to search the conformational space a simulated annealing procedure has been adopted. Globularity of the simulated proteins is forced by a harmonic constraint on the radius of gyration.We have used secondary structure information in conjunction with the well-established methodology of molecular dynamics simulations in order to implement a procedure similar to the one implemented using α atoms on the whole protein and only on core residues and the simulation time. The last column reports the RMSD from native structure for models obtained by  after addition of all atoms and energy minimization as described in the Methods Section.The simulation time, ranging approximately between one and four CPU days, required for folding each protein on a 1.533 GHz AMD Athlon processor is reported in Table The simulation time needed for obtaining structures similar to native structures increases with the size of the protein both for the increasing size of the system and for the longer simulated annealing runs needed because of increasing complexity of the free energy landscape. Unfortunately a safer scheme would employ substantially longer simulation times.This fact prompts for searching alternative ways to employ the same ideas.The results in terms of RMSD from native structure support the idea that folding may be achieved, at least in simulation, by a hierarchical approach where local secondary structure elements are formed first and later their arrangement and contacts are optimized. A similar conclusion has been reached using a different model by Maritan and coworkers . The RMS. With respect to the few applications reported in the literature so far using the same methodology [. In the present application the known secondary structure of the protein has been imposed as a constraint.  has been applied on face centered cubic lattice models of proteins where every aminoacid is represented by a single point on the lattice that can take one out of six possible positions with respect to the previous three aminoacids. It is immediately seen that the time needed for a systematic space search for such model grows exponentially with the number of free aminoacids.  is a programming paradigm that is suited for the solution of optimization combinatorial problems. In  the problem and the related heuristics are extremely natural to be programmed. Moreover, the constraint propagation allows to control the search in the huge solution's space.The purpose of the present work was to demonstrate that the protein folding problem can be approached by a well-established programming paradigm like hodology , mainly hodology ,24, the The results obtained using this approach and reported in Tables Further work is being devoted towards a more realistic modeling representation of the protein, with at least two centers of interaction per residue, and towards refinement of the potential function by including a term for rotamer preferences. This term should map on the lattice the directional preferences of each unit with respect to the previous three units. Each of the six possible next positions for each unit should be weighted by an energy term derived from database analysis.α trace, the RMSD on core residues of the resulting models, after energy minimization, compared to native structures, is as low as 4.8 Å for the thermostable domain of villin headpiece (PDB id.: 1VII), 3.6 Å for the WW domain (PDB id.: 1E0M), 2.3 Å for the coat protein-binding domain of bacteriophage P22 (PDB id.: 2GP8).Also the optimal size of non constrained parts of the chain will be determined in order to allow more possible relative orientations among constrained secondary structure elements, possibly without increasing significantly the computation time. At present, however, when the positions of all atoms are reconstructed from the lattice CIt should be also noted that both the thermostable domain of villin headpiece and the WW contain three secondary structure elements that can be arranged in different ways in order to produce a compact structure. The low RMSD is therefore significant.A comparable protocol employing a molecular dynamics simulated annealing procedure still leads to superior results for larger proteins, as expected because the protein representation is more accurate, but it takes longer execution times between one and four days on a 1.5 GHz P3 machine. could be useful for finding starting conformations for further refinement.Recent results have shown that simplified models and more refined models can be employed successfully in hierarchical modeling procedures ,10. The primary structure. This structure uniquely determines the (3D) native conformation, also known as tertiary structure. The protein structure prediction problem is the problem of predicting the tertiary structure of a protein given its primary structure. The native tertiary structure minimizes the global free energy of the protein.The sequence of aminoacids defining a protein is called sphere centered in its Cα atom; the distance between two consecutive Cα atoms is assumed to be 3.8 Å Recent results  and the value of 2.6 Å empirically determined in [contact has been developed [x, y) the energy value associated to a contact between aminoacids x and y ; this value can either be positive or negative, according to the pair x, y.We consider each aminoacid as a single e, e.g., ) show thmined in  for van eveloped ,13. Let Face-Centered Cubic Lattice  that allows realistic angles between consecutive residues. The lattice is composed by cubes of size 2, where the central point of each face and the vertices are admitted. Thus, the domain  consists in a set of triples <x, y, z> where <x, y, z ∈ >. We recall that given a point <x, y, z>, its 2-norm is: ||<x, y, z>|| = . Given two points p1 and p2, ||p1 - p2|| is known as their Euclidean distance.According to  we use t lattice, two points at Euclidean distance  are linked together, forming a lattice unit, corresponding to the distance of 3.8 Å. In this lattice, each point is adjacent to 12 neighboring points. A contact is defined between two non adjacent residues placed on two vertices of a side of a cube . This number can be considered a good approximation of the limit of 6.4 Å described above.Going back to the S = s1 ... sn, with si aminoacids, a fold of S is a function ω : {1, ..., n} →  such that: ||ω(i) - ω(i + 1)|| =  and ||ω(i) - ω(j)|| ≥ 2 for i ≠ j. The first constraint states that consecutive aminoacids have a fixed distance, corresponding to one lattice unit; the second that each aminoacid occupies a unitary sphere and that two spheres cannot overlap.In this setting, it is possible to formalize the protein folding problem as an optimization problem. Given a sequence ω of S such that the following energy is minimized [The protein folding problem can be reduced to the optimization problem of finding the fold inimized ,27:ω(i), ω(j)) is 1 if ||ω(i) - ω(j)|| = 2, 0 otherwise. To avoid solutions equivalent modulo simple symmetries, other constraints can be added on the first positions.where contact is proven to be NP-complete on various lattices ,29. Howe., e.g., )SAWfcc = 1.26N0.162(10.0364)N     (2)This formula should modify in the presence of additional constraints as mentioned later.Background Section. The complete program and related material can be found in [ where ℓ is the length of each unit and α is the cosine of the angle made by each unit with the direction of the preceding unit. The average end-to-end distance is clearly related to the average maximal dimension of the chain. Based on a survey of protein structures Huang and Powers derived the following approximated formula for the radius of gyration (in Å): 2.2N0.38 [ the radius of gyration. The default value for CompactFactor was therefore assumed to be approximately equal to  times the radius of gyration which in turn was computed by the empirical formula 2.2N0.38 [Our implementation of the protein folding minimization problem described in the above sections is based on the code briefly introduced in the found in . The profound in . In part2.2N0.38 . Note th2.2N0.38 .The auxiliary file data.pl stores the Primary and Secondary structures of the proteins that one wishes to test, as, for instance in the example reported in Figure  lattice to be even (a property of the  lattice) and we add some constraints for avoiding equivalent symmetric solutions. In what follows, we refer to predicate names as used in the code. avoid_symmetries removes redundant admissible conformations equivalent to others modulo some symmetries and/or rotations. The predicate assigns immediately three consecutive aminoacids positions (in the Tertiary list).The intrinsic complexity of the problem forces us to introduce several other constraints. For instance, we constrain the sum of the coordinates of each aminoacid in the Cαs modelling aminoacids.With distance_constraints, we also impose that two non consecutive residues must be separated by more than one lattice unit, to reflect the steric interaction between the x, y, z coordinate, is smaller than CompactFactor × N.As described above, compact_constraints imposes that, for every pair of aminoacids, the norm of the projection of their distance on each  lattice, the angle between three consecutive residues can assume values in {60°, 90°, 120°, 180°}. In real proteins, steric occupancy and energetic potential show a clear distribution of bend angles in the range 90°–150° [ lattice, it is a good approximation to exclude 60° and 180° angles, as unfeasible. This constraint allows us to restrict the search space from a number close to 10N (cf. formula (2)) to a number close to 5N.Further constraints are related to angles. In the 90°–150° ,35. WhenLattice model Section, a contact is generated by two non consecutive aminoacids with Euclidean distance less than or equal to 2. As a consequence of the constraints applied, it suffices to check for a contact when the lattice distance equals 2, since distance_constraints excludes from the domain the possibility to place two non consecutive aminoacids at one lattice unit.As said in the i, j): elements i, i + 1, ..., j of the input sequence form an α-helix; strand: elements i, i + 1, ..., j are in a β-strand; ssbond: there is a disulfide bridge between element number i and j.We also impose constraints coming from secondary structure information. Secondary structure can be predicted with good approximation . In ourIndexes that stores torsional angles defined by four consecutive aminoacid positions. Due to  lattice structure and our constraints, every four consecutive aminoacids can form only 6 discrete angles. Thus, each variable in Indexes can assume a value i from {0, ..., 5}, representing torsional angles of 0°, 60°, 120°, 180°, 240°, 300°, respectively. With these conventions, helices are approximated by sequences of indexes of the form 5, 5, 5, ... while β-strands are associated to sequences of the form 3, 3, 3, .... Note that specifying the coordinates of three points (i.e. to place and orient the protein) and the indexes, uniquely determines the conformation, ssbond, introduces a maximum distance constraint between the aminoacids i and j. The predicate energy_constrain is developed using an auxiliary symmetric matrix M. The optimal fold is reached when the sum of M elements is minimal. During the labeling phase, the information stored in M is used to control the minimization process and to cut the search tree.We use an auxiliary list called solutions_search. We describe here briefly the main features of this predicate and of its auxiliary predicates.To reduce the size of the solution's space visited during execution, we have replaced the built-in labeling predicate with an ad-hoc constraint-based solution search predicate, called solutions_search • If the Tertiary list or the Indexes list is ground , then it terminates the folding process .choose_labeling. When this procedure terminates, it calls recursively solutions_search. Termination is guaranteed by the fact that each call to choose_labeling reduces the number of non-ground variables.• Otherwise, it calls choose_labeling • If the number of variables to be instantiated is low (in our code less than 4), it calls the built-in labeling.selection_strategy. This predicate computes several subsequences of the list of Indexes. Each subsequence consists of alternations of ground elements and non-ground variables. selection_strategy selects the most known subsequence, namely the one containing the smallest ratio of variable over ground indexes, preferring the ones that include a ssbond. If in the selected subsequence there are too many variables, an arbitrary subsequence cut is done. After the subsequence is selected, the procedure labeling_new_launch is called.• Otherwise, it calls labeling_new_launch It calls the auxiliary predicate labeling_new but stops the solution search when the global runtime is greater than the input time limit. If this is the case, the best computed solution is returned.labeling_new This procedure receives the chosen sublist to be folded. Each index variable in it, is assigned an admissible value between 0 and 5. The order of values that is tried for each index is described by a pre-computed auxiliary list. For each torsional index, a frequency statistics of the 6 indexes is pre-computed and extracted from the PDB, according to the specific aminoacid sequence involved locally. We use this information to direct the search and explore first the most common torsional angles, in the hope that this selection rule reflects nature's strategy.t of variables in a phase, we collect the best known ground admissible solution, its energy and its associated potential matrix. We compare the current status to history and decide if it is reasonable to cut the search tree. In particular, we designed a heuristic that allows to control the effectiveness of the cut, adapting it dynamically to the status of the fold. Practically, when the protein is partially specified, we estimate the ratio between ground and non-ground variables in the potential matrix. If the ratio is low (i.e. the protein is poorly determined), we allow the current energy to be worse than the corresponding counterpart in the best fold so far reached. When the ratio is high  we constrain the current energy to be slightly lower than the previous best known.Moreover, the energy associated to the fold is minimized. For doing that, after each instantiation of a fixed number θ - θ0)2 kcal/(mol rad2). The reference target angles (i.e. θ0 in the previous formula) were set to φ = -139 and ψ = 135 for residues in β-strand and to φ = -48 and ψ = -57 for residues in α-helices. For all constrained residues also the ω dihedral angle was constrained at 180 degrees.In order to have a fair comparison with a similar approach using all-atom protein models we built detailed all atom models for six proteins in the studied set  and imposed, through torsional constraints, the secondary structure geometry found in the native structure. The constraining potential was 100 *  was imposed. The target radius was decreased during the simulation from a value proper of an extended conformation down to the value given by 2.2N0.38 [N is the number of residues.In order to obtain globular protein during simulation a constraint on the radius of gyration  using the Generalized Born implicit solvent model was performed. The resulting structure at the end of the simulation was energy minimized.The models obtained by t server  into an t server  and the  procedure were constrained as described above.The initial minimizations required 1500 minimization steps each, because the starting structures were built from the lattice models. The final minimization, on the structure relaxed by molecular dynamics simulation, employed 900 minimization steps. During molecular dynamics simulation the radius of gyration and backbone torsion angles corresponding to residues constrained in the


Whether inhaled corticosteroids suppress airway inflammation in chronic obstructive pulmonary disease (COPD) remains controversial. We sought to determine the effects of inhaled corticosteroids on sputum indices of inflammation in stable COPD.We searched MEDLINE, EMBASE, CINAHL, and the Cochrane Databases for randomized, controlled clinical trials that used induced sputum to evaluate the effect of inhaled corticosteroids in stable COPD. For each chosen study, we calculated the mean differences in the concentrations of sputum cells before and after treatment in both intervention and control groups. These values were then converted into standardized mean differences to accommodate the differences in patient selection, clinical treatment, and biochemical procedures that were employed across original studies. If significant heterogeneity was present (p < 0.10), then a random effects model was used to pool the original data. In the absence of significant heterogeneity, a fixed effects model was used.We identified six original studies that met the inclusion criteria (N = 162 participants). In studies with higher cumulative dose (≥ 60 mg) or longer duration of therapy (≥ 6 weeks), inhaled corticosteroids were uniformly effective in reducing the total cell, neutrophil, and lymphocyte counts. In contrast, studies with lower cumulative dose (< 60 mg) or shorter duration of therapy (< 6 weeks) did not demonstrate a favorable effect of inhaled corticosteroids on these sputum indices.Our study suggests that prolonged therapy with inhaled corticosteroids is effective in reducing airway inflammation in stable COPD. Chronic obstructive pulmonary disease (COPD) is characterized by prominent airway inflammation ,2. The iOne potential therapy for down-regulating the inflammatory process in the airways is through the use of corticosteroids, which are potent but non-specific anti-inflammatory agents. Some in vitro studies have demonstrated that inhaled corticosteroids can modulate certain aspects of the inflammatory cascade in COPD ,9; howevMEDLINE (1966–2004), EMBASE (1980–2004), CINAHL (1982–2004), and the Cochrane Databases were searched for randomized, controlled clinical trials that used induced sputum to evaluate the effect of inhaled steroids on airway inflammation in stable COPD. The search was restricted on articles published in the English language, using human participants. Subject headings included disease-specific search terms , drug-specific search terms , and laboratory method-specific search terms . We also scanned the bibliographies and reference lists of retrieved articles to supplement the electronic searches. We contacted the primary authors for additional data and/or clarification of data.1), the ratio of FEV1 to forced vital capacity (FVC), percent predicted reversibility with inhaled bronchodilator, the specific brand of inhaled corticosteroids and the dose as well as the duration of therapy. Cumulative dose of inhaled corticosteroids was calculated by multiplying the average daily dose by the total days of treatment. All formulations were converted to beclomethasone equivalent based on the recommendations from the Canadian Asthma Consensus Report [The primary objective of this meta-analysis was to compare the changes in sputum inflammatory indices among stable COPD patients before and after treatment with inhaled corticosteroids, using the control group in each individual studies as the referent. We chose sputum as the primary source of the analysis because there was a marked scarcity of quality studies which had evaluated the effect of inhaled corticosteroids from bronchoalveolar lavage fluid or tissue biopsy specimens. The inflammatory indices included total cell, neutrophil, macrophage, eosinophil, lymphocyte, and epithelial cell counts and interleukin (IL)-8 levels. Since the actions of oral corticosteroids may differ from those of inhaled corticosteroids, we excluded studies that evaluated the effects of oral corticosteroids on sputum inflammatory indices. From each selected article, two investigators  abstracted the following baseline information: the source of data, study design, inclusion and exclusion criteria, concomitant drugs, demographics of study participants including sample size, age, sex, current smoking status, pack-years of smoking history, predicted forced expiratory volume in one second , indicating that inhaled corticosteroids had a favorable effect in reducing total count compared with controls  Figure . ImportaInhaled corticosteroids had a salutary effect on neutrophil counts in the sputum. As compared with the control group, the standardized mean difference in those treated with inhaled corticosteroids was -2.16 units    Figure . InhaledTo evaluate whether the magnitude of the reduction in the inflammatory cells was modified by the absolute levels of the inflammatory cells in the sputum at baseline, we performed a stratified analysis based on the total cell counts at baseline   Figure . For preBy combining data across the clinical studies, we increased statistical power to demonstrate a salutary effect of moderate to high doses of inhaled corticosteroids on some inflammatory indices in the sputum of patients with stable COPD. Over a short term, these medications reduced neutrophil, lymphocyte and epithelial cell counts in the sputum of stable COPD patients. They had smaller (and insignificant) effect on sputum eosinophils and IL-8. They had little effect on sputum macrophages. Although the magnitudes of these reductions were relatively small, they may explain why inhaled corticosteroids decrease cough and sputum production , reduce We also found that duration of therapy and total cumulative dose, which are related constructs, made a material difference to the overall results. Short trials (less than 6 weeks in duration) were uniformly "negative"; while longer term trials (at least 6 weeks of therapy) were mostly positive. Similarly, trials that exposed the patients to higher cumulative dose were more "positive" than those that exposed patients to lower dose. This suggests that duration of therapy and total cumulative doses may be important determinants of the effect of inhaled corticosteroids on airway inflammation.Although corticosteroids delay neutrophil apoptosis and may increase neutrophil survival ,26, they1. Although there was a trend towards improvement, we did not find a statistically significant effect of inhaled corticosteroids on FEV1. Larger randomized trials have demonstrated, however, that inhaled corticosteroids significantly improve FEV1 over the first three to six months of therapy [Superficially, the present data on sputum eosinophils appear to be inconsistent with the known effect of corticosteroids in general on eosinophils. Many experiments have shown that eosinophils are exquisitely sensitive to corticosteroids ,30. The  therapy ,32-34, sIn the present review, we did not include randomized studies that used bronchoalveolar lavage (BAL) or bronchial biopsies to measure inflammatory cells in the airways. However, in one study, Balbi and colleagues  observedWe also did not include studies that used systemic corticosteroids. Barcyk and colleagues  have repAlthough in the present review, we could not adequately determine the effects of tobacco smoke exposure on the relationship between inhaled corticosteroids and airway inflammation, there is a growing body of evidence to suggest that active smoking may attenuate the effectiveness of corticosteroids in suppressing airway inflammation. Active smoking increases oxidative stress and up-regulates the production of various pro-inflammatory cytokines including Il-6, IL-8, IL-1β and monocyte chemoattractant protein-1 in airways, which may through a series of complex pathways lead to a state of steroid resistance . AdditioThere are certain limitations with the present analysis. First, although we used stringent entry criteria in order to minimize the heterogeneity in the research methods employed by each of the selected study, there were still some variations in the study design, the exposure medications, and the target population across the original studies. However, the differences in the characteristics of the studies were relatively small and unlikely to have materially affected the overall findings of the current review. We also contacted the primary authors to clarify any ambiguities or to obtain additional data, where necessary, to further minimize the "noise" inherent to meta-analyses. Moreover, to accommodate various differences in the methodology of data collection and laboratory techniques employed across the original studies, we converted the individual data into standardized mean estimates, which enhanced the comparability of data across the original studies. Second, it is possible that corticosteroid therapy could have affected the volume of sputum recovery, decreasing the total sputum cell counts in those patients exposed to this therapy. To mitigate this possibility, the cell counts were expressed as cells per volume of sputum recovered.In summary, the present meta-analysis suggests that inhaled corticosteroids when used for longer than 6 weeks can significantly reduce neutrophil counts and other inflammatory indices in the sputum of patients with stable COPD. Large randomized controlled trials are needed in the future to confirm these early findings and to determine whether these salutary effects persist longer than 3 to 4 months of therapy.COPD chronic obstructive pulmonary disease1 forced expiratory volume in 1 secondFEVFVC forced vital capacitySD standard deviationIL-8 interleukin-8DDS and SFP have received honoraria for speaking engagements from GlaxoSmithKline (GSK) & AstraZeneca, and have received consultation fees and research funding from GSK. However, no part of this work was financed by these companies. This work was funded by Canada Research Chair and a Michael Smith/St. Paul's Hospital Foundation Professorship in COPD.All the authors contributed to the design and implementation of the study. Data analyses were performed by WQG and DDS. All authors contributed to the write-up of the manuscript.The pre-publication history for this paper can be accessed here:


Order Charadriiformes (shorebirds) is an ideal model group in which to study a wide range of behavioural, ecological and macroevolutionary processes across species. However, comparative studies depend on phylogeny to control for the effects of shared evolutionary history. Although numerous hypotheses have been presented for subsets of the Charadriiformes none to date include all recognised species. Here we use the matrix representation with parsimony method to produce the first fully inclusive supertree of Charadriiformes. We also provide preliminary estimates of ages for all nodes in the tree.Three main lineages are revealed: i) the plovers and allies; ii) the gulls and allies; and iii) the sandpipers and allies. The relative position of these clades is unresolved in the strict consensus tree but a 50% majority-rule consensus tree indicates that the sandpiper clade is sister group to the gulls and allies whilst the plover group is placed at the base of the tree. The overall topology is highly consistent with recent molecular hypotheses of shorebird phylogeny.The supertree hypothesis presented herein is (to our knowledge) the only complete phylogenetic hypothesis of all extant shorebirds. Despite concerns over the robustness of supertrees (see Discussion), we believe that it provides a valuable framework for testing numerous evolutionary hypotheses relating to the diversity of behaviour, ecology and life-history of the Charadriiformes. Prosobonia leucoptera, the Canary Islands oystercatcher Haematopus maedewaldoi, and the Great auk Pinguinus impennis.The shorebirds and allies  presentet al. The higher resolution of the majority-rule tree means it is more likely to be of use in comparative studies. We therefore estimated node ages for this topology only see  and 2. WA fuller understanding of the phylogenetic affinities of fossil shorebirds will probably improve estimates of node ages for the group. For example, the extinct form Graculavidae, is represented by fossils from the Maastrichtian of New Jersey  and Cretet al. [b but used a range of methods including parsimony and Bayesian analyses. We therefore combined these trees to minimise bias. In contrast, Ericson et al. [et al. [Supertrees are still at an early stage of development and many aspects of MRP, and supertree methods in general, are not yet clearly understood. Steps can be taken to ensure that the supertree includes the most appropriate sets of sources trees, such as only using trees from explicitly phylogenetic studies. This is not always straightforward and could result in the exclusion of important information. For instance, in our shorebird supertree, we included Sibley and Ahlquist's DNA-DNA hybridisation tapestry  althoughet al.  based thn et al.  used two [et al.  should b [et al. ,33 and tOur shorebird supertree is highly consistent with recent advances in the molecular phylogenetics Charadriiformes. However, we urge caution when using the tree in comparative analyses and encourage the additional use of alternative phylogenies and branch length assumptions. It is particularly important to note that the position of some groups such as the Alcinae remains controversial and that although the majority rule tree is consistent with recent molecular studies, the strict consensus tree fails to resolve the deepest nodes.Gallinago and Vanellus are desirable. Furthermore, additional work is required to establish the true affinities of the Turnicidae. Nonetheless, it appears that shorebird phylogeny is gradually approaching a consensus view. The broad taxonomic scope and consistency of the supertree mean that is of potentially great value to future comparative studies (accepting the caveats discussed above) of the behaviour, life-history, ecology and conservation of this diverse group.The supertree presented here is, to our knowledge, the first attempt to reconstruct the phylogeny of the entire order Charadriiformes. Overall, the supertree is highly consistent with recent molecular hypotheses of shorebird phylogeny. However, it is apparent that fresh attempts to resolve both the phylogeny and estimates of age will be dependent on further gene sequencing and new fossil discoveries. The affinities of the Alcinae and the relationships between the three major shorebird clades require further corroboration, and studies of several genera such as  covering the years 1981 to 2004. We used the single key strings phylogen*, cladistic*, clado*, classif*, systematic*, and taxonom*  in the topic field, in conjunction with a major Charadriiformes taxon name (scientific or common). As supertree methods have been criticized for being biased towards historical trends, we preferred those studies that explicitly set out to derive a phylogenetic hypothesis and so exclude purely  descriptive taxonomic works. The Sibley and Ahlquist [Possible source trees were identified from online searches of Web of Science Ahlquist  DNA-DNA Ahlquist . We therAhlquist  DNA-DNA contra Monroe and Sibley [Larus thayeri [Larus glaucoides  or a spd Sibley  include d Sibley . We incld Sibley  to produd Sibley ). The oud Sibley ,68. ThisWe produced an MRP matrix of the 51 Nexus  source tThe tendency of large data sets to produce many sub-optimal trees that are close in length and topology to the shortest tree is a serious problem in phylogenetics. Standard heuristic searches frequently are trapped searching within globally sub-optimal "islands" and the tree search is often aborted before completion. Nixon  proposedWe did not calculate any measures of branch support for two reasons. First, their validity and meaning is questionable in MRP supertrees . Second,et al. [Nupharanassa tolutaria, Rupellian), Phalaropus , Burhinidae , Glareolidae , Alcinae , Stercoariini , and Larini . We took the midpoint of the range from the Fossil Record 2 [Following Purvis  and Biniet al.  we datedet al.  as the sRecord 2  as our dRecord 2 ). We assRecord 2  in termsRecord 2 . The phyet al. [A, B, and C where A and B are sister taxa and C is sister to A and B. The root is dated to 10 million years (myr) from fossil evidence, and independent molecular data provides estimates of divergence based on the number of substitutions per site. The molecular estimates of branch lengths are as follows: A, 6 substitutions; B, 8 substitutions; C, 20 substitutions; A and B are 11 substitutions from the root. A and B are therefore separated from their common node by a mean of 7 substitutions. The total length from A and B to the root is thus 18 substitutions compared to 20 for C (a mean of 19). This can be converted to date estimates such that 19 substitutions are equivalent to 10 myr. The dates of the tree are then: , C: 10)). There were no cases where multiple source trees with molecular divergence dates were able to provide estimates for the same node. We estimated relative dates from multiple nodes rather than a single dated node to minimise correlative errors in estimates.Source trees may include estimates of relative branch lengths . This allows further dating of the supertree but is problematic because different relative estimates are not comparable and cannot be applied directly to the supertree . Howeveret al. . For exaTo provide date estimates for all nodes in the tree we employed a pure birth model to date nodes for which absolute and relative dates could not be attained . Pure bidate of daughter = date of ancestor *(log daughter clade size/log parent clade size)For example, the age of a daughter node that subtends 12 taxa, estimated from its immediate ancestor dated to 20 myr and which subtends 19 taxa is:20*(log(12)/log(19)) = 16.879We applied this approach to estimate the ages of daughter nodes based on dates  of ancestral nodes. We had no ancestral node on which to base estimates of the most basal clade. In this case, we rearranged the pure birth formula and calculated the age of the ancestral node from its two daughter nodes, taking the mean as our "best estimate". Finally, to estimate the ages of nodes between daughter and ancestor nodes of known age we spaced the nodes evenly along the branches length .GHT assisted in the design of the study, carried out the phylogenetic analyses and node dating, and drafted the manuscript in partial fulfillment of a doctoral degree at the University of Bath. MAW assisted in the design of the study and with editing and revision of the manuscript. TS assisted in the design of the study, collection of source trees, and editing and revision of the manuscript. All authors read and approved the final manuscript.Estimates of node ages and node support (branch lengths.xls) Node numbers correspond to figures 2-9. Five types of estimate were used: a) absolute dates from the fossil record; b) absolute dates from molecular point estimates; c) relative dates based on branch length estimates from molecular studies; d) estimates based on a pure birth model (see text for details); and e) even spacing of nodes along branches with daughters and ancestors of known age. The numbers of characters supporting each node are provided (column D), this is equivalent to the number of source trees that share the equivalent node (see text for details).Click here for fileShorebird supertree  Shorebird supertree based on 50% majority-rule consensus of 1496 shortest trees with calibrated branch lengths. Scale bar indicates time from the present in millions of years.Click here for fileShorebird supertree  Shorebird supertree based on 50% majority-rule consensus of 1496 shortest trees.Click here for fileSource trees (source trees.xls) A summary of each tree used is given including the data type and main taxa studied. This is a brief summary and the original papers should be consulted for full details.Click here for fileMRP matrix (shorebirdMRP.txt) The MRP matrix used in the shorebird supertree analysis.Click here for fileCalibrated supertree (shorebirdsupertree.txt) The supertree in nexus format including branch length estimates.Click here for file


Natural variation rather than genetic modification - is this the way to achieve global food security? But I notice also that they may become fixed and permanent in any stock, by painting and repainting them on every individual, until at last nature adopts them and bakes them into her porcelain”—Ralph Waldo EmersonThe history of domesticated plant form and function evolves along a two-tiered track that doubles back on itself, offering panoramic vistas of natural forces intertwined with the creative force of human endeavor . For appPLoS Biology  have been selected by humans in the last 10,000 years and inevitably represent a subset of the variation found in their wild ancestors. Cultivars are recognizable because they manifest characteristics that are associated with domestication in plants. Unusual or extreme phenotypes, such as large fruit or seed size, intense color, sweet flavor, or pleasing aroma are often selected by humans and maintained in their cultivars for aesthetic reasons, while synchronous ripening or inhibition of seed shattering  are selected to facilitate harvest. These phenotypes may occur in nature but they will frequently be eliminated by natural selection before they are fixed in a population. Because of human selection, cultivars may exemplify a range of exaggerated phenotypic attributes that give them the appearance of being, on the whole, more diverse than some of the wild populations from which they were derived, but in truth, domestication usually represents a kind of genetic bottleneck. Furthermore, cultivars are grown in agricultural environments that are generally more uniform than the environments in which wild species grow, and this tends to further narrow the gene pool. Thus, while cultivars may embody a high degree of obvious phenotypic variation, this may not always be a good predictor of the extent of their genetic variation.The landrace varieties are the earliest form of cultivar and represent the first step in the domestication process. Landraces are highly heterogeneous, having been selected for subsistence agricultural environments where low, but stable yields were important and natural environmental fluctuation required a broad genetic base . LandracWild relatives and early landrace varieties have long been recognized as the essential pool of genetic variation that will drive the future of plant improvement . Early pIn today's world where automated sequencing and DNA synthesis are mundane activities, it may seem contradictory to be worrying about saving or using “old genes.” Can't new ones be synthesized to order? Can't we modify a plant at will by introducing a new gene or two into an existing variety? Why should we worry about saving populations of historically valuable genes in millions of living plant specimens at great cost to the tax-paying public?Perhaps it is not the genes themselves we are now in fear of losing. It is the information they encode in all their combinatorial complexity. After all, we are only at the very beginning of the endeavor to understand the way in which a genotype confers a particular set of attributes to a living organism. The subtleties of phenotypic plasticity in the face of a changing environment and the layers of genetic redundancy that characterize biological systems are largely mysterious. We have only just begun to consider the millions and billions of genetic trials and errors that have been evaluated by nature over evolutionary time. We cannot even begin to simulate the selective filters that have provided us with the diversity of form and function in the living world. We do know that living forms of natural diversity are needed to sustain life, and that it would be impossible to replace or recreate that diversity if it were lost at this time.As plant breeders, we know what to do with living forms of genetic diversity. If we keep our options open and learn to better utilize the reservoirs of natural variation that have been preserved in our gene banks and in the few remaining in situ populations of wild species and landrace varieties, an almost infinite array of novelty can be achieved using traditional, time-proven practices involving crossing and selection of genes that have withstood the test of evolutionary time . By restIn crosses between wild and cultivated species of inbreeding plants, alleles that were “left behind” during the domestication process may be reintroduced into the cultivated gene pool. This infusion of “new blood” renews and invigorates modern cultivars in surprising and interesting ways. It is not uncommon for some of the inbred progenies derived from these crosses to perform better than the better parent . This phThis approach has proven to be extremely successful in several crop species , variety development can go forward with the expectation that new varieties can be developed and distributed as inbred strains. This will come as very good news to people who are concerned about the infrastructural requirements needed to maintain a hybrid seed industry. Inbred variety seed can be saved from year to year without noticeable loss of vigor. Farmers are free to amplify the varieties and pass seed on to their neighbors if it proves valuable. Plant breeders living in parts of the world where germplasm diversity is highest are in the best position to explore its value. Until now, there have been few opportunities to make use of the wealth of natural diversity that abounds in many countries where people are the poorest and population is growing the fastest. This approach offers a way forward and can help people make good use of locally available resources to enhance the food security of their own nations.As we consider the implementation of smart breeding efforts in the future, we might ask, who will have access to nature's reserves of genetic diversity? How will knowledge about the patterns that govern the generation and selective elimination of that diversity help guide conservation efforts as well as current and future crop improvement efforts? What are the limits to biological variation? How far can we push those limits, and what will be the consequences of not pushing them? Who will participate in the endeavor? What will the rules of engagement be? What tools can we use to expedite the effort?What genetic characteristics will help us cope with climate change, global warming, the emergence of new pests and diseases, depleted soils, shortages of fresh water, and increasing levels of water and air pollution? What trace minerals, vitamins, and other metabolites will we need to breed into the crops of the future to fight the causes of hidden hunger, to prevent cancer, or to enhance the immune system? The combinatorial possibilities for crop improvement are almost infinite, as long as we maintain our options. Faced with a clear choice today, it is obvious that enhancing the potential for genetic flexibility in the future is a wise course of action and one we ignore at our peril.“Moreover, from our wild plants, we may not only obtain new products but new vigor, new hardiness, new adaptive powers, and endless other desirable new qualities for our cultivated plants. All of these things are as immediate in possibilities and consequences as transcontinental railroads were fifty years ago.”—Luther Burbank, 1914Luther Burbank (1849–1926) was one of America's first and most prolific plant breeders. He was inspired by Charles Darwin's Variation of Animals and Plants under Domestication (tication  to explotication .Nikolai Vavilov (1887–1943), a Russian geneticist and biologist, was one of the first to explore and actively collect wild relatives and early landrace varieties as sources of genetic variation for the future of agriculture. His botanical collecting expeditions (1916–1940) amassed many thousands of rare and valuable specimens that are preserved in the Vavilov Institute of Plant Industry in St. Petersburg, the world's first seed bank and inspiration for the International Crop Germplasm Collections (http://www.sgrp.cgiar.org/publications.html). Vavilov's concepts in evolutionary genetics, such as the law of homologous series in variation  was also well known for his plant collection expeditions and eloquent expositions about the value of wild relatives and early domesticated forms of crop plants  was an avid collector of exotic tomato germplasm. He noted that up until the 1940s, progress in tomato improvement lagged and few major innovations were achieved. The turning point, according to Rick, was the introduction of exotic germplasm. As a cultivated species, tomato had experienced a severe genetic bottleneck that led to extreme attrition of genetic variability compared to the wild species of Lycopersicon (persicon . Yet, Ripersicon , 1974. Hpersicon .


FOB1 and overexpression of SIR2 have been previously found to increase life span by reducing the levels of toxic rDNA circles in aged mother cells. We find that combining calorie restriction with either of these genetic interventions dramatically enhances longevity, resulting in the longest-lived yeast strain reported thus far. Further, calorie restriction results in a greater life span extension in cells lacking both Sir2 and Fob1 than in cells where Sir2 is present. These findings indicate that Sir2 and calorie restriction act in parallel pathways to promote longevity in yeast and, perhaps, higher eukaryotes.Calorie restriction slows aging and increases life span in many organisms. In yeast, a mechanistic explanation has been proposed whereby calorie restriction slows aging by activating Sir2. Here we report the identification of a Sir2-independent pathway responsible for a majority of the longevity benefit associated with calorie restriction. Deletion of  This study indicates that calorie restriction and Sir2 promote longevity in yeast through distinct pathways. This undermines the accepted view, and has implications for aging in higher organisms Saccharomyces cerevisiae has served as a useful model for aging research, leading to the identification of new longevity genes and pathways whose counterparts can be examined in higher eukaryotes  in the mother cell nucleus , and mutHM loci  of yeast cells can be accomplished by a reduction in the glucose concentration of growth media from 2% to 0.5% (or lower) and results in a 30%–40% increase in life span . Severalactivity . Growth activity , 2004.cdc25-10 is reported to decrease both rDNA recombination and ERC levels se  and fob1Δ. As previously reported for shorter-lived strain backgrounds . This analysis was performed in the BY4742 genetic background, which has a mean life span significantly longer than most other yeast strains commonly used for aging research . Includekgrounds , each ofkgrounds A.FOB1 increased life span individually in BY4742, we examined the effect of CR combined with deletion of FOB1. It is notable that this experiment has not to our knowledge been previously reported. We constructed a fob1Δ hxk2Δ double mutant and determined the replicative aging potential of this strain. As expected, both single mutants lived longer than wild-type mother cells (p < 0.001). However, the life span of the fob1Δ hxk2Δ double mutant greatly exceeded that of either single mutant (p < 0.001), suggesting an additional effect on longevity as a result of combining deletion of FOB1 with CR , and nearly double that of wild-type cells . Similarly, the life span of sir2Δ fob1Δ gpa2Δ triple mutant cells was significantly longer than that of sir2Δ fob1Δ double mutant cells (p < 0.001). In fact, the life spans of sir2Δ fob1Δ hxk2Δ and sir2Δ fob1Δ gpa2Δ cells did not differ significantly (p ≈ 0.4) from fob1Δ hxk2Δ and fob1Δ gpa2Δ cells, respectively. Thus, CR clearly enhances longevity in the absence of both Sir2 and Fob1, but not in the absence of Sir2 alone. While seemingly contradictory (see below), these findings demonstrate that Sir2 is dispensable for life span extension by CR, at least in the context of reduced ERC levels (as a result of fob1Δ).The observation that CR further increases the long life span of a Δ mutant , CR . The effect of growth on low glucose was even more pronounced in the sir2Δ fob1Δ double mutant, with mean life span increased by 25% on 0.5% glucose (p < 0.01) and by 60% on 0.05% glucose (p < 0.001).Genetic models of CR, such as  glucose , 2002; h glucose . Taking  glucose . Wild-tysir2Δ fob1Δ double mutant (sir2Δ fob1Δ double mutant derived from strain PSY316 (unpublished data). However, the previous experiments demonstrating that either deletion of FOB1 or overexpression of SIR2 increase life span were carried out in W303R  nor overexpression of SIR2 (p = 0.76) was sufficient to increase life span in the PSY316 background . Further, growth of SIR2-overexpressing cells on low glucose results in an additional life span increase (p < 0.001), similar to that observed for sir2Δ fob1Δ double mutant cells on low glucose. The observation that CR further enhances the already long life span of cells in which SIR2 is overexpressed reinforces our model that CR and SIR2 promote longevity by influencing different pathways , suggesting that, at least under normal conditions, Hst1 is not an important determinant of longevity.Our findings do not preclude the possibility that CR enhances Sir2 function through previously proposed mechanisms. However, the fact that the life spans of SIR2 overexpression, in particular, to increase life span in the PSY316 background supports the idea that Sir2 does not play a primary role in CR-mediated life span extension, as it is not straightforward to postulate a model whereby CR would increase life span via activation of Sir2 in a strain background that is insensitive to Sir2 dosage. Further, the inability of the fob1Δ mutation to increase life span in PSY316 provides a plausible explanation for why CR is unable to enhance longevity in the PSY316 sir2Δ fob1Δ double mutant, and suggests that either deletion of FOB1 fails to impact ERCs in this background or ERCs are not limiting for life span. While we cannot rule out the possibility that the Sir2-independent nature of CR is unique to BY4742, we note that BY4742 behaves like the majority of other strains with respect to increased life span in response to deletion of FOB1 or overexpression of SIR2, while PSY316 is the only strain (to our knowledge) that is unresponsive to these interventions , except for PSY316AR , PSY316AR fob1Δ::kanMX, and PSY316AR SIR2-ox. All gene disruptions were verified by PCR. In addition, sir2Δ mutants were verified by the sterility phenotype associated with this mutation. Strains overexpressing Sir2 were constructed by genomic integration of an extra copy of SIR2, as described  and streaked onto YPD. After 2 d of growth, single colonies were selected and patched to YPD. The next evening, cells were lightly patched to the plates used for life span analysis (4–6 strains per plate). After overnight growth, cells were arrayed onto solid medium using a micromanipulator and allowed to undergo 1–2 divisions. Virgin cells were selected and subjected to life span analysis. Cells were grown at 30 °C during the day and stored at 4 °C at night. Daughter cells were removed by gentle agitation with a dissecting needle and tabulated every 1–2 cell divisions. All life span experiments were carried out on standard YPD plates (2% glucose), except for the low glucose experiments, which were performed on YEP plates supplemented with the indicated amounts of glucose. In order to prevent introduction of bias, strains were coded such that the researcher performing the life span experiment had no knowledge of the strain genotype for any particular strain. For each experiment, each strain was randomly coded at the time of removal from frozen stock. One individual was responsible for assigning codes (K. T. K.) while a different individual (M. K. or B. K. K.) performed the micromanipulation and was unaware of the genotypes of the strains being analyzed.p-value matrices for each figure are available in p-values were calculated using the MATLAB ranksum function. Data shown in each figure and used to calculate p-values were derived from pair-matched, pooled experiments where each mutant was compared to wild-type cells examined within the same experiment(s). Strains are stated to have a significant difference in life span for p < 0.05.For statistical analysis, life span datasets were compared using a two-tailed Wilcoxon Rank-Sum test. Mother cell life span and Dataset S1p-values for a two-tailed test in which the life span data for the strain in the corresponding row were compared against the life span data for the strain in the corresponding column. Significant p-values (p < 0.05) are colored yellow. P-values were calculated using the MATLAB ranksum function.Each matrix contains the Wilcoxon Rank-Sum (46 KB PDF).Click here for additional data file.Dataset S2(16 KB TXT).Click here for additional data file.Figure S1(A) Life span extension by CR is maximized at 0.05% glucose in BY4742 mother cells. Mean life spans are shown for cells grown on 2% glucose (24.8), 0.5% glucose (28.3), 0.1% glucose (30.1), and 0.05% glucose (32.1).sir2Δ fob1Δ mother cells. Mean life spans are shown for cells grown on 2% glucose (26.0), 0.5% glucose (32.9), 0.1% glucose (40.8), and 0.05% glucose (42.0).(B) Life span extension by CR is maximized at 0.05% glucose in  (88 KB PS).Click here for additional data file.Saccharomyces Genome Database (http://www.yeastgenome.org/) accession numbers for the yeast genes and gene products discussed in this paper are CDC25 (SGDID S0004301), FOB1 (SGDID S0002517), GPA2 (SGDID S0000822), GPR1 (SGDID S0002193), HST1 (SGDID S0005429), HXK2 (SGDID S0003222), PNC1 (SGDID S0003005), and SIR2 (SGDID S0002200) The LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/) accession numbers for the non-yeast genes and gene products discussed in this paper are C. elegans Daf-16 (LocusLink 172981), C. elegans Sir-2.1 (LocusLink 177924), mouse Foxo3A (LocusLink 2309), and mouse SirT1 (LocusLink 23411).The


This review deals with the methods of identifying genes that have been activated by inner or outer impulses. The activation and subsequent expression of a gene can be detected by its transcription into a corresponding messenger ribonucleic acid (mRNA). Principles of the methods for identification of individual activated genes, as well as groups of activated genes are described, the former methods being mostly based on subtractive hybridization and serial analysis of gene expression (SAGE), the latter on microarrays. Examples of gene activation by the hormone 17beta-estradiol (E2) are given. In previous reviews, methods for the measurement of receptors and their interactions with other transcription factors and genes were described -3. In thThe life of humans and animals is influenced by the activity of a series of genes that are kept in a silent state, or are activated, depending on the temporary needs of the body. This switching on and off of each gene is executed by an assembly of transcription factors forming a transcription initiation complex (TIC).Examples of such transcription factors are estrogen receptors  that, before being incorporated into a TIC, have to be activated by E2. This hormone itself is synthesized, when an initial signal is given, by virtue of an activation of a series of appropriate genes. Via ER, E2 has manifold biological effects. Biological targets of E2 are, inter alia, blood vessel walls -8, bloodIt is evident that E2 is associated with many biological effects and that many genes must be involved. Consequently, ER must be able to bind to DNA segments, called response elements, in the neighborhood of various genes. The response elements participate, together with other transcription factors, in the formation of TICs that are specific for each gene.An important problem, currently studied in many laboratories, is to find out which genes are activated in various circumstances. The methods that solve this problem are based on a comparative  approach. A test (target) sample, containing active genes is compared with a control sample in which the genes have not been activated. Using this approach, the active genes are singled out among the multitude of inactive genes. However, the comparisons may reveal the opposite of activation, i.e., downregulation of genes.Generally, the activity of a gene is characterized by its transcription into mRNAs as the first step leading to the synthesis of specific proteins. Non-activated genes in the control tissue do not produce any corresponding mRNAs. In most methods, the mRNAs prepared from the test and control tissue are each reverse transcribed into the corresponding complementary deoxyribonucleic acid (cDNA), in order to enable a substantial increase of the material for analysis by polymerase chain reaction (PCR) . As most and Celera .The methods used for the identification of active genes are sketched below. Included are even methods that have not yet been used for the identification of E2-activated genes. It has to be mentioned that only principles, not technical details are dealt with in this review. Neither the techniques of cloning or of identification of genes by sequencing are described here. The readers who are not familiar with these techniques are advised to consult appropriate textbooks . The deActivated (expressed) genes can be found by comparison of gene contents in the test and control tissues. There are essentially two approaches for finding activated genes: (i) an individual identification, or (ii) an identification of expression profiles after hybridization to a set of known gene fragments (probes) attached to chips in microarrays.This approach means that genes are identified individually, even if several genes can eventually be picked up after cloning. There are several methods that can be used.Differential display seems to be the technically simplest method. Its name stems from the end-point that is a comparison of a side-by-side display of the test and control preparations by electrophoresis. In its basic form, total RNA of the test and control samples is separately subjected to reverse transcription into cDNA that, in turn, is PCR-amplified using arbitrarily chosen primers. The products are applied to a gel electrophoresis and the band(s) that are specific for one of the preparations are cut from the gel, further amplified by PCR (using the same primers) and eventually sequenced .In a more advanced version, mRNAs of the test and control cells are separately reverse transcribed to cDNA Fig. . Each trThe test mRNA is reverse transcribed into cDNA . This isIn another variant , the tesRsaI to obtain shorter, blunt-ended fragments. The test cDNA is divided into two portions  was used to isolate the ESTs specific for one of the libraries. For example, a fibroblast cell line cDNA library was hybridized with a hippocampus library; the common sequences were removed and the specific hippocampus sequences remained. Using the EST method, more than 2000 human brain genes were identified .The SAGE allows serial analysis of gene expression, an analysis of thousands of transcripts. It is based on the assumption that a short nucleotide sequence 10 base pairs (bp) – a tag – contains sufficient information to uniquely identify a transcript. In this respect SAGE differs from the EST approach.)  are cloned into a plasmid vector to create a SAGE library. Individual clones are then sequenced. SAGE is carried out for each sample to be compared.The principle of SAGE is as follows: mRNA is reverse transcribed into cDNA with use of a biotinylated primer, the cDNA is cleaved with a restriction endonuclease and the 3' portions are then isolated by binding to streptavidin beads . In anot) Fig. , mRNAs aThanks to the concatenation, many tags can be detected in a single clone . As eachStill, the specificity of detection of genes with use of the short tags is not absolute. There are two main problems . The firOne such method is called GLGI (Generation of Longer cDNA fragments from SAGE tags for Gene Identification) ,38,40. TMmeI. The longer tags increase the power of identification of genes, while not diminishing the sensitivity of SAGE given by the use of PCR and concatenation. Theoretical calculations showed that >99.8% of the 21 bp tags were expected to occur only once in a genome.Better of seems to be another variant of SAGE, the LongSAGE . This isSAGE was used for the investigation of differences in gene expression in various health conditions. In the studies of breast tumors , global The DNA microarray analysis is used to identify profiles of expressed genes in a given tissue and time. Thousands of known cDNA sequences or oligonucleotides are imprinted on a solid support, sometimes called a chip , using application robots. Typically, individual spots are 100–300 micrometers in size and are spaced about the same distance apart . More thsingle chip technique  produce chips by a photolithographic method in which thousands of different oligonucleide probes are synthesized in situ on the chip . A negative identification can be achieved by the use of siRNA . siRNAs are short RNA duplexes between 15 to 21 nucleotides in length. Once transfected into cells, a siRNA targets the mRNA containing an identical sequence and degrades it in a catalytic manner. The degraded message is no longer functional in translation (the biosynthesis of protein) and thus in the expression of the corresponding gene. SuperArray Corp. provides a line of validated populations of siRNAs in the form of SureSilencing siRNA kits.Quite often the gene identification obtained by an array is confirmed by other methods such as Northern blot analysis , or realThe methods described above can suit two purposes. The single-gene methods can detect and identify new, previously unknown, genes, whereas microarrays can handle a great number of known genes to establish profiles of their expression.SAGE seems to have advantages over hybridization-based methods for the studies of gene expression, such as differential display and subtractive hybridization. SAGE is superior to the EST approach in providing high efficiency in identifying the genes that are expressed at low levels and that represent a majority of genes in the human genome .Microarray techniques usually detect activation of a multitude of genes – a gene profile – that differs from the profile in control tissues/cells and thus – in medicine – may have a diagnostic and/or prognostic value. However, the microarray techniques usually require commercially produced chips as well as specialized equipment and advanced, powerful, computing facilities. Thus they are hardly affordable for small or medium-size laboratories unless they have substantial financial resources.A big question at another level remains so far unanswered: which is the biological "chain of commands" in a given tissue and time resulting in the activation of genes enabling the biosynthesis of cornerstones for gene activation, such as ligands , receptors  and other transcription factors, the entire machinery leading to gene activation and expression.