Key stakeholders regard generic utility instruments as suitable tools to inform health technology assessment decision-making regarding allocation of resources across competing interventions. These instruments require a 'descriptor', a 'valuation' and a 'perspective' of the economic evaluation. There are various approaches that can be taken for each of these, offering a potential lack of consistency between instruments (a basic requirement for comparisons across diseases). The 'reference method' has been proposed as a way to address the limitations of the Quality-Adjusted Life Year (QALY). However, the degree to which generic measures can assess patients' specific experiences with their disease would remain unresolved. This has been neglected in the discussions on methods development and its impact on the QALY values obtained and resulting cost per QALY estimate underestimated. This study explored the content of utility instruments relevant to type 2 diabetes and Alzheimer's disease (AD) as examples, and the role of qualitative research in informing the trade-off between content coverage and consistency.A literature review was performed to identify qualitative and quantitative studies regarding patients' experiences with type 2 diabetes or AD, and associated treatments. Conceptual models for each indication were developed. Generic- and disease-specific instruments were mapped to the conceptual models.Findings showed that published descriptions of relevant concepts important to patients with type 2 diabetes or AD are available for consideration in deciding on the most comprehensive approach to utility assessment. While the 15-dimensional health related quality of life measure (15D) seemed the most comprehensive measure for both diseases, the Health Utilities Index 3 (HUI 3) seemed to have the least coverage for type 2 diabetes and the EuroQol-5 Dimensions (EQ-5D) for AD. Furthermore, some of the utility instruments contained items that could not be mapped onto either of the proposed conceptual models.Content of the utility measure has a significant impact on the treatment effects that can be observed. This varies from one disease to the next and as such contributes to lack of consistency in observable utility effects and incremental utility scores. This observation appears to have been omitted from the method development considerations such as reference methods. As a result, we recommend that patients' perspectives obtained via qualitative methods are taken into consideration in the ongoing methods development in health state descriptions for generic utility instruments. Also, as a more immediate contribution to improving decision making, we propose that a content map of the chosen utility measure with patient-reported domains be provided as standard reporting in utility measurement in order to improve the transparency of the trade-offs in relation to patient relevance and consistency. National, regional and local decision makers have to determine how best to allocate scarce resource so as to obtain optimum benefit . The QuaIn the United Kingdom, The National Institute for Health and Clinical Excellence (NICE) guidance clearly states that for cost effectiveness analysis, the value of health effects should be expressed in terms of QALYs and the measurement of changes in health-related quality of life (HRQoL) should be reported directly from patients. Although the NICE guidelines state the use of disease-specific preference based measures may be considered if they are justified, the value of changes in patients' HRQoL  should be based on public preferences using a choice-based method .The equivalent body for Scotland, Scottish Medicine Consortium (SMC), also prefer the QALY and consider it to be the most appropriate generic assessment of health benefit that reflects both mortality and HRQoL effects, and which allows comparisons between interventions . LikewisQALYs are calculated by estimating the total life years gained from a treatment and then weighting each year with a score. This score ranges from 0 to 1 (or 100) representing 'worst imaginable health' and 'best health' respectively. The utility is considered reflective of HRQoL in that year. The fundamental idea is that an extra year in good health does not have the same value to patients as a year in poor health . The numThis process requires a descriptor, a valuation, and a perspective for the utility measurement. There are various approaches that can be taken for each of these. For example, a descriptor method is a description of health and its impact on HRQoL and can be developed using clinicians, patients, or people from the general population (or even a combination of these). The descriptor method can also be presented in different ways such as using a vignette or the EuroQol-5 Dimensions (EQ-5D) items. A valuation method refers to a value given to a health state description such as using a visual analogue scale or standard gamble, and a perspective can range from for example the healthcare provider or a societal perspective. The diverse approaches for descriptors, valuations and perspectives may contribute to a lack of consistency and diversity in scores, making consistent decision making more difficult.Utility instruments incorporate preferences or values attached to individual health states and express health states as a single index. They can be classified as generic and therefore suitable for use in various populations and disease-specific populations, allowing decision makers to make comparisons. Examples of generic utility measures include the EQ-5D, the Health Utilities Index (HUI) , the 15-Disease-specific instruments are developed to measure the patients' perceptions and HRQoL impact of a specific disease or health problem. The main advantages of disease-specific measures are that they tend to be relevant to the impact of a specific disease on patients, and clinicians find them useful . In addiRegulatory authorities such as the FDA and EMA have endorsed patient-reported outcomes and emphasised the need to take the patient perspective into account when developing an instrument, particularly aspects of HRQoL ,17.The recent special issue of the Value in Health summarising the ISPOR Consensus Development Workshop focuses on "moving the QALY forward" with emphasis on "how to define and refine the QALY" as a standard metric . It is gIt was recently highlighted at the QALY development workshop that the absence of a better alternative makes the QALY an indispensable tool . These gOne possible solution to developing a preferred approach is a 'reference method'  where a The broad objective of this study was to assess how qualitative methods can improve knowledge about which current utility instruments assess concepts of relevance from the patients' perspective. Alzheimer's disease and type 2 diabetes were used as examples of diseases that are prevalent and represented in the general population.A literature review was performed in two parts. The aim of part one was to identify patient qualitative research in the two diseases of interest (type 2 diabetes and AD). The focus was to gain an understanding of the symptoms and subjective experiences of patients with type 2 diabetes and those with AD. Part two aimed to identify generic and disease-specific utility instruments used in type 2 diabetes and AD. The analysis then compared the relevant domains for the diseases with the construction and validation of the instruments.A search strategy was implemented using electronic databases  to identify relevant qualitative studies from January 2003 to October 2008, using the search terms presented in Table On completion of the search all titles and abstracts were screened for possible inclusion in the study by two independent researchers. Disagreements were resolved by subsequent discussion with another researcher who performed an independent review and made a final decision.To satisfy the inclusion criteria, selected abstracts had to include an appropriate clinical term related to one of the relevant indications , at least one patient-reported outcome term , and at least one methodological term  in the title or abstract.Abstracts were excluded if they were primarily clinical, economic or quantitative in focus, the perspective was not patient focused , or the abstract did not refer to a journal article .Following pre-testing, data extraction tables were finalised to accurately extract information on the symptoms and subjective experiences of patients including patient quotes where relevant. A thematic analysis was performed on the patient quotes . Each daOR utility OR QALY [MeSH Terms]. To satisfy the inclusion criteria, papers had to include an appropriate clinical term and reference to a utility measure in the title or abstract. Limits and exclusions were similar to part one of the literature review. The extent to which patients and healthy people were involved in their development was considered. The utility instruments were then assessed for their relevance to the diabetes and AD populations.To identify utility studies, an additional search strategy was implemented from August 2006 to October 2008 using PUBMED, utilising the same indications as in Table Each item (question) of utility instruments identified from part two of the literature review was then mapped on to the conceptual models. Each item was mapped where it was considered to reflect the concept, meaning that items could be mapped to more than one concept if deemed appropriate. Partial coverage was also considered, and items that partly covered concepts were mapped and indicated in brackets.The study selection process for part one of the literature review is presented in Figure Review of these studies revealed that aches and pains -25, hungSymptoms experienced by patients were mainly related to short term memory loss -63 and iA review of the concepts of relevance to both patients with type 2 diabetes and AD indicate that there are a number of concepts that the two disease areas have in common such as activities of daily living, role functioning, emotional functioning, mental health, relationships, social functioning, and self image. However there are concepts that are relevant to type 2 diabetes such as sexual functioning that were not highlighted as relevant to AD patients, conversely patients with AD were greatly impacted by the loss of their independence where type 2 diabetes patients did not mention this. Finally the key symptoms of each disease were also quite different (fatigue in type 2 diabetes vs. memory loss in AD). Thus, this evidence highlights that while some disease areas do have common concepts there are also important concepts that may differ highlighting a potential lack of sensitivity when measuring utilities using a generic instrument.The additional literature search generated 160 abstracts. The titles and abstracts were reviewed and a total of 145 were excluded due to the search terms not being in the title or abstract, due to duplication between databases or the search terms were not the main focus of the article. A total of 15 articles were reviewed in full. Although no disease-specific utilities were identified, the following generic measures were used in both indications: EQ-5D, 15D, SF-6D, and the HUI (versions 2 and 3). A comparison of the content and measurement properties of these measures is described below.The EQ-5D was developed as a generic, HRQoL instrument designed to assess health outcome states across a wide variety of interventions on a common scale . While hThe 15D was developed to measure HRQoL and its utility, and to evaluate the efficacy and effectiveness of interventions . It was The SF-6D was developed as a simplified health state classification from the SF-36/SF-12 . It is aThe HUI consists of three systems: HUI Mark 1 (HUI 1), Mark 2 (HUI 2) and Mark 3 (HUI 3) . HUI 2 aBased on the qualitative results, Figure Further, some of the utility instruments contained items that could not be mapped onto either of the proposed conceptual models. For example, the 'hearing,' 'breathing' and 'sleeping' items from the 15D did not appear to be relevant to either indication which could lead to potential problems such as missing data or lack of sensitivity for these measures when included in a clinical trial.In general, the findings from our study have demonstrated there was incomplete and varied coverage of relevant concepts across specific disease areas, with disparities varying depending on the disease in question. There were also differences in the concepts covered within the generic utility measures discussed. Both findings point to a potential lack of consistency between measures.An evaluation of the utility instruments needs to take into account three issues: relevance of concepts to the general population, such as whether a concept specific to a disease would resonate and be relevant to a healthy individual, relevance of concepts to the patient population being assessed and comprehensiveness of the concepts included in the questionnaire from the patient and general population perspective.To our knowledge, the general population did not rate or rank which concepts were the most important to include in the four utility instruments assessed. All of the concepts included in the utility instruments were based on literature and instrument reviews, rather than using input from patients or the general population. The four instruments also differed in the concepts that were covered. It would be unrealistic to expect that a generic instrument cover each and every concept that is of importance to the general population. However, some concepts that are important to patients may also be important to the population at large. Our results for the diabetes population suggest that tiredness, sexual functioning, social or family impact, and work impact are key concepts that are not covered by many of the utility scales but are likely to be important to the general population. Similarly, cognition and independence are important concepts to AD patients and are likely to be important to the general population. Brazier et al have already demonstrated in some diseases that leaving out concepts considered relevant to patients can result in under representation of relevant health impact in the QALY and over estimation of the cost per QALY . FurtherA detailed calibration of these instruments  in AD and diabetes was beyond the scope of this study. However, the concept mapping illustrates the challenges in representing the diseases optimally and in a way that allows comparison with other diseases. It also raises some philosophical questions about how the content of utility measures should be decided upon. Some instruments may be weighted toward concepts that are of no relevance to the patient population being studied. For example, in our review, patients with AD reported being physically healthy, but the EQ-5D has a preponderance of items that cover physical health . This leaves only one item (depression) that may be relevant to AD patients and completely omits the key AD concepts of memory and independence. Thus, it is likely that in any economic analysis of current AD drugs that use the EQ-5D as the primary utility measure, the results will only allow assessment of the impact of treatment on depression thereby under representing the full utility of treatment. One could argue that the concepts covered are relevant to the general population and therefore allow for more accurate economic analyses. Yet, it could also be argued that cognition and independence are important concepts to the general population as well as to AD patients, so those concepts perhaps should have been included in a generic utility measure (as indeed they are represented in the 15D and HUI).It can also be questioned whether it is possible to measure healthy people and people with a relevant disease using the same measure. Qualitative research such as focus groups or in-depth interviews with members of the public that represent typical diseases in the general population should be conducted to ascertain the concepts that are most relevant to include in a HRQL utility measure. This could be considered in the development of reference utility measures. This is in line with recent guidance from reimbursement bodies who suggest that qualitative evidence is also important when valuing utilities. Qualitative evidence provides unique insight into the disease from the patient who has experienced the symptoms and impacts of the disease first hand. This is supported by NICE who state 'Patients and carers are a unique source of expert information about the personal impact of a disease and its treatment' .These findings are consistent with previous research. For example, one study compared three generic utility based measures  in age-related macular degeneration (ARMD) . ResultsOther studies also provide evidence to support how the choice of method for utility elicitation may lead to difference in utility assessments. For example; one study compared the SF-6D with the EQ-5D and found that there was agreement between both utility instruments demonstrating evidence of validity. However on closer reflection utility results varied, with EQ-5D scores being significantly higher in healthy populations, and SF-6D scores being significantly higher in disease samples, thus pointing to differences in scaling and a lack of calibration between utility measures .Further, a literature review of studies in a variety of disease areas  that compared preference based systems found that the EQ-5D showed larger change scores and more favorable cost-effectiveness ratios compared to the HUI 2 and HUI 3  and the The results from these studies and supporting evidence are pertinent given that generic utility measures are documented as the preferred option in reimbursement guidance documents -6 and thOur study has a number of possible limitations that deserve comment. This study was based on published literature, most of which the purpose was not to assess impact on HRQoL. In addition, the literature search was restricted to English language studies and MeSH terms were used when available. MeSH terms were not available for all search terms. Therefore valuable information could have been missed and there may still remain some concepts of importance.The proposed conceptual models of impact were developed based on existing qualitative research that had been carried out and was from the patient perspective. Consequently, they have not been supplemented with patient interviews to confirm that the proposed areas of impact are indeed important and relevant to patients and put in the context of a specific treatment of study. Having said that, the volume of available information was encouraging as a resource. Additionally, we have limited this study to two indications and the relevant concepts may be very different for others. Further research could explore the relevance of utility instruments in other indications and in the general population.Also the very nature of AD means that less patient reported information is available, and many studies rely on caregiver reports. The patient reported information that is available is primarily from mild to moderate AD patients and so the proposed models may be reflective of this. Thus the proposed model may be a reflection of the earlier stages of the condition and not the disease in its entirety.There were other disease associated factors which may influence patients' experiences with their disease. Some studies commented on the added impact of being diagnosed with and the management of diabetes if in an ethnic minority. Diabetes management itself emerged as a seemingly relevant associated factor. Coping strategies appeared to weigh heavily in AD and these included very practical adaptive techniques like using aide memoirs and writing things down, and the use of humour and laughing to cope with the emotional strain associated with the daily hassles of forgetting. Other external factors included age, gender, culture and environment. These other factors may influence the patients' experience with a condition and may have an interactive role in the proposed conceptual models that have been developed from the literature; however such factors would also be controlled for, and taken into account in a clinical trial setting.A further aspect of the development of future utility instruments is that the dominance of the QALY in the use of HTA and economic evaluation could be reduced by the use of alternative instruments or methods of measurement. Agencies such as the Institute for Quality and Efficiency in Health Care (IQWiG) in Germany and discussions at recent HTA conferences have been considering the use of alternative methods for decision making. Therefore if the QALY is to retain its place as a key method for the use of cost-effectiveness for decision making across countries generic measures need to be constantly improved. Including the patient perspective in development of future instruments and the further development of existing instruments could be a first step aiding the continual improvement of such instruments.Despite the limitations of this study, the findings illustrate the potential value of considering qualitative information in the interpretation of cost utility estimates and the need to include more qualitative research in the design of not only the next wave of utility instruments, but also in the further development of existing instruments such as the EQ-5D.Content of the utility measures has a significant impact on the treatment effects that can be observed and currently in choosing a generic utility measure there appears to be a tradeoff of content in order to achieve consistency. The amount of patient relevant information missing from the chosen generic measures varies from one indication to another, which provides, and could continue to provide a source of inconsistency between the measures. Likewise, the variability in concepts across the generic measures suggests lack of consistency even for general population research, suggesting that the potential impact and importance of content is presently under represented. With current reporting standards, we cannot know the size of this tradeoff between content and consistency and its contribution to the variance in utility findings. This issue would remain if the proposed solution of a "reference method"  for all Patient's perspectives using qualitative methods should be adopted and feature in the decision-making methods used by reimbursement bodies so that there are clearly defined criteria. This movement has the attraction of reducing the scope for controversial decisions based on utility estimates. It could also serve as a bridge to the methods endorsed by regulatory authorities such as the Food and Drug Administration (FDA) and European Medicines Agency (EMA) ,17 and aAD: Alzheimer's disease; ARMD: Age-related macular degeneration; EM: European Medicines Agency; EQ 5D: EuroQol-5 Dimensions; FDA: Food and Drug Administration; HRQoL: Health-related quality of life; HUI 1: Health Utilities Index Mark 1; HUI 2: Health Utilities Index Mark 2; HUI 3: Health Utilities Index Mark 3; MOS SF-6D: Medical Outcomes Study Health Survey; NICE: National Institute for Health and Clinical Excellence; QALY: Quality-Adjusted Life Year; SMC: Scottish Medicine Consortium; 15D: 15-dimensional health related quality of life measurePlease note that the data, models and methodology used in the research are not proprietary. With regard to corporate-sponsored research, Pfizer has commissioned Mapi Values, a health outcomes agency, to carry out research on their behalf. The contact of the sponsoring organisation is Clare McGrath.Diana Rofail works as an Associate Research Director in the Questionnaire and Validation unit of Mapi Values. She has acted as an advisor for various pharmaceutical companies regarding their clinical trials and patient reported outcomes. Diana Rofail does not have ownerships intents (including stock options) in a start up company, the stock of which in not publicly traded or in a publicly traded company. Furthermore, she is not a member of an advisory board or board of directors.Linda Abetz works as a Director of the Questionnaire and Validation unit of Mapi Values. She has acted as an advisor for various pharmaceutical companies regarding their clinical trials and patient reported outcomes. Linda Abetz does not have ownerships intents (including stock options) in a start up company, the stock of which in not publicly traded or in a publicly traded company. Furthermore, she is not a member of an advisory board or board of directors.Elizabeth Gargon worked as a Research Associate at Mapi Values. She provided support to Diana Rofail on this project in terms of the presentation and interpretation of results. Elizabeth Gargon does not have ownerships intents (including stock options) in a start up company, the stock of which in not publicly traded or in a publicly traded company. Furthermore, she is not a member of an advisory board or board of directors.Clare McGrath is Senior Director of HTA Policy at Pfizer and owns stocks and/or stock options. She has had oversight of the HE/OR contributions to licensing and HTA submissions for the past decade and previously specialized in outcomes assessment and clinical studies.Mapi Values and Pfizer would like to and intend to publish the data irrespective of the review process outcome/comments.CM conceived of the study, participated in its design and was involved in developing and critically revising the manuscript. DR participated in the design of the study and coordination, analysis and interpretation of the literature review, and developed the manuscript. EG helped to conduct the literature review, participated in the analysis and presentation of results and helped draft the manuscript. LA made contributions to design and interpretation and was involved in the revision of the manuscript. All authors read and approved the final manuscript.


Inhibitors of these original interactions are certainly the next generation of highly innovative drugs that will reach the market in the next decade. However, DB, a hand-curated database dedicated to the structure of PPIs with known inhibitors. We have analyzed protein/protein and protein/inhibitor interfaces in terms of geometrical parameters, atom and residue properties, buried accessible surface area and other biophysical parameters. The interfaces found in 2P2IDB were then compared to those of representative datasets of heterodimeric complexes. We propose a new classification of PPIs with known inhibitors into two classes depending on the number of segments present at the interface and corresponding to either a single secondary structure element or to a more globular interacting domain. 2P2IDB complexes share global shape properties with standard transient heterodimer complexes, but their accessible surface areas are significantly smaller. No major conformational changes are seen between the different states of the proteins. The interfaces are more hydrophobic than general PPI's interfaces, with less charged residues and more non-polar atoms. Finally, fifty percent of the complexes in the 2P2IDB dataset possess more hydrogen bonds than typical protein-protein complexes. Potential areas of study for the future are proposed, which include a new classification system consisting of specific families and the identification of PPI targets with high druggability potential based on key descriptors of the interaction.Here we describe this particular PPI chemical space through the presentation of 2P2Ihttp://2p2idb.cnrs-mrs.fr.2P2I database stores structural information about PPIs with known inhibitors and provides a useful tool for biologists to assess the potential druggability of their interfaces. The database can be accessed at  In the last decade, the inhibition of protein-protein interactions (PPIs) has emerged from both academic and private research as a new way to modulate the activity of proteins  are certainly the next generation of highly innovative drugs that will reach the market in the next decade.et al. analyzed the atomic interactions and profile of small molecules disrupting PPIs in the TIMBAL database, focusing on small molecules properties and comparing these results to drug-like databases As a consequence of this enthusiasm, the exponential increase of published biomedical literature on PPIs and their inhibition has prompted the development of internet services and databases that help scientists to manage the available information. There is now a growing number of structural databases dedicated to protein-protein interactions DB, which is a hand-curated database dedicated to the structure of Protein-Protein complexes with known inhibitors thereby offering complementary information to these previous analyses (2P2IDB is available at http://2p2idb.cnrs-mrs.fr). We have analyzed the protein/protein and protein/inhibitor interfaces in terms of geometrical parameters, atom and residue properties, buried accessible surface area and other biophysical parameters, such as the protein-protein dissociation constant (Kd) of a complex. The interfaces found in 2P2IDB were then compared to those of representative datasets of heterodimeric complexes from Bahadur and Zacharias http://www.bioinformatics.sussex.ac.uk/protorp/ and We describe here a chemical space, 2P2IDB present globally the same shape (planarity or eccentricity) than standard heterodimeric complexes, but their accessible surface areas are significantly smaller. More strikingly, no major conformational changes are observed between the different states of the proteins . The interfaces are also more hydrophobic than general PPIs' interfaces, with less charged residues and more non-polar atoms. Moreover, fifty percent of the complexes in the 2P2IDB dataset possess more hydrogen bonds than typical protein-protein complexes. A set of key descriptors were identified to distinguish between PPIs with known inhibitors and representative transient complexes in the protein databank. Transient protein-protein complexes are defined as protomers that, in vivo, can exist either on their own or in complex and also undergo an exchange between the free and complexed form The architecture present at the interface generally involves a globular interacting domain, a single secondary structure element  of a globular protein, or a short peptide. Complexes in 2P2IA new classification based on these parameters is proposed with potential aims for the future to identify potential new druggable PPI targets.DB) that was used to further analyze the general properties of protein/protein interfaces (PPIs) with a known inhibitor. The 2P2IDB (http://2p2idb.cnrs-mrs.fr) contains a total of 17 protein/protein complexes corresponding to 14 families and 56 small molecule inhibitors bound to the corresponding target .There are a limited number of targets in the 2P2I database at this stage due to the structural prerequisites that were used. However, it is inevitable that high throughput structural genomic programs will generate a high level of data. In addition, the development of improved methodologies for the development of small molecule inhibitors will rapidly lead to the discovery and structural characterization of disruptors of new PPI families. These new targets and their corresponding ligands will be incorporated into the database as they appear in the literature and the Protein Data Bank  was not significantly different than the natural conformational dynamics of the free target protein and was in the same range as the resolution of the crystal structures. Some local rearrangements could be observed at the binding site; however, these rearrangements do not impair the possibility to design potent inhibitors with high affinity. The fact that there is no main rearrangement between the different forms in the 2P2IDB dataset could mean that these types of complexes are easier to target. Other strategies with small molecule inhibitors binding at different sites, such as an allosteric pocket away from the interface, would be necessary to disrupt PPIs with large conformational rearrangements. These classes of inhibitors are not present in the 2P2IDB, as we only kept those ligands that are present at the interface.The formation of heterodimeric complexes can lead to large rearrangements of the two protein partners We analyzed the general characteristics of the protein/protein interfaces (PPIs) using online servers A more detailed analysis revealed that complexes from class I contained a higher proportion of secondary structure elements at the interface. Four out of 6 complexes involved mainly an alpha helix at the interface, and the other two a beta strand  or inhibitory (Ki) constants of the protein/protein complexes were compared  was significantly smaller than the standard average values of approximately 1000 Å2 observed for heterodimeric protein-protein complexes, as described in the literature by Bahadur and Zacharias 2 and 769±150 Å2 were observed for class I  was not significantly distinguishable (0.72±0.12 vs. 0.70±0.12). Similar values were observed for the two classes defined above. The Gap volume index (GVi) provides a measure of the tightness of a protein-protein complex The average planarity of interfaces in our dataset was 2.8±0.4 Å, which is a value equivalent to that of overall heterodimeric complexes (2.7±1.2 Å). Similarly, eccentricity  than standard transient heterodimers (27.0±12.5%). This result indicates that PPIs with known inhibitors are more hydrophobic than typical heterodimers, which can be correlated to the observation that small molecule PPI modulators are hydrophobic DB dataset and transient heterodimers for the four selected parameters corresponded to probabilities higher than 90% confidence according to the t-distribution table of significance.We used a student's t-test to compare the main descriptors of PPIs in our dataset to standard heterodimers in an attempt to extract the most discriminating parameters that govern the druggability of a PPI. The number of interfacial segments, accessible surface area (ASA), Gap volume, hydrogen bonds and the percentage of charged residues were selected as the key parameters that typify PPIs with known inhibitors . The difDB dataset into different families. Pockets volumes were not incorporated in the t-test study because statistics were not available to compare to the transient protein-protein dataset. However, they were used in the PCA analysis because they are known to play an important role in protein-protein specific interaction In addition to the five parameters defined above , the combined pocket volumes at the interface were used in a principal component analysis (PCA) to separate the 2P2IL/Bak complex (PDB code: 1bxl), the high values for ASA and the number of segments and low hydrogen bond content led to its classification next to the FKBP12/TGFR complex (PDB code: 1b6c). Subtilisin/Eglin C and trypsin/trypsin, which formed a separated cluster when considering the whole interface, were grouped together when considering the part of the interface that interacts with the ligand.A similar PCA analysis was performed with parameters derived from the subset of the interface that is in direct contact with the inhibitor in the protein/ligand structure . The samA web server was developed to facilitate the access to the data calculated for the different PPIs. It allows the user to search for specific complexes using different query procedures based on families, pdb codes, Uniprot numbers, cluster family or ligand properties . For eacDB dataset using the six key descriptors that were selected for the PCA analyses  is also provided. Finally, individual scores for each parameter are calculated. Lower values are indicative of complexes with high potential to become druggable targets whereas higher scores are likely to correspond to poorly druggable complexes.An interactive menu allows the user to compare the properties of a given PPI to the 2P2Ianalyses . The paranalyses  and entePPIs interfaces have long been considered to be poorly druggable because of their general properties. However, in the last few years, a growing number of PPI inhibitors have been discovered, and some of these inhibitors have even reached the preclinical stage of investigation “What makes a PPI an attractive target for the discovery and development of small molecules?” we can portray the good PPI candidates in a few points, which are summarized from existing published data, as follows: i) The target should be validated at least biologically  or, even more importantly, clinically, and the inhibition of the PPI should not be associated with toxicity; ii) Key residues involved in the interaction and defining hot spots should be characterized through mutagenesis analyses, alanine scanning or by server prediction; iii) Structural information on the PPI complex and/or unbound forms should be available to accelerate the process; bridging water molecules should be taken into account when available and because the best conformation to target for PPI inhibition is not necessarily the one found in the complex, the natural conformational dynamics of the target should be investigated through molecular dynamic simulations 3To this critical question A recent study concluded that small molecule PPI modulators are larger (average molecular weight >400 Da), more hydrophobic , with more aromatic rings (∼4 in average) and make fewer hydrogen bonds with the protein than average drugs We have gathered information available for 17 PPIs with known inhibitors whose three dimensional structural had been characterized. The interfaces were analyzed in terms of geometrical parameters, shape and chemical properties. The protein/protein complexes could be divided into two classes according to different parameters, such as the number of segments at the interface. Class I PPIs correspond to those that interact with peptide-like partners and show more secondary structure elements at the interface, whereas the class II group comprises more globular protein/protein complexes with more unstructured elements at the interface.The different PPIs were further classified by PCA analysis using descriptors that were selected based on t-test evaluations and general analyses of the interfaces. Six interfacial parameters were selected corresponding to ASA, Gap volume, percentage of charged residues, number of segments, hydrogen bonds and total volume pockets. Three clusters were defined as a result of the PCA analysis; cluster 1 corresponded to class I PPIs, while class II PPIs were subdivided into two clusters.Analysis of the part of the PPI that corresponds to the region directly in contact with the inhibitor led to similar results. However, minor differences could be observed, which suggests that parameters that define the druggability of a target are probably different from the parameters that define the chemical space of PPI inhibitors. The descriptors were selected for their ability to discriminate between whole PPIs with known inhibitors and transient dimers; additional parameters would have to be selected to predict the chemical space of the ligands that are likely to disrupt a given PPI.DB because of the limited amount of data currently available. However, the definition of what makes a PPI a potentially druggable target will become more and more reliable as the number of 3D structures increases.The number of structurally characterized complexes with known inhibitors is small. Therefore, the chemical space of PPIs is not completely covered in the 2P2IThe ZipA example, in which the ligand is lying on the protein surface in an unconventional way .The results of our study serve to expand current knowledge with new data and focuses at the interface of protein/protein complexes with prior structural knowledge. The proposed classification should lead to a better definition of potentially successful PPI targets and will accelerate the process of designing new PPI drugs. As successes in discovering PPI inhibitors accumulate, the parameters will be refined and the classification scheme updated.http://2p2idb.cnrs-mrs.fr).The whole 2P2I dataset was organized as a relational database and can be accessed through a publicly available web server  were not found in the PDB search either because the interacting partner was a peptide or because of the x-ray resolution (>2 Å).Eight protein/protein and 31 protein/ligand were retrieved by an exhaustive search of the literature ; 1z1m, 1ttv (MDM2/p53); 1tfq, 1tft (XIAP_BIR3/CASPASE_9); 1f9x (XIAP_BIR3/SMAC); 1oo9 (MMP3/TIMP1) corresponds to solution NMR structures. All other PDB codes correspond to x-ray structures.The two lists were combined to form the final dataset, which can be downloaded at The protein-protein interaction inhibition relational database was developed with MySqL. It stores information about the 17 PPIs described in this study. Scripts for interaction with the DB have been developed in PhP with the software MyAdmin.DB database can be accessed through a web-based user interface (http://2p2idb.cnrs-mrs.fr). This platform allows users to query the database to get structural information about interfaces of stored complexes. The whole database can be searched by protein family, PDB codes of the free proteins, protein-protein or protein-ligand complexes, UniProt numbers, ligand three letter codes or by cluster number.The 2P2IThe user can also provide key parameters calculated from other web resources to compare the property of a given PPI to the 2P2I dataset.http://www.ebi.ac.uk/Tools/dalilite/index.html) and are summarized into The root mean square deviations (rmsd) between free and bound states of different proteins were computed over CA atoms using DaliLite server (http://www.bioinformatics.sussex.ac.uk/protorp/). Continuous interface segment have been defined in the literature as a stretch of residues that starts and ends with interface residues and may contain intervening non-interface residues. While considering the length of the segment, only interface residues are counted Planarity, eccentricity, secondary structure in interface, Gap volume, Gap volume index, number of atoms in interface, % polar atoms in interface, % non polar atoms in interface, % neutral atoms in interface, number of residues in interface, % polar residues in interface and % non polar residues at the interfaces were calculated with the ProtorP server using default parameters (Accessible surface area (ASA) and percentage of charged residues were computed with the Naccess program with a probe radius of 1.4 Å. The size of the interface corresponded to the difference in ASA between the protein without its partner and in the complex.http://www.bioinformatics.leeds.ac.uk/qsitefinder) on the protein-protein, protein-inhibitor complexes and the equivalent free proteins Pockets at the interface were computed with the Q-SiteFinder server  using a 3.2 Å distance cutoff between the hydrogen atom and the acceptor atom and were checked manually.Hydrogen bonds were computed with the Pymol software (http://www.ks.uiuc.edu/Research/vmd/).A salt bridge was considered when an acidic residue (Asp or Glu) on one side of the interface and a basic residue  on the other side were less than 4.0 Å apart. Each putative salt bridge was then validated manually using VMD , values higher than 1.34 correspond to probabilities of more than 90% confidence. On the one hand, if t-value is positive and greater than 1.34, then the mean of the studied parameter is significantly greater in the 2P2IDB dataset than in the RCSB transient dimers dataset at 90% or higher confidence level. On the other hand, if the t-value is negative and less than −1.34, then the mean of the studied parameter is significantly less in the 2P2IDB dataset than in the RCSB transient dimers dataset.Where Varhttp://www.R-project.org) and the ade4 package Six parameters  were selected for the multivariate analysis performed according to the principal component analysis. Data were analyzed with the R software . The IQZ inhibitor ZipA surface is shown as a stick representation. .(0.11 MB PDF)Click here for additional data file.Figure S22 of accessible surface area. The X-axis represents the number of hydrogen bonds per 100 Å2. The Y-axis illustrates the number of complexes present in 2P2IDB having this number of hydrogen bonds (within ±0.1), i.e. y value at x = 0.6 indicates that there are 3 complexes having 0.3 to 0.5 hydrogen bonds per 100 Å2.Hydrogen bonds per 100 Å(0.09 MB PDF)Click here for additional data file.Figure S3Advanced query search of the protein databank. This table lists the different parameters used to parse the protein databank to search for proteins bound to a small molecule inhibitor.(0.08 MB PDF)Click here for additional data file.Table S1Root mean square deviations for the complexes in 2P2I database. The rms are computed between the unbound protein and its equivalent in the protein/protein complex; the unbound protein and its homologous in complex with the inhibitor; the protein in complex with its partner and the homologous in complex with the inhibitor. When several structures are compared, the average is shown. All RMSD were performed over CA atoms with the DaliLite web server.(0.15 MB PDF)Click here for additional data file.Table S2Secondary structure at interface. This table lists secondary structures at interface (as defined in M&M) for each complex present in 2P2IDB. Information is detailed for both the target protein and its partner.(0.16 MB PDF)Click here for additional data file.Table S3Geometrical and chemical parameters for the subset of the interface at 4.5 Å around the inhibitor. The parameters are detailed for each complex of 2P2IDB and mean and standard deviations are shown for class I, class II and the whole database.(0.16 MB PDF)Click here for additional data file.


Drosophila genetics dating back to the Morgan School. While considerable attention has been placed on the genetic novelties that duplicates are capable of introducing, and the role that positive selection plays in their early stages of duplicate evolution, much less attention has been given to the potential consequences of ectopic  gene conversion on these evolutionary processes. In this paper we consider the historical origins of ectopic gene conversion models and present a synthesis of the current Drosophila data in light of several primary questions in the field.The evolutionary impact of gene duplication events has been a theme of  Gene family expansions have jointly contributed to genome size , and to Though most duplicate alleles will eventually be lost from a population, a complex interaction between genetic drift, mutation and selection can occasionally lead to duplicate fixation and preservation . Unlike Drosophila. After summarizing commonly used methods for detecting EGC, the paper is structured into two main sections. In the first, we briefly outline the historical context in which EGC came to be studied in Drosophila, and describe how EGC research emerged from a more general analysis of repetitive DNA and concerted evolution. The second section provides an up-to-date analysis of the Drosophila empirical literature concerning duplication and EGC. We anchor the synthesis around several broad and unresolved questions:What is the relative contribution of EGC to patterns of concerted evolution?How does genomic context  affect EGC?How do selection and EGC interact to influence adaptation?Does gene conversion between duplicates bias estimates of the gene duplication rate and the tempo of paralog differentiation?A large body of theoretical work illustrates that EGC can greatly influence the evolutionary dynamics of duplicates ,19, yet Drosophila duplications. However, the empirical limitations of jointly testing for interactions between conversion, selection, linkage, and gene family size preclude a strong conclusion about the temporal duration of conversion between duplicates, or its role in promoting or constraining adaptation. We describe future analyses that may shed light on these unresolved issues.Our general conclusion is that EGC, at minimum, plays a consequential role during the early evolution of physically linked 2.Methods for detecting EGC have been developed for both divergence-  and polymorphism-based sequence data sets . Though the scope of this review does not include a detailed discussion of the methods used, it is helpful to introduce and highlight the most commonly used approaches for detecting EGC . ThroughDrosophila literature, the two most cited divergence-based approaches utilize the GENCONV software package [Within the  package  or test  package .Tests of incongruity between species trees and gene trees are based on the following logic. If phylogenetic information suggests that a given duplication event preceded speciation between two or more species, but DNA sequence data for paralogs within species demonstrate greater sequence identity than orthologs between species, then the datasets are identified as “irreconcilable”. In these cases, EGC can be invoked to explain the disagreement between phylogenetic dating of duplication events and the relative sequence identity between paralogs and orthologs . Such rA third divergence-based method is based on analysis of two types of nucleotide substitutions between paralogous and orthologous sequence alignments: 1) substitutions between orthologs that are shared between paralogs; and 2) substitutions between paralogs that are shared between orthologs. The former pattern supports a conversion model, while the latter is indicative of evolutionary independence between paralogs . ThroughThe least widely used (but most powerful) method relies on polymorphism data within a species. Alignments of the set of paralogs can be used to identify shared polymorphism. Given a low point mutation rate (as expected), parallel mutations and shared polymorphism will be rare without EGC. The actual amount of shared polymorphism between paralogs can be used to identify recent conversion events, and to estimate the rate of conversion between the paralogs . We refe3.3.1.Current debates about EGC can be traced to earlier ones over concerted evolution from the mid 1960s to the 1970s. These debates, in turn, were intertwined with emerging interest in the evolution of genome size and the underlying importance of repetitive DNA.Bar locus duplication, discovered by members of the Morgan lab [Bar duplication and the first evidence of concerted evolution in 1972 [The first empirical evidence for genome size plasticity traces back to the rgan lab –25. Duri in 1972 , advance in 1972 –28. Alon in 1972 ,29–32, w in 1972 ,33. In aFrom this earlier research on genomic content, the model most relevant to ectopic conversion is the master-slave model ,34. WorkLater modifications to the “rectifying” model attest to its appeal during this time. For example, the original master–slave model assumed that each master gene was separated by intervals comprised of repetitive slave copies, yet subsequent data indicated that unique genes (rather than copies) were likely to be physically linked . Thus, tBy the start of the 1970s, much emphasis had been placed on understanding the origins and implications of repetitive DNA, which raised questions about the evolutionary maintenance of repeats, conversion biases, interactions between selection and conversion, and the importance of chromosomal context in mediating concerted evolution. These questions remain important today.3.2.Drosophila.To improve understanding of the evolutionary dynamics of repetitive DNA, the pre-molecular biology world needed a more tractable model system. rDNA was well suited for this role ,39, and et al. [Xenopus species. They showed that individual rDNA array units exhibited very high sequence identity within compared to between species. This suggested that a “correction mechanism” between repeats resulted in “horizontal evolution” within species. Though Brown et al. [The abundant transcriptional products of rDNA loci, together with hybridization and denaturation methods of the time, provided an opportunity to estimate copy number differences and sequence divergence of repetitive units within and between species. Detailed evolutionary studies of rDNA arrays across diverse taxa gained momentum starting in the mid 1960s . Most noet al. , which pn et al.  did not Xenopus DNA data [With evolutionary models for repetitive DNA already developed, the field was situated to integrate empirical patterns of concerted evolution. One explanation for the pattern invoked the already popular model of unequal crossing over. High rates of unequal crossing over could permit the stochastic spread of identical copies throughout a given array, leading to a pattern of high sequence identity between individual copies. The alternative explanation harkened back to models of homogenization between duplicates, such as the master–slave model. Though not conclusive, initial support for unequal crossing over was provided by DNA data ,40, and DNA data ,42.Drosophila had large rDNA arrays on both the X and Y chromosome. A role for unequal crossing over during the evolution of these arrays had been suggested from studies of the bobbed mutant, which was associated with deficiencies of X-linked rDNA genes [bobbed phenotype led to the discovery of “DNA magnification”, where male germlines deficient for both X- and Y-linked rDNA could revert to the wild-type rDNA gene number [D. melanogaster species subgroup and D. hydei [Xenopus, high sequence identity between repeats was found for the nontranscribed regions . In the D. melanogaster species group, there are two pairs of HSP genes (Hsp70Aa/Hsp70Ab and Hsp70Ba/Hsp70Bb), with each pair tightly linked in a “palindromic”  orientation on the Muller E chromosome (chromosome 3R of D. melanogaster). This type of orientation has an interesting property with respect to ectopic recombination. A double-strand break in one of the paralogs can become resolved by gene conversion . HIP/HIP-R genes are X-linked, non-inverted duplicates that are confined to the D. melanogaster lineage. Like HSP genes, extensive shared polymorphisms indicate that these cofactor duplicates are undergoing conversion.As an ancient and evolutionarily conserved gene family, HSPs are good Drosophila EGC is the amylase gene clusters. The D. melanogaster species group includes a conserved set of linked paralogs (Amy-p and Amy-d) in palindromic orientation (similar to Hsp70 genes). Early work based on restriction site analysis [Another classic study system for analysis  showed tanalysis ,60, withanalysis ,62).D. melanogaster, and one or more additional clusters [D. pseudoobscura inversion karyotypes were once again limited to coding regions [Drosophila kikkawai and close relatives have two highly-divergent clusters of linked, palindromic amylase genes (Amy1/Amy2 and Amy3/Amy4), with each cluster showing high sequence identity [Amy3/Amy4 may indicate EGC or a duplication event. The Amy1/Amy2 pair, which appears to be orthologous to the melanogaster cluster, shows evidence of coding (but no noncoding) concerted evolution, which supports a model of continuous EGC [Subsequent work was extended to species outside of the melanogaster group, which carried an amylase cluster orthologous to clusters . Concert regions ,65. Drosidentity . Homogenuous EGC ,68.Idgf genes [In many ways, these classical studies are representative of the case study approach to concerted evolution. Evidence for EGC is typically associated with the analysis of small gene families that are physically linked and/or evolving under purifying selection , yet thparalogs –78, polyparalogs .On the other hand, a focus on small gene families will likely minimize the effect of gene turnover. Larger gene families are expected to be more permissive to the fixation of duplicates relative to smaller gene families because their sensitivity to deleterious dosage effects might be relatively low and their rate of copy number mutations might be relatively high. Consequently, the importance of birth/death gene turnover will likely be greatest for large gene families. Though EGC is expected to occur in such cases, disentangling EGC and gene turnover requires information about the age of individual members of a gene family, and polymorphism data to estimate the rate of EGC. Such data sets are difficult to obtain for large, repetitive gene clusters where individual copies cannot easily be distinguished ,81.4.2.Drosophila. These studies utilize different methodologies, yield different results, and emphasize EGC between gene families of different ages and degrees of sequence divergence. The perspectives of these studies seemingly reflect different evolutionary questions regarding the interplay between duplication and EGC. One perspective is geared towards understanding how EGC might govern the evolutionary fates of young duplicates. The other emphasizes broad patterns of EGC and is less concerned with the relative age or size of the gene families. Despite their differences, these studies utilize partially overlapping distributions of gene family ages, and it is within this region of overlap where some of the more puzzling differences emerge.It is currently unclear how prevalent EGC is at a genomic scale. To date, there have only been three genome-wide studies that directly addressed this question in et al. [et al. [Drosophila species . Hahn et al. applied maximum likelihood methods to infer rates of gene gain and loss along each branch of the species tree and then compared these results with those from a gene-tree/species-tree reconciliation analysis. If EGC has played a major role genome-wide, they expected that their reconciliation methods would infer multiple, parallel duplications across lineage. They estimated that approximately 17 genes were gained or lost every million years, with few signatures of EGC inferred by reconciliation analysis. The authors concluded that EGC leaves, at most, a minor genomic signature.Analysis of the long-term effects of EGC was carried out by Hahn et al.  and Caso [et al. , using gspecies  and 14.15% (in D. grimshawi). Peak conversion activity was observed for paralogs with silent divergence between dS = 0.1 and dS = 0.3. Phylogenetic reconciliation methods were also consistent with low amounts of EGC, with 1% to 3% of gene trees within the D. melanogster subgroup, and up to 15% of gene trees within deeper branches of the Drosophila tree, showing signs of EGC. The authors concluded that EGC was relevant for relatively young duplicates having silent divergence ranging between 0.1 and 0.3. Casola et al. also reapplied the likelihood-reconciliation methods used by Hahn et al. [Casola n et al. , and agaD. melanogaster, D. simulans, D. sechellia, D. yakuba, and D. erecta, to identify duplication events immediately prior to or following divergence between D. melanogaster, D. simulans, and D. sechellia. The motivation of this approach was based on the expectation that EGC should be more active and more easily estimated in young gene families. EGC was estimated with reconciliation (tree-based) methods , and site-specific tests (see above). Of 28 post-speciation blocks available for the tree-based analysis, 24 provided evidence of EGC in the D. melanogaster lineage, the D. simulans lineage, or in both. The sliding window approach identified at least one signature of EGC in every block. Likewise, the site-based test identified a signal of EGC in 29/30 pre-speciation blocks.Osada and Innan  focused et al. note that additional differences might result from the failure of Osada and Innan to account for parallel duplications between species (rapid birth-and-death rate), which potentially generate gene trees mimicking those predicted under a conversion model. They highlight estimates of high copy number variation  as suppn  as well as complex factors, such as the three-dimensional conformation of chromosomes . While tD. melanogaster and more distantly related species: e.g., [The growing availability of genomic data has shed some light on features correlated with EGC. One tractable question is how the physical distance between duplicates correlates with EGC. Several studies indicate a negative correlation between physical distance and conversion between paralogs . s: e.g., . This reDrosophila duplicates is generally higher within conversion tracts relative to sequences that flank each tract, which suggests that the underlying conditions favoring conversion biases may commonly be present in paralogs [Another question is whether EGC is biased. To date, there is no compelling evidence for this, yet the subject warrants future study. GC-biased conversion is often observed in cases of allelic (non-ectopic) conversion in mammals . GC contparalogs . Interesparalogs .6.The interaction between EGC and natural selection is central to interpretations of concerted evolution patterns, as well as inferences about the rate of ongoing EGC. The likelihood of conversion between non-allelic sequences is, in part, a function of their degree of sequence identity. For gene family members evolving under strong purifying selection, relatively high sequence identity is expected in the absence of EGC. EGC will further reduce divergence between paralogs by homogenizing (putatively) neutrally evolving synonymous sites, introns and intergenic DNA, and by promoting parallel adaptation in functionally relevant sites . For example, the interaction between EGC and natural selection may prevent the accumulation of deleterious mutations ,94–96, oWhile EGC can promote adaptation among functionally redundant genes, it may also constrain adaptive differentiation between paralogs—a process that might impact the evolution of new gene functions ,98–100. Drosophila supports both reinforcing and antagonistic interactions between selection and EGC. Positive selection between paralogs has been observed in several general contexts [Some evidence from contexts ,104,105.contexts ,75,79. TDrosophila: e.g., [versus between chromosomes. Thornton & Long [D. melanogaster genome, and found a pattern of increased divergence (Ka/Ks) when both duplicates resided on the X chromosome, but otherwise no consistent effect of intra- vs. inter-chromosome paralog orientation. To the extent that amino acid divergence has been driven by positive selection, the pattern does not indicate any constraint imposed by EGC.Conflict between EGC and disruptive selection might potentially be examined by comparing patterns of divergence between paralog pairs experiencing markedly different rates of EGC. For example, if paralogs on different chromosomes experience reduced EGC compared to closely linked paralogs , one min & Long  comparedD. melanogaster should enhance statistical power to identify and estimate the rate of EGC, particularly in small gene families or other low-copy repeat sequences. Polymorphism data will also permit discrimination between evolutionary models of genetic drift and positive selection ; these ancient duplication events may represent a filtered (and therefore biased) set of duplicates. Osada and Innan  have emprthologs ,107, ratThe second caveat concerns the potential relationship between selection and degree of dispersion between duplicates. While a negative relationship between EGC rate and distance is expected (see above), it is also possible that disruptive selection between duplicates might also covary with distance. If, for example, unlinked paralogs are exposed to different local chromatin states, or are influenced by distinct local promoter sequences, then the opportunity for disruptive selection might increase with greater dispersion between paralogs. Creative statistical and bioinformatic approaches will be required to control for possible spurious correlations between distance and adaptive differentiation.7.i.e., their ages and the distribution of inter-paralog divergence) will critically depend on whether or not EGC occurs between paralogs, as well as the long-term covariance between selection, EGC, and paralog divergence. Our understanding of the actual dynamics of EGC can have a major impact on our interpretation of: (1) the rates of duplicate birth and death, and the age distribution of duplicate genes and (2) the temporal patterns of selection during the course of duplicate evolution.EGC can influence both the temporal patterns of duplicate gene evolution, and interpretations of these patterns within the context of evolutionary theory. The inference of selection and genetic drift from empirical properties of duplicate genes  should be clock-like, and proportional to the relative age of the duplication event. EGC will downwardly bias the distribution of dS, and lead to an overestimation of the duplicate “birth” and “death” rates . However, since birth-and-death and EGC rates are largely unknown, the distribution of dS is insufficient for inferring the evolutionary rate and maintenance of gene duplicates. Exploiting genome sequence data from closely related species can circumvent this methodological limitation. Osada and Innan's [D. melanogaster/D. simulans last common ancestor (about 2.3 million years ago) suggests a duplication rate of approximately 10−9, per gene, per year, which is approximately ten-fold lower than earlier estimates based entirely on dS. Given the relatively short time interval separating these species , this estimate may differ from the true duplication rate in Drosophila, yet it should characterize the rate to an order of magnitude.The inferred rate of gene duplication is sensitive to assumptions about the degree of evolutionary independence between paralogs. Without EGC, neutral sequence divergence between duplications  and the silent substitution rate  stronger for ancient relative to young duplicates , and the correlation between dN/dS and dS may become more strongly negative.Another common observation is a negative relationship between the ratio of nonsynonymous to synonymous divergence between paralogs . dN/dS sophila; ,109): yo8.Drosophila, evidence for EGC is particularly strong in small gene families  with high sequence identity between paralogs . For larger gene families, and/or ancient paralog pairs, evidence for EGC is weaker, and is often difficult to distinguish from birth-and-death models.EGC can profoundly influence the evolutionary fates of young duplicates, as well as the patterns of concerted evolution within gene families of varying size and age. In Methodological limitations preclude a precise estimate for the rate of EGC, and are expected to cause a statistical bias towards type II error . The growing feasibility of collecting and analyzing whole-genome polymorphism datasets  will soDrosophila genomes and beyond) for calculating the ages of gene family members—should soon favor an increasingly sophisticated analysis and interpretation of the evolutionary consequences of EGC, including its interaction with mutation, selection, and linkage.The confluence of three sources of data—improved EGC estimates, rapidly accumulating CNV data that can be used to infer mutational processes for duplicates, and multispecies phylogenies (e.g., the 12


Visceral pain is the most common reason for physician visits in US. Glutamate is the major excitatory neurotransmitter and mediates visceral nociceptive neuro-transmission and hypersensitivity. Removal of extracellular glutamate is predominantly mediated by glial glutamate transporter-1 (GLT-1). The pharmacological approach to up-regulate GLT-1 by 1 week administration of ceftriaxone (CTX) has been successful to mitigate visceral nociception. The present study shows that intrathecal delivery of selective GLT-1 antagonist dihydrokainate reversed CTX-blunted visceral nociceptive response, suggesting a spinal site of action. The role of GLT-1 up-regulation in animal models of colitis was studied. CTX treatment reversed TNBS-induced visceral hypersensitivity. In addition, CTX treatment initiated one week after the onset of DSS-induced visceral inflammation also attenuated visceral hypersensitivity, revealing a potential therapeutic effect. Cephalothin, a cephalosporin antibiotic lacking GLT-1 induction activity, failed to attenuate visceral nociception. CTX-induced changes in fecal microbiota do not support a role of probiotic effects in mitigating visceral nociception/hypersensitivity. Finally, adeno-associated virus serotype 9-mediated GLT-1 over-expression was effective to mitigate visceromotor response to 60 mmHg colo-rectal distension. These studies indicate that GLT-1 over-expression is a novel and effective method to attenuate visceral nociception, and is deserving of further study as a translationally relevant approach to treat visceral pain. Visceral pain, defined as pain associated with the internal organs, is a major clinical problem affecting up to 25% of the general US population and is the most common reason for physician visits in the USA. Lower pain thresholds to aversive stimuli are common, due to visceral organ primary afferent sensitization, hyperexcitability of second-order pain-transmitting neurons, or dysregulation of descending modulation of nociception . These pβ-lactam antibiotic ceftriaxone (CTX) [http://clinicaltrials.gov/ct2/show/NCT00349622?term=Ceftriaxone&rank=4, and is being explored for other CNS disorders [A series of exciting recent findings in the subclinical study of visceral pain shows that a novel strategy to decrease synaptic glutamate by upregulating the physiologically dominant glutamate transporter GLT-1 is effective to mitigate visceral nociception –6. Evidene (CTX) , 8. Regane (CTX) , http://isorders . In thisTwo-to-three-month old FVB/N mice of both genders were used for the behavioral studies. CR57/BL mice were used for the adenoassociated virus (AAV9) studies. Mice were maintained on a twelve-hour day–twelve-hour night cycle and were housed in groups of five with water and food. All protocols were approved by the Institutional Animal Care and Use Committee in the Ohio State University and adhered to the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain.μmoles)/Kg/day in a 10 ml/kg volume for one week. Cephalothin (CLT) was administered in an equimolar dose, and in the same route and duration as CTX. Normal saline was used as control injections for these experiments. Ceftriaxone  was prepared in saline and was administrated intraperitoneally 200 mg . The Kaspar Laboratory routinely achieves >1013 viral particles from AAV preparations . For neoAmong the pseudoaffective responses commonly employed in rodents as an index for nociception is the visceromotor response to colorectal distension (CRD). CRD was performed as described previously . Brieflyμl Hamilton syringe was inserted through the L5-L6 vertebra to reach the dura at the lumbosacral spinal level. A characteristic tail flick occurred after correct placement of the needle. 5 μl of vehicle or dihydrokainate (DHK) at different concentrations  was delivered into the intrathecal space over one minute, and the animals were prepared for colorectal distension. In some experiments, one hour before colorectal distention, animals were lightly anesthetized by isoflurane. The hair in the midline of the back was shaven, and the locus of the cauda equina was determined as described previously . A 30-gaμl Hamilton syringe was carefully advanced to penetrate the allantoin-occipital membrane to access the cisterna magna. Vehicle, 0.3 or 3.0 mM dihydrokainate (DHK), was then delivered in a 5 μl volume into the cistern magna, and the animals were prepared for colorectal distension. In some experiments, one hour before colorectal distention, animals were lightly anesthetized by isoflurane. Hair was shaven on the dorsal aspect of the neck and was removed, and a 0.5 cm diameter steel bar was put underneath the neck. A 30-gauge needle connected to a 50 μl volume. After instillation (day 0), mice were held head-down by lifting up the tail for 1 min to ensure exposure of distal colon to injectate. CTX 200 mg/Kg/day or vehicle injections were administered intraperitoneally at 10 : 00 am days 0–6. Mice were utilized on day 7 for behavioral tests, myeloperoxidase (MPO) assay, and immunoblot analysis.Two groups of animals were treated with intracolonic TNBS (7 mg/ml) and another two cohorts treated with intracolonic vehicle prepared in 50% ethanol and delivered through a syringe attached to a lubricated polyethylene catheter (PE20) two centimeters proximal to the anus in a 100 Two groups of animals were administered five mililliters daily of normal drinking water and another two groups administered 4% dextran sodium sulfate (DSS) dissolved in the drinking water daily for 1 week (days 0 to 6). Mice receiving 4% DSS typically develop colitis by day 7 . All aniμl of 50 mM potassium phosphate (pH 6.0) containing 0.167 mg/ml O-dianisidine dihydrochloride (Sigma) and 0.0005% hydrogen peroxide was loaded as reaction buffer in a 96-well plate. 20 μl of supernatant sample was then mixed with reaction buffer. Two minutes later, absorbance was read at 460 nm by a spectrometer . Protein concentration was accessed by adding the 1 μl sample into 200 μl coomassie blue buffer (Pierce) in the same 96-well plate, and absorbance was read at 595 nm. The unit of MPO was defined by the change in 460 nm absorbance per mg of protein sample in two minutes.As a measure of colonic inflammation, 1 cm of the distal colon was dissected from animals and samples were minced and homogenized 30 strokes by a motor-driven grinder in 1 ml ice-cold 50 mM potassium phosphate buffer (pH 6.0) with 0.5% hexadecyltrimethylammonium bromide (Sigma). Samples subsequently underwent 30 s sonication and three freeze-thaw cycles in liquid nitrogen. After brief sonication, samples were centrifuged at 10,000 rpm for 10 min at 4°C. Supernatant was collected to measure MPO activity. 200 μg of protein sample dissected from mice lumbosacral spinal cord was sonicated, loaded in 8% SDS-PAGE gel, and transferred to nitrocellular membranes. After one-hour blocking, the membranes underwent over-night primary antibody  and one-hour secondary antibody  incubation, followed by X-film exposure with Enhanced Chemiluminescent Substrate (Pierce).Briefly, 20 t-test. Mice were trained on an accelerating rotarod  3 times per day for 3 days. The cylinder of the rotarod was 71 cm long, and its diameter was 3.2 cm. Mice were allowed to explore the rotarod for 2 min without rotation. The velocity of the rod was set to increase from 0 to 40 rpm in 5 min. Mice with stable 300 s performance on the 3rd day were used later. At the end of baseline testing, animals were divided into two groups and one group treated with CTX 200 mg/Kg/day and the other cohort administered vehicle i.p. After this one-week treatment, mice were tested three times per day for 3 days with 1-hour interval, following the same protocol. The latency to fall off the apparatus in the three trials was recorded, and the 3-day data from individual mouse was averaged. Significant difference was assessed by using Student's Multiple group differences were analyzed by ANOVA followed by post-hoc LSD analysis.μl, 3 mM) was injected into the intrathecal space (it) one hour before the graded visceromotor response to colorectal distension was elicited. In animals with GLT-1 overexpression produced by 1-week CTX, intrathecal DHK (1-week CTX + it DHK 3 mM) reversed the blunted visceromotor response produced by GLT-1 overexpression , compared to animals treated with 1-week vehicle + it vehicle Figures . To asseNotably, the 1-week vehicle + it DHK 3 mM group  produced Given the putative role of hindbrain glutamate receptors to modulate pain transmission , the rolColonic inflammation causes visceral hyperalgesia  and thisP < 0.005). 1-week CTX treatment inhibited the enhanced MPO activity produced by colonic TNBS administration (P < 0.005). These data suggest that pharmacologic GLT-1 overexpression was able to attenuate both the intracolonic TNBS-elevated visceromotor response to colorectal distension  126 mg/Kg/day) did not increase the GLT-1 protein level in the spinal cord, compared to vehicle-treated mice , a cephalosporin antibiotic not effective to enhance GLT-1 expression  was utilted mice . In contted mice , but sigted mice , These dA key question related to potential therapeutic utility of antinociceptive strategies to overexpress GLT-1 is its effectiveness after the initiation of algesia. This study utilized the DSS-induced colitis model, where 5 ml of 4% DSS in the drinking water was provided daily to two groups of mice for one week. Comparison cohorts of two groups of mice were administered normal drinking water for week one. All animals were returned to normal drinking water for week 2, and GLT-1 overexpression was produced by 1-week CTX  from day 7 through day 13 in two groups of mice , and two groups  were treated with ip vehicle daily for 1 week. In response to colorectal distension performed at day 14, DSS treated animals displayed a significantly increased (36–60%) visceromotor response to 45 and 60 mmHg colorectal distension, compared to control animals . At day An important question related to possible clinical utility of CTX treatment is to evaluate possible untoward effects caused by GLT-1 overexpression. Previous reports show a lack of effect on acute nociceptive and selective behavioral tests in rodents , 7. To aEmerging evidence suggests that changes in intestinal microflora can mediate alterations in gastrointestinal function, including visceral hypersensitivity . To asse−11 dps of AAV9-GLT or vehicle at 19–26 d, and the visceromotor response (VMR) to 60 mmHg colorectal distension (CRD) was measured in these animals at 8 weeks of age. The data show a 62% reduction in the nociceptive response to 60 mmHg pressure after transfection with AAV9-GLT-1 (n = 10) compared to animals injected with PBS (n = 10) (P < 0.05). This is correlated with a 111% enhanced glutamate uptake seen in AAV9-GLT-injected animals compared to controls  a spinal site of action of GLT-1 overexpression to attenuate visceral nociception, (2) effectiveness of GLT-1 overexpression to attenuate inflammogen-enhanced visceral nociception via both a preemptive and therapeutic approach, (3) CTX-mediated GLT-1 overexpression but not its anti-inflammatory effects mediates its antinociceptive effects, (4) translationally superior adenoassociated virus 9-mediated GLT-1 transfection attenuates visceral nociception to 60 mmHg colorectal distension, and (5) alterations in locomotor function were not produced by 1-week CTX treatment.Clear evidence by our laboratory and others demonstrates the effectiveness of 1-week CTX to enhance spinal GLT-1 expression and glutamate uptake activity , 6, 12. Intracisternal injection of doses of DHK effective after intrathecal administration (3.0 and 0.3 mM) was ineffective to reverse the reduced visceromotor response to colorectal distension produced by 1-week CTX treatment. This suggests that antagonizing brainstem glutamate transporters does not alter CTX-blunted visceral nociception. Although the volume of the fourth ventricle is larger than the intrathecal space, the concentrations of DHK given intracisternally was 1-2 orders of magnitude greater than the IC 50 of DHK (0.045 mM). These experiments support the exclusion of a role of brainstem glutamate transporters in mediating the antinociceptive effects of CTX. However, a role of midbrain, forebrain, or cerebral glutamate transporters in mediating antinociceptive effects of 1-week systemic CTX cannot be ruled out.2O2 to form hypochlorous acid, a powerful antimicrobial reagent. This unique property is widely used to detect the amount of MPO in tissue samples by measuring absorbance at 460 nm. These experiments show that GLT-1 upregulation by one-week CTX treatment effectively reduced visceral hypersensitivity in two animal models of colitis. The myeloperoxidase (MPO) assay is a well-accepted tool to measure tissue inflammatory damage in animal models of colitis. MPO is a peroxidase enzyme abundant in neutrophils and is released upon neutrophil activation by inflammatory pathogens . During To study potential effectiveness of the intervention of GLT-1 upregulation during an active inflammatory process, the use of DSS was employed due to the published unreliability of TNBS to produce consistent long-term inflammation in murine models . In bothProbiotics are emerging as potential treatments of visceral hypersensitivity . Recent Potential clinical utility of the approach to augment glutamate transporter activity depends on the lack of potential untoward effects. The rotarod test demonstrated that GLT-1 upregulation by one-week CTX treatment did not alter motor function, which is consistent with previous studies showing that CTX treatment did not change performance in the plus-maze test and open-field test, in normal mice .The adenoassociated viral vector (sc AAV9) utilized in our proposal produces long-term gene expression, thus is effective after one-time injection . This faThus, the data taken together suggests that the antinociceptive effect of one-week CTX treatment is independent of its anti-inflammatory or probiotic effects and indicates the importance of GLT-1 upregulation in reducing visceral nociception. Beneficial effects are likely related to reduced extracellular glutamate tone; thus, less activation of glutamate receptors mediating nociception, and/or second messenger events downstream are attenuated  , 26, 27.These results support consideration of novel approach of glutamate transporter upregulation in pain therapeutics. Further investigation of ceftriaxone and analogues with increased and more specific therapeutic effects seem warranted.


Chemotherapy has made an essential contribution to cancer treatment in recent decades despite its adverse effects. As cancer survivors have increased, concern about ex-patient lifespan has become more important too. Doxorubicin is an effective anti-neoplastic drug that produces a cardiotoxic effect. Cancer survivors who received doxorubicin became more vulnerable to cardiac disease than the normal population did. Many efforts have been made to prevent cardiac toxicity in patients with cancer. However, current therapies cannot guarantee permanent cardiac protection. One of their main limitations is that they do not promote myocardium regeneration. In this review, we summarize and discuss the promising use of mesenchymal stem cells for cardio-protection or cardio-regeneration therapies and consider their regenerative potential without leaving aside their controversial effects on tumor progression. Globally, cancer is the leading cause of death. There were 14.1 million new cases of cancer in 2012, and an increase of up to 22.2 million new cases by 2030 is predicted [Chemotherapy is an essential tool in cancer treatment. However, the use of anti-neoplastic agents has several adverse effects. Doxorubicin, which belongs to the anthracycline family, has been proven to be effective in different tissue-derived cancer diseases, including cancer of the breast, lung, stomach, bladder, and skin. Despite the anti-tumoral properties of doxorubicin, myelosuppression and particularly cardiotoxicity restrict its clinical use .Doxorubicin has been used in oncology treatment since the 1970s. So far, the following risk factors for doxorubicin-induced cardiotoxicity have been reported: female gender, pre-existing cardiac diseases, mediastinal radiation, cumulative anthracycline doses, and co-treatments with 5-fluorouracil, cyclophosphamide, or taxanes .Cardiomyopathy induced by doxorubicin was described at an early stage  with an incidence of 1 % to 2 % and also several years after the end of drug administration . This loIn this review, we describe the doxorubicin cardiomyopathy at molecular, histological, and functional levels and the strategies to prevent and monitor cardiac damage. Currently, the cardioprotective treatments based on medical guidelines have limitations, which drive researchers to find new ways to solve them. We discuss the potential of mesenchymal stem cell (MSC) therapy to prevent the cardiotoxicity induced by doxorubicin, its incipient and promising results, and the uncertainty about its use in patients with cancer.Pharmacokinetics studies have demonstrated that doxorubicin has a triphasic plasma clearance after intravenous injection, suggesting that doxorubicin uptake is faster than its elimination from the tissues. For this reason, the risk of toxicity depends directly on the steady-state distribution of the drug . Doxorub2+ [2+ signaling in mitochondria and sarcoplasmatic reticulum, altering the contraction cycle in cardiomyocytes, producing lipid peroxidation in cell membranes, and inhibiting transcription processes. These effects downregulate the expression of cardiac muscle-specific proteins  and mitochondrial proteins , leading the cardiomyocyte to a loss of contraction force by mechanical and energetic causes [Doxorubicin passes through cell membranes by passive diffusion. Inside the cells, doxorubicin accumulates principally in the nucleus and mitochondria (two orders of magnitude) in comparison with the cytoplasmic concentration . The dox2+ . The heac causes .+) and their progeny, reducing regenerative capacity of the heart. On the other hand, De Angelis and colleagues [Huang and colleagues , using alleagues  reportedIn vitro studies using H9c2 myoblast have shown that oxidative stress induced by doxorubicin activates AMPK (a protein kinase considered to be an intracellular sensor of the energy status) that interacts with p53, leading to bax/bad translocation from cytosol to mitochondria and promoting the release of cytochrome c and caspases activation [In regard to the mechanism of apoptosis induced by doxorubicin, there is a consensus on the main role of oxidative stress to activate cell death signal pathways. tivation ,18. On ttivation ,20. Oxidtivation .Doxorubicin also produces oxidative stress in endothelial cells, leading to an increase in endothelial permeability by reduction of nitric oxide production, pro-inflammatory cytokine secretion, and the expression of adhesion molecules . Leukocy2, or PGE2) [In this way, doxorubicin triggers a cardiac inflammatory response, in which several mechanisms of innate immune response are activated. To find the key molecules involved in doxorubicin-induced inflammation, researchers have used several strategies, including neutralizing antibodies to specific receptors  , knockouor PGE2) . The resCurrently, under a myocardium cell death process in an inflammatory microenvironment, collagen fiber synthesis is promoted, constituting the whole picture of histological markers in doxorubicin cardiotoxicity: loss of muscle fiber, sarcoplasmatic distention, vacuolization of cardiomyocytes, and fibrosis .In adult patients, the structural and functional changes induced by doxorubicin toxicity progress mainly to dilated cardiomyopathy, which is defined as an increase in left ventricle (LV) dimension, thinness of LV walls, and a severe loss of contractility. However, in pediatric patients, a restrictive cardiomyopathy described by normal dimension and wall thickness of LV and an enlargement of auricle and hardening of cardiac muscle generating diastolic dysfunction is more frequent .According to current medical guidelines, monitoring of cardiotoxicity for doxorubicin dose (in milligrams per square meter) is performed with echocardiography and multiple gated acquisition scan. A reduction of 10 % in left ventricular ejection fraction (LVEF) from 50 %  is sufficient to suspend the oncologic treatment . However2 for adults and 200 to 250 mg/m2 for children [Prevention of cardiotoxicity is managed mainly by monitoring the maximum cumulative dose  and by children . An altechildren ,35.Doxorubicin cardiotoxicity is frequently refractory to conventional pharmacologic therapies for cardiac ischemic diseases. Βeta-blockers  and angiotensin-converting enzyme inhibitors  are useful to attenuate doxorubicin-induced cardiomyopathy; however, long-term administration should be balanced with their adverse effects such as hypotension, fatigue, and dizziness since their beneficial effects are only transient .in vitro differentiation of iPSCs, improved the cardiac function in an animal model of infarcted heart, suggesting a promising future for iPSC-based therapy. iPSC therapy has the advantage of being free of ethical restrictions; however, owing to their ESC-like properties, they could be tumorigenic [ex vivo, allowing their use in cell-based therapy protocols [Cell-based therapies have a huge potential to treat cardiovascular diseases because of their regenerative properties and safety. Until 2013, approximately 2,000 patients had been enrolled in clinical trials around the world to evaluate different kinds of stem cell therapies showing promising results . In regaorigenic . As a reorigenic  reportedrotocols . The regrotocols . At presrotocols –47. Finain vivo and in vitro models, MSCs can express specific cardiomyocyte markers  [MSCs have many characteristics that make them a suitable tool for preventive or regenerative myocardium therapies (or both), including prevention of doxorubicin cardiomyopathy. MSCs are self-renewal cells with the potential to differentiate into cells of the adipogenic, osteogenic, and condrogenic lineages. Moreover, in adherin) ,49. Howeadherin) . On the adherin) , leadingMSCs secrete paracrine factors such as insulin-like growth factor, hepatocyte growth factor, endothelin-1, and basic fibroblast growth factor (with proliferative and anti-apoptotic properties), vascular endothelial growth factor and platelet-derived growth factor (with angiogenic properties), and matrix metallopeptidase-9 (with anti-fibrotic properties) ,52; all MSCs have been defined as hypoimmunogenic cells because they are not rejected by the recipient’s immune system, even if they come from a non-histocompatible individual , allowin2, and indoleamine 2,3-dioxygenase [MSCs also have anti-inflammatory properties through the activation, suppression, migration, or differentiation of specific immune system cells, including T cells, natural killer cells, B cells, macrophages, dendritic cells, and neutrophils, by the secretion of several immune regulators, including transforming growth factor-beta, IL-4, IL-6, IL-10, PGExygenase . The rolxygenase .ex vivo expansion are quite simple and secure from external contamination [In regard to oxidative stress, the main cause of doxorubicin-induced cardiotoxicity, it was reported that MSCs could manage elevated tissue oxidative stress by reducing ROS-induced apoptosis and modifying the redox microenvironment . Finallymination .In regard to the development of cell-based therapies to prevent doxorubicin cardiotoxicity or to induce the regeneration of the damaged heart, the investigation is still at pre-clinical stages. Under a regenerative therapy hypothesis, MSCs are administered after an established dilated cardiomyopathy, whereas under a preventive therapy hypothesis, MSCs are transplanted before or during doxorubicin treatment Table . It was Doxorubicin also has a toxic effect in endogenous MSCs. Oliveira and colleagues  reportedThe systemic administration of MSCs could have integral beneficial effects in patients with cancer. Zoja and colleagues  demonstrIn regard to the use of MSC therapy to prevent or revert the cardiotoxicity effect of anti-cancer drugs such as daunomycin, idarubicin, mitoxantrone (anthracyclines), 5-fluorouracil (anti-metabolite), or cyclophosphamide , we also expect a beneficial effect in cardiac function because these drugs have a common mechanism of toxicity in cardiomyocytes , which is also described in doxorubicin toxicological studies –73.in vitro [It has been postulated that the regenerative potential of MSCs may be a negative feature in patients with cancer. In fact, there is a controversial point of view about the role of MSCs in cancer. Pre-clinical studies reported that MSCs could promote or inhibit tumor growth . Many mein vitro  and incrin vitro . BMMSC-din vitro . Howeverin vitro . On the in vitro . In summex vivo-expanded , no tumoral transformation has been reported. Indeed, no association between autologous or allogeneic MSC administration and tumor formation was found in 36 clinical studies (phase I and II) reported by the Canadian Critical Care Trials Group [in vivo. Thus, a longer follow-up is required to draw a final conclusion.When human MSCs are properly ls Group . Howeverls Group , in a stThe quality of life of cancer survivors is an emergent topic in the scientific community for the consequences of the adverse events induced by chemotherapy. Therefore, doxorubicin-induced cardiotoxicity is still a relevant issue for oncologic treatment, particularly in pediatric patients. The use of cardiovascular disease therapies based on MSCs is safe, and the myocardium regeneration achieved has a promising impact on the recovery of cardiac function. In animal models, MSC administration could prevent myocardium injury induced by doxorubicin and regenerate the damaged tissue. In addition, owing to the pleiotropic effects of MSCs, their administration could have beneficial effects on extra-cardiac organs. However, in cancer applications, the use of MSCs is still controversial. More pre-clinical studies are needed to better predict the final outcome of the reciprocal influence of cancer cells and MSCs, which is dependent on the source and route of MSC administration and the state and grade of tumor growth. Additionally, clinical trials using MSC therapy may be considered in patients after surgical tumor removal, in order to prevent a heart vulnerability to cardiac diseases in cancer survivors.