The need for systematic methods for reviewing evidence is continuously increasing. Evidence mapping is one emerging method. There are no authoritative recommendations for what constitutes an evidence map or what methods should be used, and anecdotal evidence suggests heterogeneity in both. Our objectives are to identify published evidence maps and to compare and contrast the presented definitions of evidence mapping, the domains used to classify data in evidence maps, and the form the evidence map takes.We conducted a systematic review of publications that presented results with a process termed “evidence mapping” or included a figure called an “evidence map.” We identified publications from searches of ten databases through 8/21/2015, reference mining, and consulting topic experts. We abstracted the research question, the unit of analysis, the search methods and search period covered, and the country of origin. Data were narratively synthesized.Thirty-nine publications met inclusion criteria. Published evidence maps varied in their definition and the form of the evidence map. Of the 31 definitions provided, 67 % described the purpose as identification of gaps and 58 % referenced a stakeholder engagement process or user-friendly product. All evidence maps explicitly used a systematic approach to evidence synthesis. Twenty-six publications referred to a figure or table explicitly called an “evidence map,” eight referred to an online database as the evidence map, and five stated they used a mapping methodology but did not present a visual depiction of the evidence.The principal conclusion of our evaluation of studies that call themselves “evidence maps” is that the implied definition of what constitutes an evidence map is a systematic search of a broad field to identify gaps in knowledge and/or future research needs that presents results in a user-friendly format, often a visual figure or graph, or a searchable database. Foundational work is needed to better standardize the methods and products of an evidence map so that researchers and policymakers will know what to expect of this new type of evidence review.Although an a priori protocol was developed, no registration was completed; this review did not fit the PROSPERO format.The online version of this article (doi:10.1186/s13643-016-0204-x) contains supplementary material, which is available to authorized users. There is growing variation in evidence synthesis methodology to meet the different objectives evidence synthesis can support. The classic systematic review and meta-analysis are both rigorous and produce detailed information about narrow questions, but they are resource intense and the work burden limits the scope of what can be covered . To meetThese new variants on the classical systematic review are at various phases in development. Determining the unique contributions and methods of each of these new synthesis method offshoots is a challenge. Systematic reviews and meta-analyses have a standardized process for conduct and reporting, codified in the Institute of Medicine standards and Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines , 6. The Evidence mapping is the newest of these new evidence review products. In 2002, there were no published evidence maps, and as recently as 2010, only ten such publications could be identified. In addition, evidence mapping has yet to undergo the scrutiny and development of these other methodologies, and it is not clear if those authors who are using the term are using a methodology unique from other developing methods. Both evidence maps and scoping reviews set out to map the literature. A scoping review recently was described as “a form of knowledge synthesis that addresses an exploratory research question aimed at mapping key concepts, types of evidence, and gaps in research related to a defined area or field by systematically searching, selecting, and synthesizing existing knowledge” . They reOur objectives are to identify and systematically review published evidence maps and to assess commonality and heterogeneity and to determine whether additional work is warranted to try to standardize methods and reporting. We compare and contrast the presented definitions of evidence mapping, the domains used to classify data in evidence maps, and the form of the evidence map takes.The librarian on our team, RS, conducted an electronic search of ten databases , Cochrane Central Register of Controlled Trials (CENTRAL), Cochrane Database of Abstracts of Reviews of Effects (DARE), Cochrane Methodology Register (CMR), SCOPUS, and Web of Science) from inception to 8/21/2015 for publications relating to our objective by using the search terms "evidence map" OR "evidence mapping" OR "evidence maps" OR "mapping evidence" OR “evidence map*” in a search of titles and abstracts. Because evidence mapping is a relatively new method in the biomedical literature synthesis repertoire, no Medical Subject Headings (MeSH) term is available. There were no language restrictions or restrictions on study design. Additional studies were identified through reference mining of identified studies and expert recommendations.Two reviewers independently screened titles and abstracts for relevance and obtained full text articles of publications deemed potentially relevant by at least one reviewer. Full text articles were screened against predetermined inclusion criteria by two independent reviewers, and any disagreements were reconciled through team discussion. To be included, authors must have presented results  with a process called evidence mapping or figure called an evidence map. Because of the methodological focus of this review, any patient population, intervention, comparator, outcome, and setting were included.For all included publications, the following data were abstracted: use and definition of the term “evidence map,” research question or aim, search methods and years, number of citations included in the evidence map, and country of origin. The unit of analysis was also abstracted, since some maps considered all literature citations for inclusion, whereas others included systematic reviews only or aggregated all publications originating from the same study into one unit. For publications presenting maps, the domains used to classify studies in the map were also abstracted . Data were abstracted by one reviewer using a standardized form and verified by the second reviewer. The form was piloted and refined by both reviewers prior to abstraction. We applied no quality assessment criteria since we are unaware of any that exist for evidence map methods.Data were narratively synthesized in three sections, discussing key characteristics of definitions presented, domains used to classify literature in the mapping process , and what form an evidence map or evidence mapping methodology takes. Within this latter section, publications were grouped by whether they (1) presented a figure or table explicitly called an “evidence map” or called the results evidence mapping in the publication itself, (2) referred to an online database as the evidence map, or (3) said they used a mapping methodology but did not present a figure or table. No statistical analysis was planned or executed, given the focus on reporting of methods rather than a specific outcome and the heterogeneous nature of the included health topics. We created tables to summarize data on included articles to support the narrative synthesis.N = 26) [N = 8) [The total search results identified 145 titles through 8/21/2015. Reference mining and expert recommendations yielded one title each for a total of 147 titles for screening. Of these titles, we included 53 potentially relevant publications after title and abstract screening. Fourteen of these publications were rejected after full text review. Four of the excluded publications identified themselves as another type of synthesis  in their titles –22. ThreN = 26) –60, and  [N = 8) , 61–66.FN = 19), Australia (N = 10), the UK (N = 7), Canada (N = 2), and Japan (N = 1). Most publications are very recent, with no publications before 2003 , complimentary alternative medicine and nutrition-related topics account for seven publications each (18 % each), and traumatic brain injury and spinal cord injury account for four publications (10 %). Within the mental health group, four of the 11 publications come from the headspace research group, described in more detail below. Six of the seven complementary and alternative medicine publications come from the Veterans Affairs Evidence Synthesis Program, of which the authors of this review are a part. Three of the seven nutrition-related publications come from a research group based at Tufts University, and all four of the traumatic brain injury/spinal cord injury publications are a part of the Global Evidence Mapping (GEM) project. Taken together, these four research groups account for 44 % of the published evidence maps included in this review (17/39).Of the 39 included studies, eight were associated with two evidence mapping projects: GEM , 64, 65 Thus, of the 31 evidence maps with elements of a definition, the most commonly stated component of the definition was a review of evidence to identify gaps or future research needs . Another common component was that the process engage the audience and/or produce user-friendly products . In emphasizing the user-friendly aspect, one definition stated that evidence maps should “provide access to user-friendly summaries of the included studies” , while aOnly one evidence map definition explicitly stated all five of these components  . Three oN = 15). Citations occurring more than twice included Arksey and colleagues [N = 8), the GEM project [N = 7), the headspace project [N = 5), and Katz and colleagues [N = 3). The publication by Arksey and colleagues provides a description of scoping review methods but was still the most cited article. The GEM and headspace projects are both large initiatives that have funded the exploration of a very broad topic, neurotrauma and mental health disorders affecting youth, respectively. The publication by Katz and colleagues, the oldest of the publications we identified, was published in 2003, at least 4 years before any of the other identified mapping publications [Most of the 31 evidence maps did not provide any citations when referencing evidence mapping methodology (lleagues  (N = 8), project  (N = 7), project  (N = 5),lleagues  (N = 3).ications . This puOnly publications that presented an evidence map as a figure or online database were considered relevant in determining domains, or data elements used to classify literature, since these publications were the ones with data presented for such domains. Of the 34 publications, 26 presented the maps in the publication itself, and eight were associated with an online map. As in the definition section, duplicate publications from the same evidence mapping project were collapsed together, leaving a total of 26 evidence maps with domains for this synthesis.N = 12, 46 %), study design , sample size , disorder/condition , and outcomes . Some maps grouped literature in subdomains within these larger domains, such as groups of conditions and then individual conditions within those groups. The average number of domains captured in an evidence map was four, the smallest number being two  and the largest being seven .Thirteen evidence maps categorized their literature by the amount of literature relevant to a particular domain ; publications that referenced a database housing data virtually that can be queried (N = 8); and publications that employed a process or methodology leading to recommendations or synthesized result (N = 5).Three main versions of an evidence map were found: publications with a visual representation of data in the publication (Twenty-six publications explicitly include figure(s) or table(s) which are referred to as evidence maps. They display a range of potential map formats, some quite similar to a classic systematic review evidence table or literature flow diagram. These formats incorporate various numbers of characteristics and types of characteristics. Details of map formats can be found in Table N = 10, 42 %). Eight bubble plots (33 %), two flow charts (8 %), and two bar charts (8 %) were used as the main findings diagram in evidence mapping publications. The last two publications arrayed included studies on a conceptual framework (8 %) and mapped the evidence on the relationships described in the model. Most publications presented data in more than one table or graphic , either with subsets of data in each map  or with different domains covered in the different maps . Figure Most of the 24 published evidence maps used some variant of a cross-tabular format for their main findings, with counts or sums of publications arrayed across various domains  satisfied all five of these components. Thus, the heterogeneity in methods and stated definitions means that stakeholders cannot necessarily know what to expect if they commission an evidence map or seek to identify existing maps.Of all literature synthesis methods, this evidence mapping definition shares many similarities with the definition or goals of a scoping review. Both seek to review broad topics, often with the stated goal of identifying research gaps and areas for future research. In evidence mapping publications, the most often cited article for evidence mapping methods is a scoping methods publication . CompareIn terms of the “map,” the most common ways of organizing the data into a visual representation were using a cross-tabular format and categorizing literature according to interventions and/or study designs present. However, the domains chosen to display and means of presentation will necessarily vary for any particular map according to the aims of the review.Because there is no standard search term or repository for evidence maps, we may not have been able to identify all evidence maps through our search methods. Thus, our findings may be biased to represent those maps that were readily available on the ten searched databases or were cited in other sources we found.With growing numbers of publications using the term “evidence map,” clarifying scoping and evidence mapping methods is an important topic for stakeholders so that they will know what to expect when commissioning, conducting, and interpreting results of an “evidence map.” A key part of this effort will be developing reporting guidelines for these methods. This is already underway for scoping reviews , but givA key strength of the evidence mapping method is the use of visuals or interactive, online databases. Keeping these data up to date and available online may prove to be a challenge in rapidly growing fields, and new audiences may be exposed to these resources who are unfamiliar with how to best make use of the products. As evidence synthesis methods evolve to meet modern demands, they must also meet new challenges.The principal conclusion of our evaluation is that the implied definition of what constitutes an evidence map is a systematic search of a broad field to identify gaps in knowledge and/or future research needs that presents results in a user-friendly format, often a visual figure or graph, or a searchable database. However, there is diversity in methods and stated definitions, so that, as it stands now, stakeholders cannot necessarily know what to expect if they commission an evidence map or seek to identify existing maps.


The use of non-drug, non-health-service interventions has been proposed as a cost-effective alternative to help those with long-term conditions manage their illness and improve their health and well-being. Interventions typically involve accessing activities run by the third sector or community agencies and may also be described as non-medical referral, community referral or social prescribing. To be effective, patients need to be “transferred” from the primary care setting into the community and to maintain their participation in activities. However, it is not currently known how and why these approaches enable which people under what circumstances to reach community services that may benefit their health and well-being.Database searches and extensive searching of grey sources will be carried out in an attempt to find evidence associated with referral and retention in social prescribing. After initial scoping searches, two main phases of searching will be conducted: (a) will focus on the identification of programme theories to illustrate how approaches to social prescribing work for different people and in different contexts and (b) will consist of targeted searches to locate evidence to refine these candidate theories into configurations of the contexts in which populations and the main mechanisms outcomes are achieved. Inclusion criteria will initially be broad in order to develop a clear picture of the ways in which social prescriptions might operate but may iteratively become more focused in response to initially identified evidence, for example, in terms of the population group.An expert advisory group consisting of professionals working in a range of organisations involved in social prescribing will be convened to check the approaches in the review and provide real-life experience of social prescribing. Findings from the review will be disseminated to commissioners, published in a peer-reviewed journal and used to help refine an intervention model for an outdoor nature-based group intervention.This realist review will explore why mechanisms of social prescribing work, for what groups of people and their impact on enrolment, attendance and adherence to programmes. The use of realist approaches to detail the social prescribing process is novel and will offer insights into effective transfer of patients.CRD42016039491PROSPERO  The use of non-drug, non-health-service interventions has been proposed as a cost-effective alternative to help those with long-term conditions manage their condition and improve their health and well-being , contribSocial prescribing interventions potentially enable healthcare providers to respond to a more holistic range of patient needs, reduce frequency of attendance to GPs, reduce social isolation and aid return to work. It is also argued that social prescription models extend the traditional boundaries of primary care and facilitate opportunities for contact with dedicated professionals with increased time allocation and more specific expertise –14. The Information service—this is an information-only service, with display boards and directory access. There is no face-to-face contact.Information and telephone line—leaflets and notice boards advertise the service, and patients self-initiate a telephone discussion with a worker. Again, there is no face-to-face contact.Primary care referral—primary healthcare workers assess patients and refer them to a social prescribing service. This is based upon an appointment leading onto non-clinical issues and is opportunistic.Practice-based generic referral worker—groups are held in general practice, and patients can be referred by health workers or self-refer. This may mean that there is a process of triage and signposting. The surgery can act as a “one-stop shop”.Practice-based specialist referral worker—as above the specialist, worker works from primary care practices but may offer specific services such as Citizens Advice. Direct advice may then be offered as well as referral or signposting onwards.Non-primary care based—referrals from practice-based staff are sent to a referral centre (Bromley User Group Outreach Centre). This could be an outreach service, set in the community, offering a one-to-one service. Other organisations also use this service and facilities.For social prescription interventions to be successful, patients need to be successfully “transferred” from the primary care setting to the relevant resource and to maintain participation for an appropriate period of time. By primary care, we include community mental health and general practice services. The routes through which people are referred are likely to influence the uptake of these services and therefore their success. For instance, without an appropriate, supportive structure for this transition, the suggestion  to pursue an activity is unlikely to be taken up by the patient , 17. WorOthers have suggested that effective delivery would need to consider the following: “link” workers, resource directories, developing of primary care organisational links, increased referrals to organisations, cultural differences between sectors and diversity of referral routes , 12, 14.A recent rapid review found little good quality evidence about social prescribing, largely due to a paucity of controlled study designs . HoweverWe plan to undertake a realist review to explain why different methods of referral and retention in social prescribing activities do (or do not) work in certain circumstances for certain populations as well as the uncertainties relating to methods of referral and retention . A realiWhat are the main factors or mechanismsAre there methods of referral that are particularly useful and appropriate for different groups of people (we are particularly interested in those with mild to moderate depression)?Which approaches to referral are likely to be appropriate, or inappropriate, within NHS primary care settings?What is the best practice for people with mild to moderate depression to receive nature-based interventions? .The primary objective of this review is to explain why different methods of referral and encouraging adherence to social prescribing activities do (or do not) work in certain circumstances for certain populations. The review will have two phases in approaching the objectives listed below: first, programme theories in the literature will be identified, and second, targeted searches will be used to seek suitable evidence to refine these theories see the “” sectionAs we will explore the complex nature of social prescriptions, the methods of the realist review methodology are most suitable . RealistInitial scoping searches will be conducted to develop familiarity with the various models of social prescribing relevant to NHS primary care settings. Subsequently, iterative and progressively more focussed searches will then be used through two main phases.First (a) is the identification of programme theories, which will be used to illustrate how approaches to social prescribing are thought to work for different groups of people in different contexts. Since remaining involved in the activity is important, as well as being referred to it, we will consider contextual factors such as the suitability and acceptability of the types of activity to which people are referred. We will use this range of candidate theories to develop a series of “if, then” statements around mechanisms which we will refine through discussion.Second (b), following the development of key programme theories, targeted searches will be undertaken to seek evidence suitable to refine these candidate theories.Search terms for (a) will focus on types of prescribing models and also the setting . Search terms for (b) will depend on the results from the first phase and will be discussed both within the review team and the broader expert advisory group for sense-checking and completeness.MEDLINEEmbaseCINAHLGreenFILEPsycInfoElectronic databases to be searched in both phases will be as follows:The purpose of the searches is not to be exhaustive, but rather to purposively identify a range of models which, coupled with the fact that we anticipate the literature for this topic to be diverse and dispersed, means we will use extensive searching of grey sources. The precise approach will develop alongside the project, however we anticipate using top-level website searches , selected organisation contacts through the expert advisory group, and searches of, for example, OpenGrey.We will also undertake forward and backward citations chasing on included studies, hand-searching of key journals, and study author contact at both stages where appropriate.In order to develop a clear picture of the range of ways in which social prescriptions might operate, we will include evidence, in the initial phase (a—as above), which is of relevance and contributes to theory building and testing through provisions of descriptions of how potential participants are identified, nature of referral model, people involved in the referral model, context for referral, nature and type of support offered following referral and so on. These sources of evidence are likely to include but not limited to editorials, opinion pieces, communications, primary studies, process evaluations and systematic reviews. Screening will likely be iterative, with disagreements providing articulation of study richness and relevance, on which prioritisation of studies will be based should it be required.In the second phase (b), we will include any studies which include adults (>18) in a primary care setting. By primary care, we incorporate community mental health services, home care, community pharmacies and services in general practices. Referral can be through any health professional or dedicated link worker based in general practice. Initially, we will not restrict evidence to those with mild to moderate depression in order to obtain information on all potentially relevant social prescribing models, but we may iteratively focus down the population groups in response to initially identified evidence. For example, we may focus on a group exhibiting key personality traits associated with depression  where there is a particularly rich seam of evidence.Social prescribing programmes which have sought to transfer patients between primary care and organisations delivering community-based activities. As above, we will initially cast a wide net to ensure that we can learn from all available prescribing models, including exercise referral, arts based practice and nature-based referrals.As a range of evidence and study designs will be included, comparator criteria will only be applied to comparative designs, for example, in randomised controlled trials which compare social prescribing to a non-social prescribing activity. Relevant comparator activities will be those delivered within or through primary care (i.e. patients are not referred out to a community-based activity), and examples will be specific to conditions but may include prescriptions for drugs, cognitive behavioural therapy or physiotherapy. Only the processes of prescription and acceptance, attendance or adherence will be compared, rather than any effect the treatment or intervention has on the patient’s health.As stated, in the first phase (a), we will include evidence that provides descriptions of social prescribing processes; we anticipate that important details will be provided in non-empirical studies.We will include, in the second phase (b), quantitative studies including randomised controlled trials (RCTs), controlled before-and-after studies, interrupted time series, cohort studies and case-control studies. However, any study providing a detailed account of a programme will be considered.We will include qualitative studies from any discipline or theoretical tradition that uses recognised methods of data collection and analysis. We will also include qualitative evidence linked to quantitative intervention studies (“sibling studies”).Quantitative or qualitative process evaluations will be included.Where insufficient controlled evidence is located, we will include uncontrolled studies .Primary care professionals’ awareness and prescribing of a social prescription activity and patients’ acceptance of the prescription (enrolment)Patients’ initial participation in the activity (engagement)Patient’s ongoing engagement and/or completion of the prescription (adherence)We anticipate that the outcomes will be diverse and context specific, and so we are unable to produce an exhaustive list at the outset. The outcomes will relate to the process of social prescribing and are likely to encompass the following:“Contexts” will be central to the interpretation of the outcomes, and their impact to the firing of particular mechanisms will be of interest to the review .effectiveness of the social prescriptions activities on health outcomes will not be considered in this review.The We will explore relevant outcome measures with our expert advisory group and with the full-review team.Titles and abstracts (where available) will be screened by one reviewer, and where these appear to fit the inclusion criteria, the full text will be screened by two reviewers. Disagreements will be resolved through discussion and, where required, a third reviewer.We will assess the rigour (i.e. whether the method used to generate that particular piece of data is credible and trustworthy) of relevant evidence in the following ways: in the first phase (a) and in keeping with other realist reviews, we will use a hybrid classification tool in the first instance , which cIn the second phase (b), we will use standard quality assessment tools suitable to judge the plausibility and coherence of the method used to generate data in the included studies. For randomised studies, we will use the Cochrane Collaboration’s tool for assessing risk of bias , and forAppraisal of studies at both stages will be undertaken by two reviewers independently, with any disagreement being resolved through discussion and where necessary a third reviewer.The exported files from the searching will be uploaded and de-duplicated in Endnote X7 . SearcheIn the first phase (a), data will not be formally “extracted” but rather, in keeping with a realist approach , the revThe second phase (b) will build on the first phases to ensure relevant data is captured and, in method, will constitute more traditional data extraction. Data will be extracted into bespoke forms developed for the review. Data will be extracted by one reviewer and checked by another. We will extract data which helps clarify and explain the mechanisms and refine programme theory. Extracted data will likely relate to full details of the nature of the programme  and any theory informing it, setting/provider, and outcomes.An expert advisory group will be convened to check our approaches and to develop “everyday” theories about how social prescribing is thought to work, or not work, in different contexts for different groups of people. This advisory group will meet at least once in person over the course of the project. It will comprise general practice staff involved in social prescribing, community/third sector groups providing activities that are socially prescribed and specialist academics in the field.We aim to build an understanding of the process of social prescribing by identifying how specific outcomes are generated by relevant mechanisms which are triggered in particular contexts. We will seek recurring patterns across the data.Juxtaposition of sources of evidence—for example, where evidence about implementation in one source enables insights into evidence about outcomes in another source.Reconciling of sources of evidence—where results differ in apparently similar circumstances; further investigation is appropriate in order to find explanations for why these different results occurred.Adjudication of sources of evidence—on the basis of methodological strengths or weaknesses.Consolidation of sources of evidence—where evidence about mechanisms and outcomes is complementary and enables a multi-faceted explanation to be built.Situating sources of evidence—where outcomes differ in particular contexts; an explanation can be constructed of how and why these outcomes occur differently.We will use the same approach to synthesis for both phases (a) and (b), following the realist methodology , 32:JuxtWe will organise studies according to mechanism and contextual factors such as type of activities, type of participants, modes of referral and support. We will further explore mechanisms and complex outcomes through tabulation of data and, where appropriate, through the derivation and iterative development of a conceptual framework/logic model . We willWe will submit the results of the review to a high-impact, peer-reviewed journal. Any write-up will follow the Realist And Meta-narrative Evidence Syntheses: Evolving Standards (RAMESES) publication guidelines .We will also produce a briefing document for commissioners outlining our findings. This will be a two-page summary of the purpose, aims, findings, and implications of the review that are relevant and user-friendly for the commissioners’ respective organisations.In addition, the findings will help to refine an intervention model about nature-based interventions (NatureScript) for those with mild to moderate depression. This approach is being offered by a number of GPs in the South West under the Dose of Nature scheme, as well as through other third sector providers, such as the Conservation Trust’s Green Gym.GP, general practitioner; NHS, National Health Service; NIHR, National Institute for Health Research; PCT, Primary Care Trust; PenCLAHRC, Peninsula Collaboration for Leadership in Applied Health Research and Care; PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses; RAMESES, Realist And Meta-narrative Evidence Syntheses: Evolving Standards; RCT, randomised controlled trial.


In their comparative analysis of Randomised Clinical Trials and observational studies, Papanikoloau et al. (2006) assert that “it may be unfair to invoke bias and confounding to discredit observational studies as a source of evidence on harms”. There are two kinds of answers to the question why this is so. One is based on metaphysical assumptions, such as the problem of causal sufficiency, modularity and other statistical assumptions. The other is epistemological and relates to foundational issues and how they determine the constraints we put on evidence. I will address here the latter dimension and present recent proposals to amend evidence hierarchies for the purpose of safety assessment of pharmaceuticals; I then relate these suggestions to a case study: the recent debate on the causal association between paracetamol and asthma. The upshot of this analysis is that different epistemologies impose different constraints on the methods we adopt to collect and evaluate evidence; thus they grant “lower level” evidence on distinct grounds and at different conditions. Appreciating this state of affairs illuminates the debate on the epistemic asymmetry concerning benefits and harms and sets the basis for a foundational, as opposed to heuristic, justification of safety assessment based on heterogeneous evidence. •Benefit vs. harm: distinction of epistemic/practical constraints for safety assessment.•Evidence standards in relation to distinctive epistemologies.•Hypothesis testing vs. inductive methods of hypothesis confirmation.•Probabilistic hypothesis of causality in safety assessment.•Epistemological rationales for the acceptance of “lower level” and heterogeneous evidence. Evidence standards are supposed to provide “quick and dirty rules” for assessing the quality of evidence, as a function of the greatest possible reduction of confounding and bias. Hence, randomized controlled studies are followed by comparative studies which are not randomized (e.g. cohort or case–control studies), and these are followed by reasoning about pathophysiologic mechanisms underlying the observed outcome. Expert judgment is regarded as the weakest form of evidence and put at the bottom of the hierarchy . On the other side, cases falling under the heading of “uncertain inference”, i.e. situations in which the conclusion is highly probable or plausible, but fallacious from a strictly logical point of view. The former generally fall into the category of deductive inference, whereas the latter are generally labeled with the umbrella category of “induction”. An additional form of uncertain inference falls under the heading of inverse induction .0). The underlying epistemology is hypothetico-deductive (0 ➔ E). If the outcome shows a statistically significant difference (¬E), then the hypothesis of lack of treatment can be rejected (¬H0), following classical modus tollens:In classical hypothesis-testing, the result is expressed as the probability of observing the experimental result or more “extreme” results in the sample space , if the treatment makes no difference  to some alternative cause. Blinding, intervention and randomization are essential instruments in warranting this causal claim see also  and evidto the extent that other conditions warrant for lack of bias and confoundingas alternative to randomization, blinding and intervention. The focus on internal validity is evident also in allowed deviations from evidence hierarchies in specific cases, i.e. where “lower level evidence” such as case reports and observational data are considered sufficient evidence for causal claims Non-deductive methods abandon the goal of outright hypothesis acceptance or rejection and track uncertainty while updating the degree of confidence in a given hypothesis upon new evidence by also taking into account background knowledge. This allows them to be more flexible with regard to the kind of evidence which is allowed to inform hypothesis confirmation and the methods for amalgamating it.Within this framework, the two somewhat contending paradigms are constituted by probabilistic approaches to hypothesis confirmation (e.g. Bayesian epistemology) and abductive reasoning , as well as on the probability measure associated to each hypothesis prior to collecting the evidence, P(hi), and by updating it through conditionalization . This distinguishes them sharply from frequentist statistics where the p-value measures instead the probability of observing the evidence obtained in the experiment (or “more extreme results”) if the hypothesis under investigation is false.Bayesian epistemologies  insist oIn the Bayesian paradigm the main requirement is that all available evidence is used : this isIn the IBE paradigm, hypotheses are justified by their explanatory power: the greater the amount of data the hypothesis is able to explain, the greater its plausibility. Thus, explanatory power is considered to be truth-conducive . This pais there any other way of explaining the set of facts before us, is there any other equally, or more, likely than cause and effect?” (emphasis added). Thus, Hill both refers to explanatory power and hypothesis likelihood as reliable grounds to justify causal judgments.The first important advocate of alternative approaches to statistical hypothesis testing was Sir Austin Bradford Hill with his most cited President's Address  inauguraIn recent times other authors have endorsed similar proposals. The next section presents a case study which illustrates the different epistemologies underlying, often implicitly, the distinctive stances in evaluating evidence for causal assessment. Such debates may be clarified by making the respective epistemic framework explicit. Furthermore, with the help of this case study, four points will be illustrated as to show why non-deductive approaches fare better in safety assessment.The case of acetaminophen and asthma emerged with a study by Possible biological pathways underlying the causal hypothesis have also been investigated. when the study findings are considered together with other available data, there is substantive evidence that acetaminophen use in childhood may be an important risk factor for the development and/or maintenance of asthma” . An even stronger commitment to the hypothesis of causal association is expressed by The evidence gathered so far in support of the hypothesis of causal association between acetaminophen and asthma has generated two opposite stances. On one side, a series of authors show some reluctance in accepting such evidence as a sufficient basis for practice change and for establishing a causal relationship between acetaminophen and asthma, on grounds that it does not result from randomized clinical trials . ParticuMcBride justifies his claim by appealing to the consistency of interdisciplinary evidence: 1) strength of the association displayed in comparative studies; 2) robustness of association across geography, culture and age; 3) dose–response relationship; 4) coincidence of time trends in acetaminophen use and asthma increase; 5) lack of other equally strong causal explanations; 6) relationship between asthma epidemic and per-capita sales of acetaminophen across countries; and 7) plausible mechanism. McBride explicitly warns against the use of acetaminophen in children with asthma or at risk for asthma and claims that if further evidence is required, then this is for documenting product safety rather than the contrary.1)The threshold for practice change is determined by the risk–benefit balance: the higher the risk in comparison to the therapeutic effect, the weaker may be the hypothesis of the causal link in order to change the marketing status of the pharmaceutical product . For ins2)effects of causes, rather than causes of effects, with the latter rather than the former being the focus of risk detection. Indeed when testing efficacy one is interested in ascertaining what kinds of effects a given treatment produces and in proving that no other causes is at play. Instead the main point of risk detection is to explain observed adverse events and to identify their causes in the first place. As Following 3)Concerning side-effects, most information comes gradually through “lower level” kinds of evidence, and there comes a point where the signal strongly suggests causation without demonstrating it. In such cases, one cannot pretend to know nothing about the side-effects of intervention until one doesn't have an RCT proving causality; yet this is exactly what the epistemology underpinning evidence hierarchies asks one to do.4)RCTs deliver limited and purposely decontextualized information; they have been developed in order to test fertilizers on plant growth . The cauThe dissent concerning the best course of action among scholars is ultimately caused by differing epistemological views which are left implicit. Those recommending the performance of placebo-controlled RCTs are in line with the rationales underlying evidence hierarchies. Thus they insist on the elimination of any suspicion of confounding, especially confounding by indication . On the In sum, the complexity of the causal structure in biological domains, the need for flexible decision tools which require the possibility to act upon uncertainty, and the focus on risk/benefit balance, strongly suggest a paradigm change in the evaluation of evidence for harm.Different epistemologies grant different methodological actions and grant “lower level” evidence on distinct grounds and at different conditions, thus it is worthy and also crucial to bear in mind the criteria underlying the evidence constraints one imposes on oneself. Appreciating this state of affairs illuminates the debate on the epistemic asymmetry concerning benefit and harm assessment and sets the basis for a foundational, as opposed to heuristic, justification of evidential support for pharmaceutical harm. Particularly, granted that knowledge about the drug risks comes from different sources and grows cumulatively over the course of time, probabilistic methods of causal assessment which rely on evidence amalgamation are better suited to the purpose. Instead, hypothesis testing is ill suited for risk management and pharmacosurveillance in that it has been developed in order to address the issue of false positives, rather than false negatives, which are the main concern in safety assessment. In sum, categorical approaches are too rigid for safety assessment on the basis of probabilistic hypotheses and of heterogeneous evidence which possibly fails to meet their strict desiderata.The authors declare that there are no conflicts of interests.


R. Soc. B 281, 20140558. (doi:10.1098/rspb.2014.0558)) and the subject continues to be an area of very active research and high policy relevance.A summary is provided of recent advances in the natural science evidence base concerning the effects of neonicotinoid insecticides on insect pollinators in a format (a ‘restatement') intended to be accessible to informed but not expert policymakers and stakeholders. Important new studies have been published since our recent review of this field (Godfray  They are used in different ways, but particularly as seed treatments where the chemical is absorbed by the growing plant and is distributed through all tissues at concentrations that can kill insect herbivores. However, neonicotinoids are also translocated to nectar and pollen where they can be consumed by pollinating insects. Numbers of pollinators have declined in agricultural landscapes and there is concern that the introduction and widespread use of neonicotinoids is partly responsible.In December 2013, the European Union (EU) instigated partial restrictions on the use of neonicotinoid insecticides on crops that might be used as food by pollinating insects. This move is strongly opposed by many in the farming community and there has been a vigorous debate focusing on the scientific evidence that neonicotinoids harm pollinators, as well as the environmental and economic costs and benefits of the restrictions.To try to assist the debate we produced a ‘restatement’ of the underlying natural science evidence base in a form that was intended to be accessible to informed but not expert policymakers and stakeholders . Our avohttp://www.efsa.europa.eu/en/data/call/150522). Much new research has been published on the topic (we review over 80 studies here) including the largest replicated field study to date [Since the restatement was published the debate about restricting neonicotinoid use has continued unabated. Farming organizations have successfully applied for ‘120-day derogations’ from the restrictions in several European countries  on the grounds of lack of alternative pest-management options, moves that have been criticized by environmental non-governmental organizations. The EU is committed to review the restrictions in 2015–2016 and through the independent European Food Safety Authority opened a call for evidence  because of the significant advances in the science; (ii) because of the continuing need for policy-neutral evidence summaries in this highly contested area, especially in the run up to the review of the EU restrictions; and (iii) in response to a request to do so by the UK Government Chief Scientific Adviser.2.The literature on pollinators and neonicotinoids published since our restatement was completed was reviewed and a first draft evidence summary produced by a subset of the authors. All authors reviewed and revised the document, and agreed on the categorizing of the different evidence components using the same scheme we adopted earlier, and which is explained in paragraph A2 of the restatement update (appendix A). The second draft was sent to a series of stakeholders or stakeholder groups including scientists involved in pollinator research, representatives of the farming and agrochemical industries, non-governmental organizations concerned with the environment and conservation, and UK government departments and statutory bodies responsible for pollinator policy. The document was revised in the light of much helpful feedback from over 40 stakeholders (see acknowledgements). Though many groups were consulted, the project was conducted completely independent of any stakeholder and was funded by the Oxford Martin School (part of the University of Oxford).3.The update to the restatement of the natural science evidence base concerning neonicotinoid insecticides and insect pollinators is given in appendix A, with an annotated bibliography provided as the electronic supplementary material.4.The new evidence and evidence syntheses that have been published in the last 18 months (between February 2014 and August 2015) significantly advance our understanding of the effects of neonicotinoids on insect pollinators. Nevertheless, major gaps in our understanding remain, and different policy conclusions can be drawn depending on the weight one accords to important (but not definitive) science findings and the weightings given to the economic and other interests of different stakeholders. The natural science evidence base places constraints on policies that claim to be consistent with the science, but does not specify a single course of action.We also raise an issue here that arises from our original study but is not directly relevant to the evidence base on the effects of neonicotinoids on pollinators. In introducing the subject we wrote ‘Neonicotinoid insecticides are a highly effective tool to reduce crop yield losses due to insect pests’, and in the restatement itself listed a small number of papers in the scientific literature to support this statement . It has per se.The efficacy of neonicotinoids is clearly an important issue, and we believe few would doubt that in some circumstances  they are highly effective and in other circumstances they do not justify the costs of their purchase. We did not attempt to review this subject and should have been more careful to say we were not commenting on efficacy https://clinicaltrials.gov/ct2/home and https://www.clinicaltrialsregister.eu) provides a model for how the latter might be achieved.Though a meta-analysis of efficacy would be very informative it would also be very difficult. Efficacy studies are largely conducted by industry, the sector that benefits most from the data, and are not the type of science usually funded by public organizations. Typically, the studies are not published in the peer-reviewed literature (though they are often made available to regulators) and some are kept confidential for commercial reasons. Efficacy trials are expensive and it seems unlikely that they will ever be publicly funded at scale. It is an interesting topic for debate whether industry would benefit in the long run from placing more of its data in the public domain as well as putting in place measures to increase public confidence in studies they fund themselves. The recent movement in the pharmaceutical sector to set up trial registries (see


Background: Recently, the International Agency for Research on Cancer (IARC) Programme for the Evaluation of Carcinogenic Risks to Humans has been criticized for several of its evaluations, and also for the approach used to perform these evaluations. Some critics have claimed that failures of IARC Working Groups to recognize study weaknesses and biases of Working Group members have led to inappropriate classification of a number of agents as carcinogenic to humans.Objectives: The authors of this Commentary are scientists from various disciplines relevant to the identification and hazard evaluation of human carcinogens. We examined criticisms of the IARC classification process to determine the validity of these concerns. Here, we present the results of that examination, review the history of IARC evaluations, and describe how the IARC evaluations are performed.Discussion: We concluded that these recent criticisms are unconvincing. The procedures employed by IARC to assemble Working Groups of scientists from the various disciplines and the techniques followed to review the literature and perform hazard assessment of various agents provide a balanced evaluation and an appropriate indication of the weight of the evidence. Some disagreement by individual scientists to some evaluations is not evidence of process failure. The review process has been modified over time and will undoubtedly be altered in the future to improve the process. Any process can in theory be improved, and we would support continued review and improvement of the IARC processes. This does not mean, however, that the current procedures are flawed.Conclusions: The IARC Monographs have made, and continue to make, major contributions to the scientific underpinning for societal actions to improve the public’s health.http://dx.doi.org/10.1289/ehp.1409149Citation: Pearce N, Blair A, Vineis P, Ahrens W, Andersen A, Anto JM, Armstrong BK, Baccarelli AA, Beland FA, Berrington A, Bertazzi PA, Birnbaum LS, Brownson RC, Bucher JR, Cantor KP, Cardis E, Cherrie JW, Christiani DC, Cocco P, Coggon D, Comba P, Demers PA, Dement JM, Douwes J, Eisen EA, Engel LS, Fenske RA, Fleming LE, Fletcher T, Fontham E, Forastiere F, Frentzel-Beyme R, Fritschi L, Gerin M, Goldberg M, Grandjean P, Grimsrud TK, Gustavsson P, Haines A, Hartge P, Hansen J, Hauptmann M, Heederik D, Hemminki K, Hemon D, Hertz-Picciotto I, Hoppin JA, Huff J, Jarvholm B, Kang D, Karagas MR, Kjaerheim K, Kjuus H, Kogevinas M, Kriebel D, Kristensen P, Kromhout H, Laden F, Lebailly P, LeMasters G, Lubin JH, Lynch CF, Lynge E, ‘t Mannetje A, McMichael AJ, McLaughlin JR, Marrett L, Martuzzi M, Merchant JA, Merler E, Merletti F, Miller A, Mirer FE, Monson R, Nordby KC, Olshan AF, Parent ME, Perera FP, Perry MJ, Pesatori AC, Pirastu R, Porta M, Pukkala E, Rice C, Richardson DB, Ritter L, Ritz B, Ronckers CM, Rushton L, Rusiecki JA, Rusyn I, Samet JM, Sandler DP, de Sanjose S, Schernhammer E, Seniori Costantini A, Seixas N, Shy C, Siemiatycki J, Silverman DT, Simonato L, Smith AH, Smith MT, Spinelli JJ, Spitz MR, Stallones L, Stayner LT, Steenland K, Stenzel M, Stewart BW, Stewart PA, Symanski E, Terracini B, Tolbert PE, Vainio H, Vena J, Vermeulen R, Victora CG, Ward EM, Weinberg CR, Weisenburger D, Wesseling C, Weiderpass E, Zahm SH. 2015. IARC Monographs: 40 years of evaluating carcinogenic hazards to humans. Environ Health Perspect 123:507–514;  Important advances in human health have come from the recognition of health hazards and the development of policy actions to address them . GovernmIARC Monographs on the Evaluation of Carcinogenic Risks to Humans of the International Agency for Research on Cancer (IARC) are a prominent example of such an expert review process. The goal of the Monograph Programme is to assess carcinogenic hazards from occupational, environmental, and lifestyle exposures and agents, thus providing an essential step in the societal decision-making process to identify and then control carcinogenic hazards. For these evaluations, IARC assembles groups of scientists with a range of relevant scientific expertise  to review and assess the quality and strength of evidence from informative publications and perform a hazard evaluation to assess the likelihood that the agents of concern pose a cancer hazard to humans . Indeed, as recently as April 2014, the IARC Monographs program has been a subject of a review by the Advisory Group to recommend priorities for IARC Monographs during 2015–2019 . The AdvHere, we focus on current IARC processes and practices because these have been the focus of recent criticisms. The authors of this Commentary are scientists from a wide range of disciplines who are involved in designing and conducting studies that provide data used in hazard evaluations, such as those performed by IARC. Many  of us have served on IARC Monograph Working Groups, but none are current IARC staff. We first discuss the history of IARC, and describe how the IARC evaluations are performed in order to foster evidence-based policy. We then describe why unbiased evaluations, based on the evidence and free of conflicts of interest, are necessary for public health decision making. Finally, we discuss the recent criticisms of the IARC approach.History of the IARC Monographs. Shortly after IARC’s establishment, its parent entity, the World Health Organization (WHO), asked IARC to prepare a list of agents known to cause cancer in humans. IARC recognized the need for a systematic process to determine which agents should be listed. Such a process was launched in 1972 by Lorenzo Tomatis, then Chief of the Division of Carcinogenicity of IARC . The classification categories are described in the preamble to the Monographs . CarcinoThe IARC Monograph process. The process for the preparation of an IARC Monograph is clearly described in the Preamble, which is published as part of each Monograph . For example, in 2013 IARC sought nominations for agents to be evaluated in 2015–2019  and availability of sufficient literature for an evaluation of carcinogenicity, as well as advice from the Advisory Groups. The large majority of evaluations concern specific compounds, but there are also monographs on various occupations or industries, for example, aluminum production, insecticide applicators, firefighters, manufacture of leather goods, leather tanning and processing, welding, painters, petroleum refining, and pulp and paper manufacturing. Some individual exposures that occur in these settings have also been evaluated.http://monographs.iarc.fr/ENG/Meetings/) before the WG is convened, and anyone can send comments. Members are typically scientists who have conducted research relevant to the agent under review, but not necessarily on the specific agent. Selection procedures are evaluated yearly by the Scientific and the Governing Councils. The IARC Section of Monographs also has an external Advisory Board, made up of independent scientists, that periodically peer reviews its activities. In addition to Working Group members, invited specialists, representatives of health agencies, stakeholder observers, and the IARC Secretariat also attend meetings.The next step is the selection of members of the Working Group (WG). IARC staff review the literature to identify Working Group candidates and specialists in relevant areas of expertise; they also seek names of possible candidates from the scientific community and advisory groups. The list of potential members, including disclosure of relevant conflicts of interest, is posted on the IARC website . Working Group members are also responsible for writing the IARC Monograph, which must both review the literature and explain why the Working Group came to their specific conclusions.The procedures used to evaluate the scientific evidence are described in the Preamble to the Monographs . It is iIARC assessments of carcinogenicity are based on, and necessarily limited to, scientific evidence available at the time of the review. The evidence comes from epidemiologic studies, animal bioassays, pharmacokinetic/mechanistic experiments, and surveys of human exposure. The aim is to include all relevant papers on cancer in humans and experimental animals that have been published, or accepted for publication, in peer-reviewed scientific journals and also any publicly available government or agency documents that provide data on the circumstances and extent of human exposure. To that end, the search of the literature takes a comprehensive approach. Papers that are found not to provide useful evidence can be excluded later in the process. IARC staff first use previous IARC Monographs (if available), database searches using relevant text strings, and contact with investigators in the field to identify potentially relevant material. Thus, the initial assembly of the literature is performed by individuals who are not engaged in the actual evaluation. Working Group members are then assigned various writing tasks and are instructed to perform their own literature searches to identify any further papers that might have been missed. In addition, all of the papers assembled by IARC are made available to the full Working Group before they meet, and any member can recommend other papers not previously identified that they think should be considered. Finally, papers can be recommended by stakeholder representatives before or during the Working Group meeting.At the meeting of the Working Group, the assembled documents are reviewed and summarized by discipline-related subgroups.However, any member of the Working Group has access to all of the assembled literature. The summaries are distributed to all subgroups, and information from all disciplines is discussed in plenary sessions prior to assigning the agents to a specific carcinogenicity category.http://monographs.iarc.fr/ENG/Monographs/PDFs/index.php).Because new findings continually emerge in the literature, agents are reconsidered when IARC and IARC Advisory Groups judge that there is sufficient additional information that might alter a previous evaluation. Thus, conclusions regarding human carcinogenicity of particular substances may change as new evidence becomes available. For some agents, this reevaluation has resulted in progression toward greater certainty regarding their human carcinogenicity, whereas for others the progress has been moved toward less certainty. Such movements are expected in an open, transparent, and evidence-based process. A comprehensive update of all Group 1 carcinogens was recently accomplished in Volume 100 A through F  or to preferentially report positive findings over negative or inconclusive findings  (on bias) . This crFalse-positive findings may occur by chance, particularly when many combinations of exposures and health outcomes have been examined in a single study without strong prior expectations of association; this happens often, for example, in genome-wide association studies where thousands of gene–disease associations are evaluated. Chance, of course, operates in all disciplines and in both observational and experimental studies. However, there are well-known statistical techniques to reduce the probability of declaring chance findings as “positive” . IndepenFalse negatives are more difficult to address, and perhaps they occur more frequently than false positives because of low statistical power, nondifferential misclassification of exposure and/or outcome, and incomplete follow-up, which tends to reduce the observed difference in risk between the exposed and nonexposed populations . A new pThere are difficulties in conducting epidemiologic studies of agents that are relatively “weak” carcinogens, or for stronger carcinogens where exposure is very low because bias and confounding can obscure weak positive associations . In geneFalse-positive and false-negative findings in individual studies may arise by chance or bias, including bias due to confounding . HoweverDiscontent with IARC Monograph processes. The IARC Monograph evaluation process has been criticized and it has been alleged that “a number of scientists with direct experience of IARC have felt compelled to dissociate themselves from the agency’s approach to evaluating carcinogenic hazards”  have been classified as carcinogenic to humans (Group 1), 69 (7%) as probably carcinogenic (Group 2A), 283 (29%) as possibly carcinogenic (Group 2B), 504 (52%) as not classifiable regarding their carcinogenicity (Group 3), and 1 (< 1%) as probably not carcinogenic to humans (Group 4). Thus, even for this highly select group of agents , more than one-half were “not classifiable” or “probably not carcinogenic,” and a further 29% were placed into the category of possibly carcinogenic to humans. This distribution, based on nearly 1,000 evaluations in which fewer than one in five agents were classified as carcinogenic or probably carcinogenic to humans, does not support a conclusion that the process is heavily biased toward classifying agents as carcinogenic ; U.S. Environmental Protection Agency (http://www.epa.gov/); National Academy of Sciences (http://www.nasonline.org/); the American Conference of Governmental Industrial Hygienists (ACGIH) Threshold Limit Values and Biological Exposure Indices (http://www.acgih.org/); the Nordic Expert Group for Criteria Documentation of Health Risks from Chemicals (http://www.av.se/arkiv/neg/); Institute of Occupational Medicine (http://www.iom-world.org/); World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) Expert Reports; European Chemicals Agency (https://echa.europa.eu); http://oehha.ca.gov/prop65/background/p65plain.html); Health Canada Bureau of Chemical Safety (http://www.hc-sc.gc.ca/ahc-asc/branch-dirgen/hpfb-dgpsa/fd-da/bcs-bsc/index-eng.php); Scientific Committee on Occupational Exposure Limits (SCOEL), European Commission, Employment, Social Affairs and Inclusion ; European Food Safety Authority (http://echa.europa.eu/). Assessments from these groups typically come to conclusions similar to those from IARC. This further indicates broad agreement within the scientific community regarding evidence on carcinogenicity in the scientific literature and expands the number of scientists who do not have a “vested interest” but who have generally agreed with those conclusions.There has been very broad involvement of the scientific community in the IARC Monograph Programme through participation in the Working Groups and service on the IARC Governing and Scientific Councils and ad hoc Advisory Board for the Monograph Programme. The long list of scientists who are coauthors of this paper attests to the strong support that IARC has in the scientific community. Many exposures that IARC has evaluated have also been independently evaluated by other institutions, such as the U.S. National Toxicology Program  at one time went through a quite lengthy period of contentious debate , 2008. Acarcinogenic to humans.Group 1: The agent is This category is used when there is sufficient evidence of carcinogenicity in humans. Exceptionally, an agent may be placed in this category when evidence of carcinogenicity in humans is less than sufficient but there is sufficient evidence of carcinogenicity in experimental animals and strong evidence in exposed humans that the agent acts through a relevant mechanism of carcinogenicity.Group 2.sufficient, as well as those for which, at the other extreme, there are no human data but for which there is evidence of carcinogenicity in experimental animals. Agents are assigned to either Group 2A (probably carcinogenic to humans) or Group 2B (possibly carcinogenic to humans) on the basis of epidemiological and experimental evidence of carcinogenicity and mechanistic and other relevant data. The terms probably carcinogenic and possibly carcinogenic have no quantitative significance and are used simply as descriptors of different levels of evidence of human carcinogenicity, with probably carcinogenic signifying a higher level of evidence than possibly carcinogenic.This category includes agents for which, at one extreme, the degree of evidence of carcinogenicity in humans is almost probably carcinogenic to humans.Group 2A: The agent is limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in experimental animals. In some cases, an agent may be classified in this category when there is inadequate evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in experimental animals and strong evidence that the carcinogenesis is mediated by a mechanism that also operates in humans. Exceptionally, an agent may be classified in this category solely on the basis of limited evidence of carcinogenicity in humans. An agent may be assigned to this category if it clearly belongs, based on mechanistic considerations, to a class of agents for which one or more members have been classified in Group 1 or Group 2A.This category is used when there is possibly carcinogenic to humans.Group 2B: The agent is limited evidence of carcinogenicity in humans and less than sufficient evidence of carcinogenicity in experimental animals. It may also be used when there is inadequate evidence of carcinogenicity in humans but there is sufficient evidence of carcinogenicity in experimental animals. In some instances, an agent for which there is inadequate evidence of carcinogenicity in humans and less than sufficient evidence of carcinogenicity in experimental animals together with supporting evidence from mechanistic and other relevant data may be placed in this group. An agent may be classified in this category solely on the basis of strong evidence from mechanistic and other relevant data.This category is used for agents for which there is not classifiable as to its carcinogenicity to humans.Group 3: The agent is inadequate in humans and inadequate or limited in experimental animals.This category is used most commonly for agents for which the evidence of carcinogenicity is inadequate in humans but sufficient in experimental animals may be placed in this category when there is strong evidence that the mechanism of carcinogenicity in experimental animals does not operate in humans.Exceptionally, agents for which the evidence of carcinogenicity is Agents that do not fall into any other group are also placed in this category.An evaluation in Group 3 is not a determination of noncarcinogenicity or overall safety. It often means that further research is needed, especially when exposures are widespread or the cancer data are consistent with differing interpretations.probably not carcinogenic to humans.Group 4: The agent is evidence suggesting lack of carcinogenicity in humans and in experimental animals. In some instances, agents for which there is inadequate evidence of carcinogenicity in humans but evidence suggesting lack of carcinogenicity in experimental animals, consistently and strongly supported by a broad range of mechanistic and other relevant data, may be classified in this group.This category is used for agents for which there is


Community-acquired pneumonia (CAP) remains a major cause of morbidity and mortality worldwide among children. The growing number of guidelines have been accompanied with a growing concern about variance and conflicts among guideline recommendations. There is a need to critically appraise clinical practice guidelines (CPGs) in order to ensure safe and effective practices.A literature search was systematically conducted in English and Chinese major academic databases (from January 2000 to March 2015). CPGs related to CAP in children were evaluated by four independent assessors, according to AGREE II instruments. Standardized domain scores were calculated for each guideline. Inter-rater reliability was assessed by intraclass correlation coefficient. The software used for analysis was SPSS 17.0.A total of 10 guidelines met the inclusion criteria and were appraised. Scope and purpose (69.03 %) and clarity of presentation (83.33 %) achieved relative high scores, while the scores of the other four domains were low: stakeholder involvement (42.78 %), rigour of development (44.95 %), applicability (37.60 %), and editorial independence (23.74 %). 3 guidelines were strongly recommended as a result of the overall scores were greater than 60 %.The qualities of CPGs for CAP in children were generally acceptable with several flaws. Stakeholder involvement, rigour of development, applicability and editorial independence should be considered and well described in the future development of CPGs. Community-acquired pneumonia (CAP) is defined as an acute infection of lower respiratory tract occurring outside hospital. It remains a major cause of morbidity and mortality among children in developing and developed countries. In developing countries, CAP has an incidence of 0.29 episodes per child-year and a mortality rate of 1.3–2.6 % . In certGuidelines for the management of CAP in children have been increasingly produced and disseminated in recent years. Generally, globalization and uniformity between centers worldwide in patient management has become a trend. However, acceptance of evidence-based medicine (EBM) varies around the world. The growing number of guidelines has been accompanied with a growing concern about variance and conflicts among guideline recommendations. Therefore, there can be huge differences between countries or regions in respect to guideline qualities. Hence, it is urgent to identify existing high quality guidelines and make guideline developers follow methodological standards while developing guidelines.The appraisal of guidelines for research and evaluation II (AGREE II), consisting of 6 domains covering 23 key items, is initially developed from AGREE and updated in 2010. The purpose of this instrument is to evaluate the strengths and limitations of guidelines; to provide methodology for development of guidelines; and to present guidance for reporting recommendations. It is a widely-used instrument and remains the current golden standard for guideline appraisal , 6.The quality of CAP guidelines has not been systematically evaluated till now. Thus, the aim of this study was to access the available guidelines for the treatment of children with CAP and to determine the methodological quality and reliability of guidelines.A literature search was systematically conducted in English and Chinese major academic databases (from January 2000 to March 2015). English databases were MEDLINE, EMBASE, Guidelines International Network (GIN), National Guideline Clearinghouse (NGC), Chinese databases were Chinese Biomedical Literature Database (CBM), China National Knowledge Internet (CNKI), VIP Database and Wanfang Database. Key words were “community-acquired”, “pneumonia”, “children”, “pediatric”, “guideline”, “guidance”, “recommendation”, “management”, and “consensus”.Guidelines published in Chinese or English, which addressed diagnoses and treatments of CAP for children under 18 years of age were included. Guidelines that are systematic reviews, editorials, short summaries, and other literature explaining or evaluating guidelines were excluded. If guidelines have been fully updated, the latest version were evaluated and the old versions were excluded.Two of the investigators (Z.W.X and L.B.Z) searched the database, selected guidelines independently and determined if they warranted inclusion in this study and then extracted general characteristics of the guideline. Disagreements were resolved by consulting a third expert opinion (A.D.S). The following information was extracted, such as title of guidelines, country or region, publication date, affiliation or organization, developed method, quality of evidence and filled in an established standard table on Microsoft Excel 2013.The AGREE II instrument was used to assess the methodological quality of included guidelines. It contains 23 key items of 6 domains, such as scope and purpose, stakeholder involvement, rigour of development, clarity of presentation, applicability, editorial independence, and overall guideline assessment. Each item was scored on a scale of 1–7, with 1 being strongly disagree and 7 being strongly agree. Each domain was calculated by summing up all the scores of the individual items in a domain and then standardizing as follows:  / .Quality assessment were performed independently by four trained reviewers . They conducted an independent review of the quality of each eligible guideline. Disagreement between reviewers was resolved through consensus or by consulting an independent expert adjudicator (L.B.Z). Final average appraisal scores and standard deviations for each domain and scaled domain percentages were calculated.Intraclass correlation coefficient (ICC) was used as a measure of agreement between reviewers. The ICC values was carried out by SPSS v.17.0 and applied to each guidelines.Finally, the overall guideline assessment was evaluated. A guideline would be “recommended” if overall scores were greater than 60 %, “recommended with modifications” at scores between 30 % and 60 %, and “not recommended” at scores less than 30 %.P values < 0.05 were considered statistically significant. All statistics were calculated using SPSS v.17.0.Standardized domain scores were calculated according to methods described in the AGREE II user manual. A descriptive statistical analysis was used to summarize the data. Subgroup comparison between evidence-based guidelines and non evidence-based guidelines were performed by t-test. All tests were two-sided, and A total of 827 records were identified through comprehensive database search and 22 additional records more were identified through Google search engine. 56 were exclude due to duplication, and the remaining 771 were screened by titles and abstracts. 88 were screened by full text articles for eligibility. 42 review articles, 6 guidelines before 2000, 13 interpretation of guidelines, 9 outcome on empirical treatments, 2 old version, 1 involving other disease guideline, 2 adult guidelines, 2 old version  and 3 other foreign languages  were excluded. Finally, 10 guidelines were identified and involved in the analysis . The detThis domain addressed the overall objectives, health issues and target population. The score of this domain was relatively high , indicating most guidelines met the criteria of this domain.This domain assess whether the relevant professional group is represented, the patients’ views and preference has been sought, and the definition of target users of the guideline are clearly defined. The score of this domain was 42.78 % (range: 9.70–76.39 %). A few guidelines , 16, 17 This domain is the core of guideline methodology and mainly focus on the method of evidence search, grading, summarizing and the formulation of the recommendation. The score of scope was 44.95 % (range: 17.19–79.69 %).This domain evaluates the presentation and the format. The score of this domain was 83.33 % (range: 69.44–94.44 %). Most guidelines recommendations were specific. Different options for management of the condition were clearly presented, and key recommendation was easily identifiable.This domain discusses the organization barriers, cost implications and monitoring criteria. The assessment result was 37.50 % (range: 17.71–61.46 %). Most guidelines failed to consider it during the guideline development.This domain focuses on the funding issues and competing interests of all contributing members. The score of this domain was 23.74 % (range: 0–87.50 %). Both evidence-based guidelines and non evidence-based guidelines achieved low scores in this domain.ICC for each guideline is listed in Table 3 CPGs , 8, 17 wOf the guidelines involved, 4 were evidence-based guidelines which processed details of evidence scale, on the other hand, the remaining 6 were more consensuses than evidence-based guidelines. Compared with non evidence-based guidelines, evidence-based guidelines obtained significant higher scores in nearly all domains except “editorial independence”. Table This study discussed a qualitative evaluation of the CPGs on CAP in children with the AGREE II guideline assessment. Eventually, 10 guidelines were included. In this study, we demonstrated that the scores of CAP guidelines were higher than an average international quality level , especiaIt was concluded from our results that CPGs tackling CAP on children account for a small amount compared with adults. A great number of countries or associations ignored to develop guidelines which are targeted to this particular group. Pediatric patients are tend to be more vulnerable in nature, therefore, it is essential to ensure that guidelines are developed under strict criteria. On the other hand, a great number of organizations failed to translate the CAP guidelines into English and hindered the efficiency of health science communication. Efforts are needed to translate guideline into a universal language and to assist health professions in better mastering strictly developed guidelines and to rectify discrepancies and promote evidence-based practices to enhance patient outcomes.In our study, it should be noted that “scope and purpose” and “clarity of presentation” achieved a relative high scores among the six domains, which indicated that most guidelines fully satisfied this criteria. Most guidelines could fully describe the overall objective and their specific clinical issues. For “stakeholder involvement”, relevant professionals in the development process were involved in most guidelines and target population was declared. However, the guideline developers did not seek the preference of target populations resulting in a decrease in score of this domain.“Rigour of development” was considered to be one of the most crucial domain for the assessment of guideline development. In our study, this domain varied in guidelines, for evidence based guideline, it obtained higher scores, while consensus not. A strong recommendation should not solely be based on expert opinion. Guideline developers should adhere to a standardized evidence-based evaluation system. In this study, only 4 , 16, 17 Our study found that “applicability” scored low among the six domains. A great number of guidelines lack of describing facilitators and barriers to its application, advice and/or tools on how the recommendations can be put into practice. Guideline would rate high if the potential resource implications of applying the recommendations had been considered, such as a summary document, a quick reference guide, and educational tool. In order to ensure the feasibility of clinical practice, it is also suggested that a pilot test for the applicability of new guidelines should be performed before its publication. These items should be taken into consideration when the following guidelines are developing.Editorial independence was also neglected in a great deal of guidelines. Only 3 , 10, 17 A strength of our study is that we used AGREE II instrument to assess the methodological quality of guidelines related to CAP in children, which has no related appraisal research on this particular group. Furthermore, our team consisted of pediatric clinical experts and pharmacist with extensive experience in clinical guidelines appraisal.There are limitations within this study that should be noted. Firstly, the guidelines involved are confined to English and Chinese. Secondly, the literature search only identified published guidelines in pediatric pneumonia. It is possible that some guidelines may have been missed by the search, especially those that combined pediatric and adult recommendations. Thirdly, the AGREE II instrument is a tool that assesses the methodological rigour and transparency in which a guideline is developed , 23. It Overall, this study appraised quality scores of the CPGs on CAP in children. The appraisal results showed acceptable. The developers are suggested to pay more attention to rigour of development, applicability, and editorial independence during the development process. The AGREE II instrument should be adopted by guideline developers to improve the applicability of guidelines.AGREE II, the appraisal of guidelines for research and evaluation II; CAP, community-acquired pneumonia; CBM, Chinese databases were Chinese Biomedical Literature Database; CNKI, China National Knowledge Internet; CPGs, clinical practice guidelines; EBM, evidence-based medicine; GIN, Guidelines International Network; GRADE, Grading of Recommendations Assessment, Development and Evaluation; ICC, intraclass correlation coefficient; IDSA, Infectious Diseases Society of America; NGC, National Guideline Clearinghouse; SIGN, The Scottish Intercollegiate Guidelines Network


Agrocybe aegerita, i.e., Agrocybe aegerita lectin 2, or AAL2, that could bind terminal N-acetylglucosamine with higher affinities and specificity than other currently used probes. In this paper, we report the crystal structures of AAL2 and its complexes with GlcNAc and GlcNAcβ1-3Galβ1-4GlcNAc and reveal the structural basis of GlcNAc recognition by AAL2 and residues essential for the binding of terminal N-acetylglucosamine. Study on AAL2 may enable us to design a protein probe that can be used to identify and purify O-GlcNAcylated proteins more efficiently.O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification that plays essential roles in many cellular pathways. Research in this field, however, is hampered by the lack of suitable probes to identify, accumulate, and purify the O-GlcNAcylated proteins. We have previously reported the identification of a lectin from the mushroom  Lectins are a large group of non-enzyme proteins that recognize and specifically bind to carbohydrate moieties that are either free or conjugated on the surfaces of proteins or lipids . They arO-linked addition of N-acetylglucosamine (O-GlcNAcylation) on the serine and threonine residues of cytosolic and nuclear proteins is a reversible post-translational modification that plays essential cellular roles. Recent studies have shown that O-GlcNAcylation has extensive cross talk with phosphorylation, another essential protein modification, and serves as a nutrient sensor for the modulation of numerous signaling and transcription pathways , 5. It wAgrocybe aegerita, designated as AAL2, in its apo- and complex forms with GlcNAc and GlcNAcβ1-3Galβ1-4GlcNAc, as part of our endeavors to develop a system to facilitate the identification and purification of O-GlcNAcylated proteins.In this paper, we report the crystal structures of a lectin from A. aegerita. It consists of 407 amino acid residues, with a molecular weight of 43.17 kDa. It was the second lectin purified from A. aegerita to be characterized by x-ray crystallography by our groups, after A. aegerita AAL, an antitumor lectin that recognizes and binds Thomsen-Friedenreich antigen [in vitro as well as inhibiting hepatoma growth in tumour-bearing mice in vivo [1–3 over 1–3, which lacks the terminal GlcNAc [Griffonia simplicifolia lectin-II), which have been widely used in glycan studies for their GlcNAc-binding activities [Lectins from fungi (mainly from mushrooms) have attracted wide attention in recent years owing to their diverse bioactivities , includi antigen . AAL2 wa in vivo . Glycan l GlcNAc . ITC asstivities , 16. AALPsathyrella velutina Lectin) [To illustrate the mechanisms of how AAL2 specifically recognizes non-reducing terminal GlcNAc, we performed structural and surface plasmon resonance (SPR) affinity characterizations on AAL2 and its binding with GlcNAc and GlcNAcβ1-3Galβ1-4GlcNAc, as well as its divalent metal cation binding properties. These results showed that AAL2 has homologous sequence and similar structure with previously characterized fungal lectins PVL ( Lectin) , and it Psathyrella velutina, was reported in 2006 [The crystal structure of PVL, a fungal lectin from  in 2006 , which wAAL2 was amplified from A. aegerita and cloned into pET30a expression vector between NdeI and HindIII restriction sites, without any tag. The recombined plasmid was transformed into Escherichia coli host strain BL21 (DE3) competent cells for expression. The AAL2 protein expression and first-step purification with a GlcNAc-coupled Sepharose 6B affinity column has been described in detail previously [The gene of eviously , 18. TheAAL2 was mixed with GlcNAc (from Sigma) and GlcNAcβ1-3Galβ1-4GlcNAc trisaccharide , respectively, at AAL2:ligand molar ratios of 1:10, and the mixtures as well as pure AAL2 sample were used in crystallization condition searches. Crystallization was performed using the hanging-drop vapor diffusion method, by mixing protein and reservoir solutions with equal volume of 1μl and equilibrated against 400μl reservoir solutions at 293K. Crystals of AAL2 were obtained under the condition of 30% PEG 8000 (w/v), 0.2M sodium acetate and 0.1M sodium cacodylate pH 6.4. Crystals of AAL2-GlcNAc complex were obtained with the solution containing 25% PEG 4000 (w/v), 6% Tacsimate pH 6.0 and 0.1M MES pH 6.0. The crystals of AAL2-trisaccharide complex were obtained in solution containing 2.3M ammonium sulfate and 0.1M sodium acetate pH 4.4.All the three types of crystals were mounted on cryoloops (Hampton Research) directly from the crystallization drops and flash-cooled in a nitrogen-gas stream at 95 K. X-ray diffraction data for AAL2 was collected on an in-house FR-E Bluemax/R-AXIS IV++/Varimax HF (Rigaku) diffractometer at wavelength of λ = 1.5418Å; the data for AAL2 complexed with GlcNAc was collected on beamline 1W2B at Beijing Synchrotron Radiation Facility  at λ = 1 Å; and the data for AAL2 complexed with GlcNAcβ1-3Galβ1-4GlcNAc was collected on an in-house MicroMax 007 Saturn944+ VarimaxHF (Rigaku) diffractometer at λ = 1.5418 Å. Each dataset was collected with a total of 180 images using an oscillation of 1° per image.MOSFLM v.7.0.4 [SCALA v.6.0 from the CCP4 v.6.0.2 program suite [http://www.ncbi.nlm.nih.gov/BLAST) identified a structurally characterized protein PVL that has 60% sequence identity with AAL2 [AutoMR program of the Phenix suite v.1.8.2 [AutoMR program in Phenix, with the structure of solved AAL2 as search models. The carbohydrate ligands were added and adjusted manually according to the electronic density map displayed in Coot [Refine program in Phenix alternated with manual rebuilding using the graphical program Coot.The data were indexed and integrated using the program  v.7.0.4  and thenam suite . A sequeith AAL2 . The str v.1.8.2  with the in Coot . The strThe SPR experiments were carried out on a BIAcore T100  using CM5 sensor chips at 298 K. AAL2 protein solution was diluted to a concentration of 20μg/ml with fixing buffer (pH 5.0) and subsequently immobilized by amine coupling onto the chips. The immobilized level of AAL2 reached 3000 response units. Flow path 1 was used as an activated blank control for subtraction. Interaction experiments were performed in a running buffer of 50mM Tris-HCl pH 7.5, 150mM NaCl, 0.005% Tween 20 at a flow rate of 30μl/min. The ligands in sample buffer were employed as the flowing phase respectively. Each interaction experiment was carried out with gradient concentrations from low to high programmed to inject at a 30μl/min flow rate under a sensorgram. Binding curves were corrected, aligned and fitted kinetically using the BIAcore T100 evaluation program .2, CaCl2, MnCl2, CoCl2, and NiSO4, respectively, to concentrations of 10mM. All the samples were incubated at 277K overnight and then applied to a HiTrap Desalting Column again to remove free metal ions. The metal content of the five samples as well as the protein solution not processed with EDTA were determined on a Varian Inductively Coupled Plasma—Atomic Emission Spectrometry (ICP-AES) . SPR experiments were also performed on EDTA-treated sample in buffers containing 5mM CaCl2 to find out if the metal binding state of AAL2 would affect its affinity to ligands.The purified AAL2 was loaded on a HiTrap Desalting Column  with buffer containing 50mM Tris-HCl pH 7.5, 150mM NaCl, and 5mM EDTA to remove metal ions from the protein. The flow-through fraction of protein was incubated in 277K for 1 hr. and then loaded on a HiTrap Desalting Column once again with the buffer of 50mM Tris-HCl pH 7.5 and 150mM NaCl to remove EDTA and EDTA-coupled metal ions. The flow-through fraction of protein was then evenly divided into five parts, and was added solutions of MgClPsathyrella velutina Lectin) (Kd = 214 μM) [To measure the affinities between AAL2 and glycan ligands, we performed Surface Plasmon Resonance (SPR) experiments. The ligands we used were GlcNAc monosaccharide and GlcNAcβ1-3Galβ1-4GlcNAc, a trisaccharide showing very high affinity toward AAL2 in glycan array analysis. In SPR experiments, the experimental curves obtained automatically from different sensorgrams showed that both GlcNAc and GlcNAcβ1-3Galβ1-4GlcNAc could be captured efficiently by the sensor chip surface through binding to AAL2 and quickly dissociated to the baseline level . The dat 214 μM)  and BLL  214 μM) . AAL2 waThe structures of AAL2, AAL2-GlcNAc and AAL2-GlcNAcβ1-3Galβ1-4GlcNAc were determined at resolutions of 2.0Å, 2.1Å, and 2.0Å, respectively. The data collection and refinement statistics are summarized in There are two virtually identical AAL2 monomers arranged around a two-fold pseudoaxis of symmetry in an asymmetric unit in both the structures of AAL2 and AAL2-GlcNAc, and one AAL2 monomer in AAL2-GlcNAcβ1-3Galβ1-4GlcNAc, while the protein exists as monomers in solution as determined from its elution volume on a Superdex 200 10/300 GL column. Discussions thereafter will be focused on monomer A unless specified otherwise.As expected from the sequence homology, the structures of AAL2 and AAL2-GlcNAc complex are very similar to those of the corresponding structures of PVL, with root-mean-square deviations (RMSDs) of 0.7Å for Cα atoms.The overall structure of AAL2 monomer is a single domain with the shape of cylindrical torus with a diameter of about 51Å and a height of about 27Å, showing a very regular, seven-bladed β-propeller fold formed by the blades packing in a circular array . Each blThe four β-strands of each W-motif are numbered 1–4 according to their spatial arrangement from AAL2 center to its surface . The firWhat is notable is that the seven sequence repeats observed in AAL2 do not match exactly with the seven blades, but instead have a shifting of one β-strand . WI consThe structure of AAL2-GlcNAc complex revealed six binding sites out of the seven W-motifs, with the exception of motif WI. All the bound GlcNAc molecules have virtually identical orientations in the structure.Glycan ligands are bound in shallow pockets located on the entrance side, between the connecting segment and the 2–3 loop of each W-motifs (except for WI) . ElectroBecause the six binding pockets of AAL2 for glycan ligands are almost identical, we chose motif WIII for detailed discussion and the descriptions also applied to other W-motifs unless specified otherwise. The carbohydrate binding pockets of AAL2 are very similar to those of PVL, as implied by the sequence homology.The binding pocket for GlcNAc consists of two parts. The major part is formed by the segment connecting WII and WIII, including the side chains of Asn104, Trp112 and His117 and main chain atoms of Gly110 and Gly111. The other part is formed by the main chain atoms of Gly136, Glu137, and Lys138 from the 2–3 loop of WIII. Among the residues mentioned above, Asn104 is located at the beginning of the connecting segment and it is either Asn or Asp in the corresponding position of each blade, and offers its OD1 and ND2/OD2 to form hydrogen bonds with O3 and O4 hydroxyl groups of GlcNAc, respectively. The O3 hydroxyl of GlcNAc moiety also forms a hydrogen bond with NE1 of Trp112. The N-acetyl group of GlcNAc forms two hydrogen bonds with the main chain of the protein: N2 donates one to O of Gly110, and O7 carbonyl accepts one from N of Glu137 . The hydGlcNAcβ1-3Galβ1-4GlcNAc ligands are bound to the same pockets of AAL2, with the non-reducing terminal GlcNAc (1’-GlcNAc) adopted the same position as GlcNAc. The polar interactions between AAL2 and the trisaccharide are summarized in Tachypleus tridentatus binds GlcNAc in a manner almost identical to that of AAL2, although the binding sites of these two proteins do not share the same architecture  while in GalNAc it is axial (up from the "plane" of the ring). If a Gal moiety were to take the position and orientation of GlcNAc as in the complex structures, the key hydrogen bonds between Asn104 and O4 hydroxyl of GlcNAc would be disrupted . AAL2 diIn addition to glycans, AAL2 also binds two cations per molecule as observed in the structure of AAL2-GlcNAc complex . Similar2+, Ca2+, Mn2+, Co2+and Ni2+for their binding affinities. The binding assay results are shown in 2+ was not shown. But there was indeed zinc in the purified native protein samples. Calcium is the main element AAL2 binds, which is the reason why we modeled cations in the AAL2-GlcNAc complex as Ca2+. The preference of the divalent cation type may lie in the diameter fitting the size of the negative charged pocket. SPR assay were also carried out and illustrated that the adding of Ca2+ did not affect the glycan affinity of AAL2. Although it has been reported that Ca2+ binding property is a common phenomenon observed in propeller structures and it was believed that the binding of Ca2+ play a role of stabilizing the overall structure [To identify the cation-binding preferences of AAL2, we tested Mgtructure , similarIn this study, we have demonstrated that AAL2 has relatively high affinities and exclusive specificity towards terminal nonreducing GlcNAc and illustrated the structural basis of its ligand specificity.Although further studies are required to reveal the antitumor mechanisms of AAL2, the high affinities and exclusive specificity of this lectin toward nonreducing GlcNAc moieties strongly point to the possibilities that AAL2 may exert its functions through interactions with O-GlcNAcylated proteins. Many tumor suppressors and oncogenes that involved in transcription regulations, such as c-Myc, p53, and retinoblastoma, are modified by O-GlcNAcylation. The extensive cross talk between O-GlcNAcylation and phosphorylation involving these proteins underlines the roles played by O-GlcNAcylation in tumor pathogenicity , 6, but The studies reported here, on the other hand, have demonstrated that AAL2 is a very promising candidate to replace WGA in glycan research for the enrichment and purification of O-GlcNAcylated proteins. WGA lectin weak affinity chromatography has been the most frequently used method in the enrichment and purification of O-GlcNAcylated proteins , but thiThe results reported here illustrated the structural basis of how AAL2 recognizes and binds terminal GlcNAc moiety and laid a solid foundation for us to reveal the antitumor mechanisms of this lectin and to develop a better reagent for glycan studies involving O-GlcNAcylated proteins.