The use of research evidence as an input for health decision-making is a need for most health systems. There are a number of approaches for promoting evidence use at different levels of the health system, but knowledge of their effectiveness is still scarce. The objective of this overview was to evaluate the effectiveness of knowledge communication and dissemination interventions, strategies or approaches targeting policy-makers and health managers.This overview of systematic reviews used systematic review methods and was conducted according to a predefined and published protocol. A comprehensive electronic search of 13 databases and a manual search in four websites were conducted. Both published and unpublished reviews in English, Spanish or Portuguese were included. A narrative synthesis was undertaken, and effectiveness statements were developed, informed by the evidence identified.We included 27 systematic reviews. Three studies included only a communication strategy, while eight only included dissemination strategies, and the remaining 16 included both. None of the selected reviews provided “sufficient evidence” for any of the strategies, while four provided some evidence for three communication and four dissemination strategies. Regarding communication strategies, the use of tailored and targeted messages seemed to successfully lead to changes in the decision-making practices of the target audience. Regarding dissemination strategies, interventions that aimed at improving only the reach of evidence did not have an impact on its use in decisions, while interventions aimed at enhancing users’ ability to use and apply evidence had a positive effect on decision-making processes. Multifaceted dissemination strategies also demonstrated the potential for changing knowledge about evidence but not its implementation in decision-making.There is limited evidence regarding the effectiveness of interventions targeting health managers and policy-makers, as well as the mechanisms required for achieving impact. More studies are needed that are informed by theoretical frameworks or specific tools and using robust methods, standardized outcome measures and clear descriptions of the interventions. We found that passive communication increased access to evidence but had no effect on uptake. Some evidence indicated that the use of targeted messages, knowledge-brokering and user training was effective in promoting evidence use by managers and policy-makers.The online version contains supplementary material available at 10.1186/s12961-021-00780-4. Knowledge translation (KT) seeks to address the challenges involved in the use of research evidence by different and diverse stakeholders, in order to close the gap between the evidence generated and the decisions made by these stakeholders (KT for action). In recent years, KT processes targeted at healthcare providers and patients have been addressed by a number of publications –3. HowevDespite conceptual differences, the term “knowledge translation” has frequently been used interchangeably with the term “evidence-informed decision-making” , as wellIn this context, diffusion, dissemination and implementation have been described in research findings by identifying and overcoming barriers through specific multifaceted interventions (“make it happen”) , 9 alongDissemination focuses primarily on communicating research results by targeting and tailoring the findings and the message to a particular target audience (“helping it happen”). Finally, in this taxonomy, implementation involves systematic efforts to encourage the adoption of the research findings by identifying and overcoming barriers through specific multifaceted interventions (“make it happen”) , 9.For the purposes of this review, we focused on communication and dissemination interventions/strategies/approaches  targeted towards policy-makers and health managers. As mentioned before, dissemination refers to identifying the appropriate audience and tailoring, targeting or framing the message to that audience. This could include different research products such as evidence-based guidelines, publications, research reports, pamphlets, videos and websites, disseminated through different interventions/strategies/approaches depending on the target group. Furthermore, conceptual frameworks underlying each intervention/strategy/approach and tools used in its implementation could be identified.What is the effectiveness/impact of the different knowledge communication and dissemination interventions/strategies/approaches targeted to health policy-makers and managers?This overview of systematic reviews (SRs) adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . An SR pStudies were selected based on the following inclusion criteria:SRs including primary studies of any design providing information on the effectiveness of dissemination strategies were included. When no SRs were identified for a specific intervention/strategy, a focused search for primary studies in PubMed was conducted.As no relevant primary studies were located , SRs focused on a different audience  were assessed as indirect evidence, which means this study remained focused on SRs only.Studies including only managers or policy-makers as the target audience or studies including a larger group  but in which data for different target audiences was reported separately were included. Because the definitions of managers and policy-makers are not always precise, we used a broad definition including all types of decision-makers such as hospital directors or administrators, health administrators, department chiefs, health planners, and programme directors or managers.Techniques to communicate evidence through (1) tailoring the message, (2) targeting the message, (3) using narratives, (4) framing the message and (5) using a multicomponent approach.Strategies for disseminating evidence to (1) increase the reach of the evidence, (2) increase people’s motivation to use and apply the evidence, (3) increase people’s ability to use and apply the evidence, and (4) use a multipronged approach with any of the dissemination strategies described above.Intervention(s), exposure(s): The definitions of KT communication and dissemination strategies were adapted from the classification proposed by McCormack et al.  and are The communication or dissemination support could be physical materials , electronic , Internet-based , interpersonal , through media  or social media , and others .The strategies could be defined as single or combined. The combined or multicomponent strategies refer to different combinations of single strategies—targeted messages + knowledge brokers; education + information service + free access to databases; small group workshops + one-to-one consultations, etc.We did not have restrictions on types of comparisons.We included outcomes related to the effectiveness of communication and dissemination strategies targeted at managers or policy-makers.Our primary outcomes were use or uptake of research results, decision-making, adherence to research knowledge (i.e. change in knowledge/awareness) and behavioural change.Secondary outcomes were those related to understanding, perception and persuasiveness. We considered only objective understanding and not self-reported understanding. Perception referred to how effective an intervention was perceived to be. Persuasiveness considered how likely participants were to make a hypothetical decision in favour of an intervention. Costs and cost-effectiveness of strategies were also considered when data were available. Additionally, we considered barriers to uptake of research evidence through communication or dissemination strategies, underlying theories behind the strategies and research gaps.A comprehensive electronic search of 13 databases and a manual search in four websites was conducted. The databases searched were MEDLINE (PubMed), EMBASE, Health Systems Evidence, the Cochrane Library, CINAHL (EBSCOhost), PsycINFO (EBSCOhost), the Campbell Collaboration, EPPI-Centre database of health promotion research (BiblioMap), WorldWide Science, Education Resources Information Center (ERIC), DoPHER [database of promoting health effectiveness reviews], Epistemonikos and PDQ-Evidence. The manual search was carried out in Google including Google Scholar, Rx for Change, International Initiative for Impact Evaluation (3ie) and Institute of Education Sciences (IES). The reference lists of included studies were also searched to identify other relevant literature not picked up by our search.Searches were conducted between 30 January and 4 February 2021, and supplementary searches (reference lists) were performed until 24 February 2021. Databases were searched using keywords from both keyword areas—combined using “AND”. Keywords were searched for in the title and abstract fields. Results were downloaded into the EndNote reference management programme (version X9) and duplicates removed. The internet search utilized the search terms: “policy-makers” OR policymak* OR manager; “research utilization” OR “research communication” OR “information dissemination”; “knowledge translation”; “research use”; and “research uptake”; combined with “systematic review”.Search terms and strategies for the electronic searches are presented in Additional file SRs in English, Spanish or Portuguese (because the reviewers are familiar with these languages) during the past 15 years were included. We included both published and grey literature.Searches were conducted and titles and abstracts were screened independently by two review authors (EC and TP) according to the selection criteria. The full texts of any potentially relevant papers were retrieved for closer examination. The inclusion criteria were then applied against the full-text version of the papers independently by two reviewers (EC and TP).Disagreements regarding the eligibility of studies were resolved by discussion and consensus. All studies which initially appeared to meet the inclusion criteria but on inspection of the full-text paper did not meet the inclusion criteria are listed in Additional file Information extracted from the included studies considered the type of synthesis document , objectives, study design and number of studies included, date of last search, participants (type of decision-maker), settings, country of study, intervention/strategy and domain , outcome measures, findings, barriers, research gaps, underlying theory and financing source. We limited data extraction to SRs assessing the effectiveness of strategies targeted at policy-makers or managers.Data were extracted by two reviewers (EC and TP). Each reviewer extracted 50% of the included studies, and then 40% of the extractions were cross-checked. Doubts or disagreements were resolved through discussion and consensus. During the process of extracting data from the included SRs, reviewers completed a matrix containing the different types of interventions and the outcomes measured in each included SR.Additionally, we disaggregated the formats or tools used to facilitate or support the communication and dissemination strategies. Classification was done by two reviewers (EC and TP).The methodological quality of the included SRs was evaluated using AMSTAR 2 (A MeaSurement Tool to Assess systematic Reviews 2) . ScoringFindings from the SRs were synthesized using tables and a narrative summary. Meta-analysis was not possible because the included studies were too heterogeneous in terms of the populations, strategies/interventions tested, and outcomes measured. Few SRs informed effectiveness measures; therefore, to inform the main results, we developed effectiveness statements using four categories and standardized language as proposed by Ryan et al. , 14. TheTwenty-seven SRs met the inclusion criteria for the overview , 15–40. Details of the characteristics of the included SRs  and AMSTAR 2 scores are presented in Additional file Regarding AMSTAR 2, six SRs had high ratings –19, 23, Of the 27 SRs, two only included randomized controlled trials , 23, sixFour reviews included only low-income countries , 36, 37,Twelve SRs mentioned or were informed by a theory or framework , 39, 40,Three studies included only a communication strategy , 16, 23,Regarding strategies to communicate evidence, we found 12 SRs reporting “Targeting the message”, 11 reporting “Tailoring the message”, four reporting a “Multicomponent approach”, three reporting “Using narratives and other forms of art”, and three reporting “Framing the message”. There was just one study reporting “Using different presentation formats”.Regarding strategies to disseminate evidence, we found the following: “Increase people’s motivation to use and apply the evidence” 18 studies, “Increase reach of the evidence” studies, “Increase people’s ability to use and apply the evidence” 12 studies, and for the “Use a multipronged approach” six studies.Eight studies used “physical or printed tools”, for example, drawing, painting, research summaries, journal prints and bulletins; 13 used an “electronic tool”, such as simulation games, multimedia, television appearances, entertainment education (prime-time network TV storyline), short films, videos, PowerPoint presentations and handouts. Seventeen studies reported “tools based on Internet”. Highlighted mong these were specific websites , access to a repository of SRs, web-based training programmes, structured seminar series, networks, access to research and to databases, web-based information and communication, dissemination of SRs through the website, and series of emails with links to full references, abstracts and summaries. Regarding “interpersonal” communication or dissemination skills—understood as any interaction between two or more people face-to-face or not —we found 20 studies reporting targeted messaging and knowledge brokers, engagement with arts-based projects including elements of training or skills-building, knowledge-brokering (the most frequently mentioned), small group workshops, one-to-one consultations, collaborative approaches, structured seminar series to promote interaction between policy-makers and researchers, conference technology to support knowledge sharing, networks to support knowledge production and exchange, organization-wide capacity-development initiatives, grant-funded collaboration involving policy-makers, deliberative dialogues, interactions and interpersonal connections with staff, tailored exchanges within and across departments and disciplines, face-to-face exchange, KT platforms (KTPs), and research engagement actions such as increasing the interaction between decision-makers and researchers. Three studies reported the use of “media or social media”. They included, among others, media and public opinion, television appearances, entertainment education (prime-time network TV storyline), short films, magazines and journal prints, narrative action reflection workshops, conference technology to support knowledge sharing, communities of practice, booklets with testimonies, advocacy summaries of personal experiences, wikis, blogs and online forums. For more details see Additional file Many reviews included data for more than one primary or secondary outcome; therefore, the number of outcomes measured may exceed the number of included SRs. For greater detail see Additional file Thirteen studies assessed the use or uptake of research results, 14 studies assessed decision-making or changing behaviours, six studies assessed intention to use or apply evidence, 14 studies assessed change in knowledge, and five studies assessed changes in awareness.Understanding was assessed by nine studies, perception by seven studies and persuasiveness by three studies, and cost was reported by a single study as a research gap.Additionally, the included studies assessed outcome measures that were not included in our protocol. These included learning (six studies), attitudes/beliefs (four studies), skills or competencies (three studies), discussion regarding the evidence (two studies), health outcomes (two studies), engagement (two studies), policy changes (one study), value of research evidence (one study), scaling-up of intervention (one study), acceptability (one study), research culture (one study), intention to act (one study), sustainability of evidence-informed policy-making (EIPM) (one study), research coproduction (one study) and credibility (one study). More details are provided in Additional file The effects of interventions are presented below by each strategy according to the adopted taxonomy . Some reFourteen reviews assessed the effects of a communication strategy , 39, 40,There was little to no difference in the effect of evidence summaries compared with complete SRs on the use, understanding, belief or perceived usefulness of evidence. However, summary of findings tables and graded entry summaries were perceived as slightly easier to understand than complete SRs . The eviOne additional review targeted at healthcare providers and recipients of care provided indirect evidence regarding the effects of different ways of framing the message on the use of evidence . The negTwenty-four reviews assessed the effects of a dissemination strategy , 17–40, Thirteen reviews provided some information, but it was insufficient for making definitive effectiveness statements, and the other eight reviews were used to inform research gaps. Interventions aimed mainly at improving the reach of evidence did not seem to have a significant impact on the use of evidence in decision-making processes. On the other hand, interventions designed to motivate target audiences to use and apply evidence (e.g. knowledge brokering) or to enhance recipients’ ability to use and apply evidence (e.g. training workshops with an interactive component) may have a positive effect on decision-making processes in some organizations, but the evidence was inconsistent. Finally, multifaceted dissemination strategies could lead to changes in knowledge about evidence but not in its use in decision-making .Nine studies reported barriers to the communication and dissemination processes , 32, 40.Finally, barriers related to research itself included the type of research being considered, the need to refine methodologies used for conducting reviews, the need to consider diverse contexts, the perceived credibility of findings, timeliness of research and the relevance of research in day-to-day decision-making , 27, 40.Eighteen studies reported research gaps , 17–40. Also highlighted was the need for qualitative studies to better understand the knowledge, beliefs and attitudes of policy-makers towards narratives  or theirThe description of the interventions was also noted as a gap—for example, the need to include the frequency and duration of the intervention, its components , 32, theThe need for valid and standardized measurements of results and impacts of strategies on policies was highlighted –22, 32, Limited descriptions of frameworks or theories that guide the development of the intervention , 16 wereThere is no doubt about the relevance of the use of evidence in the formulation, implementation and evaluation of policies and the growing scientific production in health policy research .We identified 27 SRs, but only four , 29, 30 The other nine reviews only provided evidence to inform research gaps. An additional review  providedSurprisingly, although evidence summaries  were perceived as easy to understand, they did not have a relevant effect on the understanding, knowledge, beliefs, perceived usefulness or use of evidence in decision-making. However, this finding was only based on two studies comparing different types of evidence summaries with complete SRs; therefore, more research is needed to determine whether evidence summaries are in fact effective, and also what formats and types of evidence summaries  are optimal for decision-makers. Regarding dissemination strategies, those interventions aimed mainly at improving the reach of evidence do not seem to have a significant impact on the use of evidence in decision-making processes. However, interventions designed to motivate target audiences to use and apply evidence (e.g. knowledge-brokering) or to enhance their ability to use and apply evidence (e.g. training workshops with an interactive component) could have a positive effect on decision-making processes in some organizations, although the evidence was inconsistent. Multifaceted dissemination strategies (e.g. combining improving access to evidence + workshops or multiple technology-enhanced interventions) could lead to changes in knowledge about evidence but not in its use in decision-making. SRs, evidence briefs and other synthesis documents have been established as a powerful tool for policy-makers and managers –45. HoweRecent research also suggests some aspects to consider in the decision-makers themselves. A recent study points out that managers should demonstrate basic competencies consisting of knowledge, skills and attitudes necessary to perform managerial tasks and also proposes new competencies that include evidence-informed decision-making, where managers should know how to evaluate and apply the evidence . SomethiRegarding the use of theories, frameworks or models to inform the communication or dissemination processes as KT strategies, we found 13 SRs that used or mentioned one or more different frameworks. We assume that such variability depends on the context, key components, objectives of KT, primary target audience, levels of use or characteristics of the users. This variability and high number of frameworks was also reported in various studies as creating confusion for users regarding which to select . While cIn contrast to the substantial evidence available on the effectiveness of communication and dissemination strategies targeting healthcare professionals and consumers –3, the bThis could be related to differences in complexity between clinical and managerial/policy decision-making environments, with a more prominent role for evidence in the former and a greater influence of other factors  in the latter making less intensive intervention—focused only on the access to evidence—more effective in clinical environments.Our findings also mirrored, to some extent, those from a similar review of the efficacy of interventions applied to increase decision-makers’ use of research in various decision arenas . AlthougMany SRs in this area address questions not only about effectiveness but also about barriers, facilitators and mechanisms through which interventions affect outcomes , 31, 32.There are a number of known barriers and enablers influencing the potential use of research evidence in decision-making at both the individual and organizational levels. Many other factors and mechanisms are still not sufficiently studied –55. ThisHowever, most of the evidence evaluating these multipronged strategies is based on case studies or similar methodologies, which makes it difficult to draw definitive conclusions about their effectiveness . LikewisFinally, a relevant and current issue is the use of evidence by decision-makers and managers in times of crisis, such as the current COVID-19 pandemic. In this context of public health emergency, there are three common challenges: (1) obstacles or barriers, for example, arising from uncertainty about risks of insufficient treatment or mistrust in the government, which will ultimately shape decisions and response measures; (2) variability in the way decision-makers acquire, interpret and apply evidence; and (3) limited capacity for decision-making because decision-makers often face competing demands on their time, limiting their ability to consider and act on available evidence .We could also add that the current situation of COVID-19 is not only a “pandemic” but also an “infodemic”. Sufficient levels of knowledge are required not only to access, understand and act on relevant health information, but also to examine and assess the excessive and potentially misleading information in the wake of this infodemic. The forms of communication in this scenario are extremely relevant –64.The main strength of our overview is that we followed a systematic and transparent process to locate and assess the evidence in this field using reliable methods and frameworks previously used by others, such as the taxonomy for classifying the communication and dissemination strategies , the AMSHowever, our overview has a number of limitations. First, because of the project time frame, we have taken a “rapid overview” approach, implying that only part of the study selection and data extraction processes were performed in duplicate, which could have introduced bias and potential errors in our overview. Second, because we did not find an established intervention classification in the field , we chose to adapt one previously developed by others . AlthougAt the level of evidence synthesis, it is worth mentioning that most of the included reviews used combined strategies, but there was scarce information regarding details of characteristics such as the intensity, frequency, or duration of the interventions, as well as the way in which the information was presented. Researchers with ample experience in KT processes for policy recently designed a checklist to facilitate this. The checklist contains recommendations focused on items that quickly determine the relevance of the information and the key messages .Future research should examine the effectiveness of communication and dissemination strategies targeted at managers and policy-makers using sound methods and a more standardized and agreed upon set of outcomes, incorporating theoretical inputs into the intervention design.This is especially urgent for interventions where we did not find sufficient direct evidence such as the use of different framings of the messages from research and the use of narratives to communicate research to decision-makers. Likewise, a better description of interventions in terms of type, intensity and duration will be needed in order to make reviews of primary studies more useful and relevant. More evidence is also needed on how the results or impacts of different interventions are measured to improve the uptake of knowledge. Many of the results evaluated were more subjective (self-reported) than objective. Further, the role of technology in this area should be explored in more detail. The use of technology-enhanced interventions is a promising area, especially in an era of remote work using digital interfaces.Finally, an approximation or consensus on what would be the most appropriate framework for these strategies and contexts in which communication or dissemination activities are carried out would be desirable.Because of the relative dearth of evidence about the effects of communication and dissemination strategies targeted at health managers and policy-makers for promoting the use of research evidence, it is not possible to make strong recommendations regarding the use of specific interventions. However, the targeting and tailoring of messages seems to be a key “ingredient” to be used in communication strategies, and the use of interventions that not only enhance recipients’ ability to use and apply evidence, but also motivate them to use and apply evidence , seems to be a key component of any dissemination strategy.In addition, strategies to communicate and disseminate more broadly, not just push and pull mechanisms, should be considered. Although we have not found evidence of effectiveness for multifaceted strategies on changing behaviour, we think that combining them with those that promote, for example, knowledge coproduction processes, where decision-makers and researchers are interacting from the beginning, could increase the probability of translating research into policy. Some of these strategies have been promoted by KTPs such as those proposed by the Evidence-informed Policy Network (EVIPNet), and other initiatives such as the Structured Operational Research and Training IniTiative (SORT IT), which also include mentored capacity-building and knowledge coproduction and its implementation in the decision-making process.On the other hand, the use of passive dissemination strategies aimed only at increasing the access and reach of the evidence does not seem to have a relevant effect on the knowledge or use of such evidence in decision-making processes. Additionally, when selecting any communication/dissemination strategy, a number of factors influencing the use and application of evidence should be carefully considered (what has been described as barriers and facilitators in this overview), identifying their presence in the specific settings where interventions will be implemented.Considering the complexity of the decision-making environment in which managers and policy-makers perform their work, knowledge communication and dissemination strategies are just a single component of a multilevel evidence ecosystem/infrastructure that should be built in order to increase the role of evidence in decision-making.Knowledge communication strategies based on the use of targeted and tailored messages, and knowledge dissemination strategies that not only enhance recipients’ ability to use and apply evidence but also motivate them to do it, seem to be the more effective in promoting the use of evidence by managers and policy-makers. Passive dissemination strategies aimed only at increasing the access and reach of the evidence does not seem to have a relevant effect. However, there is a relative dearth of evidence about the effects of interventions targeting this audience compared to those targeting healthcare providers, consumers and patients. Also, more studies are needed that are informed by theoretical frameworks or specific tools, using robust methods, standardized outcome measures and clear descriptions of the interventions.Additional file 1. Search terms and results.Additional file 2. Excluded studies with reasons.Additional file 3. Characteristics of included studies.Additional file 4. Evidence rating scheme.Additional file 5. AMSTAR 2 quality assessment.Additional file 6. Interventions and tools.Additional file 7. Results and evidence statements.Additional file 8. Frameworks/theories/models from included SRs.


Escherichia coli BAP (EcBAP) did own an extra enzymatic activity of oxidizing Phi to Pi. We further revealed EcBAP could be used as a dominant selective marker for Agrobacterium-mediated tobacco transformation. Although the involved Phi selection for transformed tobacco cells surprisingly required the presence of Pi, it showed a considerable transformation efficiency and dramatically accelerated transformation procedure, as compared to the routine kanamycin selection and the well-known PtxD/Phi system. Moreover, the EcBAP transgenic tobaccos could metabolize toxic Phi as a phosphorus (P) fertilizer thus underlying Phi-resistance, and competitively possess a dominant growth over wild-type tobacco and weeds under Phi stress. Therefore, this novel BAP/Phi-coupled system, integrating multiple advantages covering biosafe dominant selective marker, plant P utilization and weed management, can provide a PTDH-bypass technological choice to engineer transgenic plant species, especially those of great importance for sustainable agriculture.Selective markers are generally indispensable in plant genetic transformation, of which the frequently used are of antibiotic or herbicide resistance. However, the increasing concerns on transgenic biosafety have encouraged many new and safe selective markers emerging, with an eminent representative as phosphite (Phi) in combination to its dehydrogenase . As bacterial alkaline phosphatase (BAP) can resemble PtxD to oxidatively convert toxic Phi into metabolizable phosphate (Pi), herein we harnessed it as the substitute of PtxD to develop an alternative Phi-based selection system. We first validated the  Selective markers are generally required for plant transgenic studies to enable the putative transformants regenerated under a selection pressure, and can be overall grouped into two categories, the negative selection marker and the positive/dominant selection marker ,2. The fPhosphite (Phi) is the second phosphorus (P) reserve in Earth, but confers a higher solubility, a lower chemical reactivity with soil components, and a better thermo/photo- stability, as compared to phosphate (Pi) . MoreovePseudomonas stutzeri, which can efficiently catalyze the oxidative conversion of Phi to Pi and the reduction of cofactor NAD+ to NADH [PtxD-transformed cells can convert Phi into metabolizable Pi, and thus survive, thrive and dominantly regenerate into green shoots, as compared to the non-transformed ones. When using Phi as a P fertilizer/herbicide, the PtxD transgenic plants can metabolize toxic Phi into nutritional Pi, while the wild weeds are retarded by Pi deprivation and die. This might reduce the competitive use of the soil Pi by weeds, largely save the current Pi consumption, and also prevent Pi overuse-incited water eutrophication and algal blooms [The above properties and advantages of Phi had evoked the establishment of a PtxD/Phi-coupled selection system for plant transgenics ,24. In t to NADH . PtxD ca to NADH –28. This to NADH ,29. Duril blooms ,18–22. MThis pleiotropic PtxD/Phi selection system has been shortly exploited to develop transgenic variants for both monocot and dicot plant species including Arabidopsis , tobaccophoA-encoded bacterial alkaline phosphatase (BAP) in E. coli (termed EcBAP) had an additional ability beyond its main phosphate hydrolysis activity, i.e. oxidizing Phi irreversibly to Pi with hydrogen emission [EcBAP, which was now presented herein. First, we focused to test whether or not EcBAP could replace PtxD as a Phi-metabolized dominant selective marker for plant genetic transformation, and then evaluated the Phi-resistance of EcBAP transgenic plants under Phi stress. We expected to establish a trinary (selective marker / plant P utilization / weed control) system based on EcBAP/Phi combination, resembling the well-known PtxD/Phi suite, so as to provide an alternative technical choice for plant genetic engineering.In this context, we noticed the report of Yang and Metcalf (2004) that the emission . The duaemission ,41. BAP emission , rightlyemission . Nearly E. coli DH5α genome as the template, we deliberately amplified the coding DNA of EcBAP lacking its SP (1–21 amino acids (aa)). For efficient expression in plants, a Kozak sequence was introduced in the forward PCR primer EcBAP-5Bm, thus imparting EcBAP a high translation potential. This amplicon was directly subcloned by dual enzyme (BamH I/Sac I) digestions in a binary plasmid pBI121 to generate the plant expression vector pBI(EcBAP)  is not required for its activity. Herein, using I(EcBAP)  for subs protein .EcBAP gene from pBI(EcBAP) onto the backbone of pET32a(+) via designated enzyme (Nde I/Xho I) digestions. This recombinant EcBAP (~ 47.2 kDa) has a fused C-terminal His6-tag that is suitable for affinity chromatography purification.To preliminarily verify the Phi-oxidizing activity of EcBAP, we further constructed its prokaryotic expression vector pET(EcBAP) by transplanting the E. coli strain BL21(DE3). Due to the omission of SP, the recombinant EcBAP protein was restrictively located in the cytoplasm. The cell lysates obtained by ultrasonic disruption were analyzed by SDS-PAGE. As seen from E. coli. However, most of the EcBAP protein expressed at 37°C was accumulated in the inclusion bodies with a poor solubility (less than 30%), while its solubility increased significantly (up to 50%) when expressed at 25°C. This is in consistence with the classical viewpoint that the low temperature induction can improve the solubility of target proteins recombinantly expressed in E. coli [The inducible expression of prokaryotic vector pET(EcBAP) was conducted in a routine  E. coli .The recombinant EcBAP protein expressed at 25°C could be efficiently purified by His-tag specific immobilized metal affinity chromatography (IMAC) under Pi-free conditions, and almost eluted at one time by 200 mM imidazole solution.in-situ react with Phi to produce Pi that was immediately fixed by calcium ion. The newly formed insoluble calcium phosphate was then incubated with ammonium molybdate and consequently converted to insoluble phospho-molybdate complex which could be distinctly stained by methyl green to develop blue bands under weak acidic condition  for measuring Pi amount. As observed from -1 EcBAP (E. coli periplasm [We further used a Pi/ammonium molybdate (AM)/malachite green (MG)-based spectrometric assay to estimate the Phi-oxidizing activity of recombinant EcBAP. Ten reactions were applied by incubating 0.5–5 μg  of purified EcBAP with Phi, the newly generated Pi was reacted with AM/MG/T (Tween-20) mixture to form a green-blue conjugate that has a maximum absorption at the wavelength of 660 nm -selected EcBAP transgenic (termed EcBAP(Kan)) tobaccos using NPTII selective marker originally from the binary vector pBI121. The main selection process (T3+T4 stages) averagely lasted 50 days for Agrobacterium-mediated tobacco transformation of pBI(EcBAP) under 100 mg·L-1 Kan selection on standard MS medium ) medium containing Phi of different concentrations, using wild-type (WT) tobacco as the control Figs  and S4A.We further implemented this test on standard Pi-containing MS medium Figs  and S4B.Taken these results together, a few of implications can be deduced. Phi is toxic to WT tobacco, which is more serious with the increase of usage. Consequently, the leaves of WT tobacco cannot differentiate into regenerative plants in the presence of Phi even on the normal Pi-containing MS medium. Contrastively, EcBAP(Kan) transgenic tobacco can detoxify Phi likely by genetically introduced EcBAP, thus accounting for its outperformance over WT tobacco to tolerate Phi with a better differentiation/regeneration at any Phi stresses. Nevertheless, probably due to the relatively weak Phi-oxidizing activity of EcBAP, the Phi inhibition at high dosage can not be timely alleviated, and the converted Pi from Phi may also not be enough to satisfy the cellular demands in case of Phi as the sole P source. Plausibly, these would explain why the leaf explant regeneration of EcBAP(Kan) transgenic tobacco occurs only at the conditions of low Phi concentrations (e.g. 1–3 mM) and an accompanying supply of Pi from standard MS medium. In conclusion, replenishing Pi generally benefits the leaf differentiation under Phi stress no matter what WT or transgenic tobacco, and seems indispensable for further shoot regeneration happened only on EcBAP(Kan) transgenic tobacco.EcBAP as the selective marker gene and following a routine procedure (EcBAP transformation (T3+T4 stages) and saving 20 d on average. This time course is also much shorter  than that (8–9 weeks) of PtxD/Phi-directed tobacco transformation [EcBAP (termed EcBAP(Phi)) transgenic tobacco plants were strictly identified by multiplexed PCR  under a suitable selection pressure of 2.5 mM Phi on standard MS medium, using rocedure . When corocedure , Phi-resormation . Moreoveexed PCR , with a exed PCR . As expeexed PCR . Conclus18S rRNA  transgenic tobacco plants were cultivated in greenhouse under normal conditions. Three transgenic lines (termed EcBAP(Phi)- 1, 2, or 6) were chosen for transcriptional expression analysis. As shown in 18S rRNA . Concomi-1 Kan, the equivalent of which seemingly sit at a concentration of about 1 mM Phi.These EcBAP(Phi) transgenic tobacco plants were finally cultivated to flowering and seed-setting, with no aberrant morphology during the full growth. After seed harvest, we performed an evaluation about their Phi-resistance, by judging the seed germination and seedling growth on standard MS or MS (-Pi) medium containing different concentrations of Phi, using WT tobacco as the control . For an In all, these results demonstrate that the EcBAP(Phi) transgenic tobacco has an obvious tolerance to Phi toxicity, and the presence of Pi can mitigate Phi inhibition to promote seed germination and seedling growth to some extent. Probably, an associated and acceptable explanation might still points to the inherent but mild Phi-oxidizing activity of EcBAP introduced in transgenic tobaccos.-1 Pi, or +120 mg·L-1 Phi). After 20 days of standard cultivation in a plant growth chamber, the seed germination and seedling growth status were appraised. As seen in We firstly examined the Phi-tolerance of two surrogate weed species, Bermuda grass and Tall fescue, by sowing the seeds on the filter papers soaked with 0.1 x MS . After 15 days of cultivation in greenhouse, the growth status of mixed seedlings was reciprocally compared  transgenic tobacco line  on a solid matrix composed of perlite, vermiculite and little gravel, individually watered with 0.1 x MS  with the removal of signal peptide. The recombinant EcBAP could be robustly expressed in E. coli cytoplasm, despite its prototype as a secretory enzyme in periplasm. The bad solubility of recombinant EcBAP could be remarkably improved by lowering the induction temperature  to cells and allowing high accumulation  transgenic tobaccos , and assEcBAP-transformed cells differentiate/regenerate more vigorously into shoots under Phi selection. Furthermore, we would also like to compare our results with the PtxD/Phi selection system previously used in the same tobacco transformation [Therefore, we followed this antecedent ‘transformation simulation’ and conducted the routine tobacco transformation directly using EcBAP as the selective marker, in which the selection was kept under a suitable pressure of 2.5 mM Phi on standard MS medium . The maiormation . Thereinormation . Presumaormation ,32.EcBAP transgenic tobaccos. As compared to WT tobacco, all examined transgenic lines showed an evident Phi-resistance, with a remarkably weaker inhibition on seed germination and seedling growth under any concentrations of Phi no matter on standard or Pi-free MS medium  expressed in transgenic tobacco has the chance to oxidatively form the authentic ‘active dimer’ [Taken together, we can deduce that EcBAP is suitable as a dominant selective marker to generate Phi-resistant transgenic plants that can convert the herbicidal Phi into nutritional Pi for their own use. The triple roles of Phi as a transformation selection agent, P fertilizer and herbicide are concentrically governed by the Phi-oxidizing activity of heterologous EcBAP in its transgenic plants. This enzymatic activity reported first on the native EcBAP , was defin vitro  and in v by dual digestions with BamH I and Sac I  to create the plant expression vector pBI(EcBAP) that was further verified by sequencing with primers 35sPro-Fw, NosDw-Rv  by primers EcBAP-5Nd, EcBAP-3Xh  digestions to construct the prokaryotic expression vector pET(EcBAP).Subsequently, the cBAP-3Xh  and PhusE. coli expression is the major source for obtaining large amount of recombinant proteins and usually uses the Novagen pET System. Herein, E. coli strain BL21(DE3)  harboring vector pET(EcBAP) was grown to an exponential OD600 of 0.6 in LB medium (containing 100 mg·L-1 ampicillin) at 37°C, then induced by 0.5 mM isopropy-β-D-thiogalactoside (IPTG)  at 37°C for 4 h or 25°C overnight. A 14 mL culture was precipitated by centrifugation to collect bacterial cells that were subsequently resuspended in 4 mL of 100 mM PBS buffer (pH 7.4) and ultrasonificated for lysis. Each aliquot (e.g. 16 μL) of the crude cell lysate (T) was then partitioned by centrifugation into the supernatant (S) and pellet (P) that was resuspended in the same initial volume of PBS buffer. Meanwhile, a 150 μL cell culture taken before induction was precipitated and resuspended in 16 μL PBS buffer as the uninduced sample (UI). Finally, all these samples were subjected to 12% SDS-PAGE. The solubility of recombinant proteins was estimated by gel band grey-densitometry via the program “Quantity One” .2O.As the product of Phi oxidation is Pi, this enzymatic activity in EcBAP can be evaluated by the known Pi-specific gel activity staining and spectrometric assay. To avoid any Pi contamination during the purification and activity-analysis of the recombinant EcBAP, all used reagents were required to be free of Pi and the involved equipments (e.g. electrophoresis apparatus) were washed repeatedly with ddHThe recombinant expression of EcBAP at 25°C was conducted as mentioned above. The precipitate of a 14 mL induced culture was thoroughly washed with 5 mL Pi-free lysis buffer , and then resuspended in 4 mL of the same lysis buffer for ultrasonic disruption. Afterwards, the recombinant EcBAP protein in the supernatant of cell lysate was purified by using the routine Ni-NTA IMAC and the column of HisTrap FF crude , and finally eluted in 200 mM imidazole solution. The purification quality was evaluated on samples with a gradient of aliquots  by 12% SDS-PAGE. The concentration of purified EcBAP was determined by using a BCA Protein Assay Kit .2O; incubate in a reactive solution  for 30 min; transfer into 1% ammonium molybdate solution and gently shake for 10 min, repeat this step once; transfer into the staining solution  and mildly shake for 30 min; finally rinse with ddH2O and store therein.Afterwards, the gel activity staining analysis of recombinant EcBAP was performed according to a modified protocol of Stochaj and Berkelman (2006) . At firs2HPO3·5H2O, pH 7.0), 0–50 μL  of the purified EcBAP solution and ddH2O to a total volume of 500 μL, and incubated in a water bath of 37°C for 30 min. Then, 1 mL of the AM/MG/T mixture was added in each reaction for color development. Half hour later, the absorption at the wavelength of 660 nm (OD660) of these reactions were measured in a spectrometer . The value of OD660 was used to calculate the Pi yield for each reaction, according to a standard Pi curve. The enzymatic activity of recombinant EcBAP was determined by Pi production versus the protein amount of EcBAP, and finally averaged.Meanwhile, a quantitative malachite green-based spectrometric assay on the enzymatic activity of recombinant EcBAP was also carried out, as previously described  with smaNicotiana tabacum L. cv Xanthi) [2) of aseptic WT tobacco leaves were incubated for 5 min in the culture of A. tumefaciens strain LBA4404 harboring the pBI(EcBAP) expression vector, then washed three times with sterile ddH2O, and placed on T1 co-cultivation medium under 28°C and darkness for 3 d. Afterwards, leaf explants were transferred to T2 bacteria-removing medium (containing 500 mg·L-1 carbenicillin (Car)) for 5 d, then to T3 selection medium supplemented with 100 mg·L-1 Kan or 2.5 mM Phi. After a period, the resistant calli with emerged small shoots were translocated to T4 subculture medium for regeneration of more shoots. Finally, the green shoots of qualified size were excised from the explants and cultivated in T5 rooting medium for growing into plantlets. From T2 to T5 stage, the culturing in a plant growth chamber was maintained at a normal condition of 26°C, 75% humidity, 1500 lux light intensity, and a regime of 16 h light / 8 h dark. The T1–T5 mediums were prepared using standard Murashige-Skoog (MS) medium  and detailed in 2HPO4·12H2O and Na2HPO3·5H2O, respectively.The Agrobacterium-mediated leaf disc transformation of tobacco ( Xanthi)  was prev Xanthi) ), and thEcBAP transgenic tobacco plants, with reciprocally combinatorial primer-pairs derived from the coding region of EcBAP and the backbone of vector pBI121, e.g. EcBAP-5Bm/EcBAP-3Sc, 35sPro-Fw/EcBAP-3Sc, and EcBAP-5Bm/NosDw-Rv  of Kan/or Phi- selected NosDw-Rv .EcBAP transgenic (i.e. EcBAP(Kan)) tobacco plantlets growing in glass bottles, then cut into small pieces (0.5 cm × 0.5 cm), and pairwise laid on MS or MS (-Pi) medium containing Phi of different concentrations . After 7 days, 1 month, and even 2 months of cultivation in a plant growth chamber with normal condition settings, the differentiation/regeneration status of these leaf explants under Phi stress on standard MS or MS (-Pi) medium were photographically recorded and compared between WT and EcBAP(Kan) transgenic tobacco.To preliminarily evaluate a candidate selective marker, leaf explant regeneration test under the corresponding selection stress can be used as a ‘transformation simulation’ to assess its resistance . This taEcBAP expression in its transgenic tobaccos was only qualitatively analyzed by the classic RT-PCR. First, total RNA from the main tissues  of three Phi-selected EcBAP transgenic tobacco lines (EcBAP(Phi)-1, 2, 6) were extracted using the Trizol reagent . By using M-MLV RTase  and random primers, aliquots of the RNA samples were then reversely transcribed as the cDNA templates for PCR examination of EcBAP transcriptional expression in the main tissues of tobacco by primer-pair EcBAP-iFw/EcBAP-iRv -1, 2, 6) were sowed individually at four delineated quarters of each petri-dish containing standard or Pi(-) MS medium supplemented with 100 mg·L-1 Kan or a serial concentration  of Phi. These petri-dishes were horizontally placed for seed germination in a plant growth chamber under normal conditions. Meanwhile, a similar test was performed with only small variations, i.e. evenly dot-sowing the different seeds with marked boundaries in one line on each petri-dish and placed them vertically. After 2 weeks, the seed germination and seedlings growth status were photo-recorded and compared between EcBAP transgenic lines and WT tobacco.To examine the acquired traits (i.e. Phi-resistance) imparted by the transgene -1 Pi, or +120 mg·L-1 Phi). The holding glass plates (15 cm x 15 cm) were placed in a plant growth chamber under normal conditions. After 20 days of cultivation, the seed germination and seedling growth on each plate were photo-recorded for both horizontal and vertical views.For a Phi-based weed control test, the basal Phi-tolerance of two candidate wild weed species  was preliminarily assessed. Their seeds were pairwise sowed on each filter paper individually soaked with 0.1 x MS  transgenic tobacco line  were mixed at a ratio of 4:1:1, and evenly sown in plastic plates containing solid matrix composed of perlite, vermiculite and little gravel. Each suite of sowing was individually irrigated with 0.1 x MS . After 15 days of cultivation in greenhouse, the growth of mixed seedlings on each plate was photo-recorded and compared.To probe the herbicidal role of Phi for future field cultivation of EcBAP transgenic individuals among a number (30) of regenerated tobacco plantlets, as did for PtxD [EcBAP transgenic tobaccos (on petri-dishes) and weed control simulation test (in greenhouse), were conducted with at least two replications.The solubility of recombinant EcBAP was estimated by band grey-densitometry on SDS-PAGE gels from three separate expression experiments. The Phi-oxidizing activity of recombinant EcBAP was measured as the mean±SD from malachite green-based spectrometric assays on triplicate groups of enzymatic reactions. Positive transformation efficiency using Kan or Phi selection was determined as the percentage of PCR-identified for PtxD . In addiS1 FigEcBAP gene was subcloned in plant binary vector pBI121 between CaMV 35S promoter (termed Pro(CaMV 35S)) and nos terminator (termed Ter(nos)) by BamH I/Sac I digestions. The kanamycin (Kan)-selective marker gene NPTII was controlled by nos promoter (termed Pro(nos)) and Ter(nos).The (TIF)Click here for additional data file.S2 FigEcBAP gene. The codons of additional residues (MA in red letter) were introduced for initiating translation.The N-terminal SP (1–21 aa) was truncated in the deduced protein of the cloned (TIF)Click here for additional data file.S3 FigEcBAP transgenic tobacco plantlets by multiplexed PCR with three primer-pairs EcBAP-5Bm/EcBAP-3Sc, EcBAP-5Bm/NosDw-Rv, and 35sPro-Fw/EcBAP-3Sc. M: DNA marker; Arrow-heads indicate the target PCR bands. The original gel image of this figure (B) is available in (A) The procedure of tobacco transformation of plant vector pET(EcBAP) via Agrobacterium infiltration and Kan selection. (B) Identification of Kan-resistant (TIF)Click here for additional data file.S4 FigSmall leaf pieces (0.5 cm × 0.5 cm) of WT and EcBAP(Kan) transgenic tobacco were pairwise laid on (A) MS (-Pi) or (B) standard MS medium, containing Phi of high concentrations . After 7 days, 1 month, and even 2 months, the differentiation/regeneration status of these leaf explants under Phi stress were photo-recorded and compared between WT and EcBAP(Kan) transgenic tobacco.(TIF)Click here for additional data file.S5 FigExperiments were performed using three primer-pairs, i.e. (A) EcBAP-5Bm/EcBAP-3Sc, (B) EcBAP-5Bm/ NosDw-Rv, and (C) 35sPro-Fw/ EcBAP-3Sc. M: DNA marker; Arrow-heads indicate the target PCR bands. The original gel image of this figure is available in (TIF)Click here for additional data file.S6 Fig18S rRNA  with a correct product (552 bp) by primer-pair Nt18S-iFw/Nt18S-iRv; (C) RT-PCR of EcBAP with a correct product (408 bp) by primer-pair EcBAP-iFw/EcBAP-iRv. R: root; S: stem; L: leaf; Arrow-heads indicate the target PCR bands. The original gel images of this figure (A–C) are available in (A) The extracted total RNA; (B) RT-PCR of (TIF)Click here for additional data file.S7 Fig-1 Pi, or +120 mg·L-1 Phi), and cultivated in a plant growth chamber under normal conditions for 20 days, then photo-recorded for both horizontal and vertical views.Seeds of two selected weed species, Bermuda grass (W1) and Tall fescue (W2), were laid on the filter papers wetted with 0.1 x MS Click here for additional data file.S8 FigThe blue-circled bigger seedlings in (TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


Clinicians commonly try to use mechanism-based knowledge to make sense of the complexity and uncertainty of chronic pain treatments to create a rationale for their clinical decision-making. Although this seems intuitive, there are some problems with this approach.The widespread use of mechanism-based knowledge in clinical practice can be a source of confusion for clinicians, especially when complex interventions with different proposed mechanisms of action are equally effective. Although the available mechanistic evidence is still of very poor quality, in choosing from various treatment options for people with chronic pain, an approach that correctly incorporates mechanistic reasoning might aid clinical thinking and practice.By explaining that not all evidence of mechanism is the same and by making a proposal to start using mechanism-based knowledge in clinical practice properly, we hope to help clinicians to incorporate mechanistic reasoning to prioritize and start choosing what may best work for whom. Modern concepts of pain science point out that chronic pain is a multidimensional experience driven by a complex interaction of various degrees of biological, psychological and social factors. These factors are ultimately associated with large inter-individual variability of symptoms and clinical manifestations , making . Therefore, to answer and choose better what works for whom, clinicians will benefit from some guidance on using mechanism-based knowledge in clinical practice. To do that, we need to better understand the mechanisms by which treatments work  and for whom it may work  while using valid conceptual models to aid clinical reasoning, that for now on will be referred in this text as mechanism-based knowledge.Not all findings of mechanistic studies should be used to explain the interventions’ mechanisms of action or for whom the intervention may work. For example, the results of cross-sectional or case-control studies should not be used to explain how or why an intervention may or may not work. Although clinicians may benefit from using conceptual models to make sense of patient’s complaints and to assist their clinical reasoning, not all frameworks are robust enough to be incorporated in clinical practice. Several frameworks have not been properly investigated yet or does not provide a good explanation of the phenomenon of interest to aid clinical reasoningThe widespread use of mechanism-based knowledge in clinical practice can be a source of confusion for clinicians, especially when complex classification systems and interventions with very different proposed mechanisms of action are equally effective. For example, although active interventions , behavior modification  and strategies to improve self-management  are effective for people with chronic musculoskeletal pain, all of them produce small effects on pain and disability , 5. Besi“Should we just choose among the available evidence-based intervention based on clinical expertise and patient preferences, whatever their proposed mechanism of action and moderating factors, or should we take the evidence of mechanisms of action and moderating factors into account when deciding among which intervention to choose?”The use of mechanistic reasoning in clinical practice is important. However, it may increase even further the complexity of treatments offered and the number of clinical tests used with no clear indication that it will improve patients‘outcomes . This raWhile some clinicians choose among different evidence-based interventions based on what they observe during the evaluation process, others might be comfortable to just combine interventions with different proposed mechanisms of action expecting additive effects (even when there is no such evidence). But clinical encounters are brief, and patients usually presents with more than one complaint. Therefore, interventions need to be prioritized and assertive. The available evidence of mechanisms of action and moderating factors for pain interventions is still of very poor quality . HoweverIf several effective treatment options with different proposed mechanisms of action are available, clinicians should look for the interventions that maximize the likelihood of improving patient’s symptoms. To do that, clinicians need to understand the mechanisms by which treatments work and for whom it may work . When tIn summary, when multiple interventions with different proposed mechanisms of action are equally effective for a given outcome, clinicians may benefit from considering the evidence from mechanisms of action and moderating factors in decision making to improve the patients’ probability of success.Clinicians are overloaded with the number of available pain conceptual models and evidence showing that complex interventions with very different proposed mechanisms of action are equally effective for chronic pain. This can be misleading and confusing, since the clinical presentation of chronic pain is complex and heterogeneous. We need more research on interventions’ mechanism of action and moderators of effect to explore whether an approach that correctly incorporates mechanistic reasoning can improve clinical thinking and practice. To assist clinicians on how to select good mechanistic evidence to improve their decision making, we described 5 key steps that can be used to identify and interpret high quality mechanistic studies to be applied in clinical practice (Table 2).Until better mechanistic evidence is available, clinicians should use the available evidence about mechanisms of action and moderating factors to optimize treatment prescription with caution, as we tend to give more credibility to stories that make sense . Clinicians should be aware that mechanistic evidence is not a proof of treatment efficacy, no matter how much it makes sense . SimilaCurrent available mechanistic evidence does not support a mechanistic reasoning to clinical decision-making in choosing between various treatment options for people with chronic pain. However, clinicians do use mechanistic evidence (or what they think that are good mechanistic evidence) for clinical reasoning and for justifying their interventions (for themselves and for patients). By explaining that not all evidence of mechanism is the same and by making a proposal to identify, interpret and start using mechanism-based knowledge in clinical practice properly  we hope to help clinicians to properly incorporates mechanistic reasoning in practice. This may lay the foundation for the future, when higher-quality evidence are available.


The aim of this study was to test the feasibility and reliability of the Animal Welfare Indicators (AWIN) protocol for welfare assessment of dairy goats when applied to semi-extensive farming conditions. We recruited 13 farms located in the NW Italian Alps where three assessors individually and independently applied a modified version of the AWIN welfare assessment protocol for goats integrated with some indicators derived from the AWIN welfare assessment protocol for sheep. The applied protocol consisted of nine individual-level  and seven group-level  animal-based indicators. On most farms, the level of welfare was good. Many of the considered welfare problems  were never recorded. However, oblivion, severe lameness, hair coat condition and abscesses were detected on some farms, with percentages ranging from 5 to 35%. The mean percentage of animals with normal body condition was 67.9 ± 5.7. The level of synchronization during resting was on average low (14.3 ± 7.2%). The application of the whole protocol required more than 4 h/farm and 3 min/goat. The inter-observer reliability varied from excellent  to acceptable , but insufficient for hair coat condition, improper disbudding, synchrony at grazing, QBA. Differences in background of the assessors and feasibility constraints  can affect the reliability of data collection. Extensive training seems necessary for properly scoring animals when applying the QBA, whereas the FHAT to evaluate the Human-Animal Relationship of goats at pasture seems promising but needs to be validated. Indicators that evaluate the synchrony of activities require to be validated to identify the best moment to perform the observations during the day. Welfare assessment in extensive production systems has received a lower interest in research than in intensive husbandry systems . This isThere are at least three issues that need to be considered in case of welfare assessment in extensive conditions: (1) although valid indicators for welfare assessment in intensive/indoor husbandry systems are already available, many of them still need to be tested for validity under extensive conditions and, in some cases, new indicators need to be identified –7; (2) tCompared to other species , goats Even if there is a need for assessing the welfare of goats in extensive systems, the scarcity of the research on this topic  makes itIn 2011–2015, an EU-funded project on Animal Welfare Indicators (AWIN) developed on-farm welfare assessment protocols for sheep, goats, horses, donkeys and turkeys, possibly using animal-based indicators , 15. Desex novo. Unfortunately, the research performed by Leite Oliveira et al.  TO3 territory of Pinerolo-Collegno . From the database, we selected farms presenting the following characteristics: (i) raising goats for dairy or dual purposes; (ii) making use of outdoor grazing in spring and autumn in proximity to the winter housing buildings; (iii) keeping goats only, with no coexistence with other domestic species; (iv) breeding prevalently Alpine and Valdostana breeds ; (v) voluntary acceptance of the farmer. Only 13 farms satisfied all the inclusion criteria and were therefore included in the survey. None of the selected farms bred animals for dual purpose; hence, the assessment was only performed on dairy animals.2, but large differences were found among the farms . The average available pasture area/goat was equal to 343.47 m2 . The goats had access to pasture for 90–250 days/year for 4–12 h/day, except in one farm where they had permanent access to the outdoor grazing area. During summer, the goats were taken to alpine ranges from June to October for a total period of 90–180 days.These farms housed the goats during the winter in indoor pens or, on three farms, in tie stalls. During spring and autumn, the animals were housed during the night and they were released in flat to medium/steep slope areas (ranging from 470 to 920 m a.s.l.) near the farms after the morning milking, giving them the opportunity to graze in meadows, but also to browse the surrounding bushy and woody areas. In some farms, only bushes and woods were available for foraging. In nine farms, woods were also used as shelters to protect the goats from wind, sun, and rain. When woods were not available, the goats had no shelters. On average the total area available for spring and autumn pasture was equal to 20,872.73 mThe total number of goats in our farm sample ranged from 12 to 77 with a mean (±SD) of 31.2 (±20.74) goats. Lactating goats ranged from 8 to 77 animals, with a mean (±SD) of 17.70 (±18.71) goats. The average age of lactating goats ranged from 24 to 78 months. Goats were in their mid-lactation stage and were milked twice a day. In 12 farms, the goats were manually milked, while one farm was provided with a mobile milking unit. Besides making use of fresh grass and bushes available in the grazing area, once or twice a day, during milking, eight farms provided supplementary feed consisting of hay, chestnuts, alfalfa, bran, whole or flaked barley, and whole or flaked corn. Three farms provided supplementary feed consisting of whole, flaked or mash corn, with or without mineral supplementation, in the winter period only. In two farms, no supplementary feed was delivered. Fresh and clean water was always available inside the barn. At pasture, water was available through streams (five farms) or watering tanks (four farms); water was not available at pasture in four farms. The distance of water from the pasture area ranged from 0 to 1,500 m.Claw trimming occurred once a year in six farms, when necessary in five farms, every 6 months in one farm and every 4 months in another farm.All the farms produced cheese from pure goat milk in small dairies adjacent to the farms. The cheeses were sold at the farm shop, at local markets or were destined to the small-scale organized distribution.In each farm, the welfare assessment protocol was tested in the period April–July 2019, during the spring grazing period. In order to test protocol reliability, the assessment was carried out by three assessors who had different background and level of experience with dairy goats. The three assessors were students of the MSc in Animal Science at the University of Turin . Assessor A also had a M.Sc. in Veterinary Science and in Biostatistics, worked as a veterinarian in the Public Health Service and had more than 10 years of experience with dairy goats. Assessors B and C had no specific experience with dairy goats. The three assessors received a common training before the beginning of the study, including both theoretical and practical sessions, and received the AWIN protocol  as trainFarmers were contacted by telephone to illustrate the research and gather essential information about the farm routine, in order to identify the best time for welfare assessment, which depended mainly on milking time.On each farm, the protocol was applied simultaneously and individually by the three assessors, without any kind of interaction among them. All the assessors were unknown to the farms. The assessments took place mostly under sunny weather (77%), with some cloudy days but never when raining. Visibility was always good. Wind was almost absent. Ambient temperatures ranged from 9 to 24°C, with an average of 18°C. Relative humidity ranged between 21 and 90%, with an average of 54%.The protocol included 13 out of the 18 original indicators from the 2nd level AWIN welfare assessment protocol for goats , plus foAssessor A evaluated all the 13 selected farms. Due to unforeseen circumstances, assessors B and C only assessed 12 farms. A minimum of two assessors per farm was always guaranteed.For individual-level indicators, absolute and percentage frequencies of animals without welfare problems were calculated. The prevalence of group-level indicators was calculated as the proportion of goats with absence of welfare problems out of the number of assessed goats. For indicators collected with the scan sampling method, we considered the proportion of goats in the scan with the highest number of animals synchronized during grazing and resting or presenting thermal stress or oblivion, out of the total number of goats. FHAT results were reported as the proportion of farms where goats avoided, accepted the contact or spontaneously approached the farmers out of the total number of farms. Principal Component Analysis  was used to explore results from the QBA. Data was analyzed using the statistic software IBM SPSS v. 26.0 for Windows .The above-mentioned welfare outcomes are presented in the results referring to data collected by Assessor A. This choice is due not only to the fact that this was the only assessor who evaluated all the farms, but also because this assessor had more experience with goats than the others. Therefore, data collected by Assessor A were used as reference and compared with data collected by the other two observers in order to assess inter-observer reliability (IOR).IOR was only calculated for the 11 farms where all the three assessors were present. For categorical data  IOR was calculated over all animals, regardless of farm, using the S index , 27. Thipo is the rate of observed concordance and M is the number of categories.where The percentage of concordance agreement was calculated for pairs of assessors against Assessor A (A vs. B and A vs. C) and for the three assessors together (A vs. B vs. C). The S index was only calculated for the three assessors together. For continuous data (group-level indicators), intra-class correlation (ICCs) coefficients were calculated . According to Bateson and Martin , we adopW. The results were interpreted according to Martin and Bateson  scores attributed by the three observers to each farm on the first two PCs was analyzed by using the Kendall Correlation Coefficient  Bateson , 30, wheAccording to several authors , 29, a gAt the end of the assessment, the assessors were asked to report the major constraints experienced during the application of the protocol.The results of the application by Assessor A of the welfare assessment protocol for dairy goats in semi-extensive conditions are shown in Two descriptors (Bored and Frustrated) were scored 0 in all the farms by Assessor A; hence, they were removed from the analysis. PCA was performed on 11 out of 13 descriptors. The analysis identified four main PCs with eigenvalues >1 . The first two PCs together explained 62.57% of the total variance among the farms . The IOR calculated for individual-level observations is reported in W (0.597). The agreement on PC2 was acceptable .The agreement among assessors for QBA was considered unacceptable for PC1, due to the rather low Kendall's The collection of individual-level indicators took about 3 min/goat and the time required to perform the whole protocol was about 4 h/farm. The assessors reported the presence of some constraints that reduced the feasibility of both individual- and group-level indicators. For individual-level indicators, narrow pens and low-light conditions affected the quality of the assessment and extended the time needed for the inspection of the animals; for group-level indicators, open ranges, the presence of thick vegetation cover and the presence of guardian dogs were the major limitations.This paper reports the results of the application of the AWIN welfare assessment protocol for goats in semi-extensive husbandry systems. The original protocol was partly modified to be adapted to the different context.The assessment showed that in our farms some welfare problems were completely absent  or almost absent . However, a comment is necessary for hair coat condition and abscesses. The average situation for both these indicators was good also in comparison with Italian and Portuguese intensive farms , 31 and Potential welfare issues were highlighted regarding body condition and resting synchronization. The percentage of animals presenting a normal body condition was low  if compared to the results obtained in extensive double-purpose systems in Portugal 89.4%; ] and in 9.4%;  anThe second indicator that poses some welfare concerns is synchrony at resting, which was very low in our study with only on average 14.3% of goats that lied down simultaneously, and even some farms where the animals never rested at the same time. When a sheltered area, such as a thick vegetation cover, was available, most of the goats (95.1%) used it for resting. According to Zobel et al. , hiding The AWIN welfare assessment protocol for goats  uses theQBA studies ground on contrasting expressive qualities where contexts are previously selected for their divergent characteristics . In our According to Kaufman and Rosenthal , IOR is Most of the indicators collected on goats at pasture showed acceptable reliability; however, IOR was not sufficient for QBA  and synchrony at grazing. Investigating the reasons for this result, we identified some issues related to the background and training of the observers, and to feasibility constraints. Regarding background and training, assessor A had a sound experience with goats, whereas assessors B and C only had a limited experience. This may have affected the effectiveness of training that possibly did not bridge the gap among the assessors and in turn affected the results of the observations. Furthermore, the assessors reported several hurdles during the collection of the indicators that may have affected the IOR and reduced the feasibility of the protocol. The application of the whole protocol under semi-extensive conditions took more time than the application in intensive farming conditions. In fact, the average estimated time required in intensive conditions is 90 min/farm and 30–45 s/goat , whereasA further complication that affected the feasibility of our assessment, and in turn its reliability, is that observations performed when the animals were at pasture required the use of binoculars, in particular in unfenced pastures where animals could stray far away. If animals graze in areas with thick vegetation cover, observations can be difficult, as the vegetation reduces the visibility. As to visibility, the farms were visited on purpose only on good weather days, as we supposed that rain or fog could worsen the reliability of the results. Hence, we suggest checking the weather forecast before scheduling the farm visits.In addition, the assessors reported difficulties to perform the individual assessment during milking in narrow and dark pens, especially because milking frequently occurred very early in the morning, under suboptimal lighting conditions. Hair coat condition, abscesses and udder asymmetry were considered the hardest indicators to be collected. A relatively low IOR was reached for improper disbudding in some cases 0.20), whereas a good IOR was obtained in other cases (0.91); the assessors did not report any specific constraints, probably because this indicator was only applicable on one farm. The assessors reported some difficulties to detect severely lame goats due to the different flooring on which the animals walked: in some cases, they walked on concrete floor, but in others, they reached the pasture on gravel roads with variable slopes. Different surfaces  and, in this case, also different slopes, may affect the reliability of the observation, as suggested by other authors . Althoug, whereasFurther studies are needed to test the validity of the FHAT in goats, but all the assessors reported that this test is easy to be conducted with goats at pasture and the agreement among assessors was perfect for the three possible reactions of goats to the farmer. However, it is probably advisable to register the goat reaction toward a familiar human when the farmer really gathers the flock, in order not to affect the routine and management of the farm, as maybe goats would react differently if handled out of the normal routine. In any case, the validity of this indicator in goats still has to be confirmed and requires further consideration.All the indicators used in this attempt to adapt an already existing welfare assessment protocol for goats are animal-based measures. However, no practical animal-based indicator was found to cover the “absence of prolonged thirst” criterion. This lack is common to most of the evaluations conducted at pasture . HoweverIn conclusion, this research showed that most of the indicators selected to assess the welfare of goats in semi-extensive conditions could be applicable, even if most of them were originally developed for intensive conditions. For most indicators, an acceptable level of reliability was reached; however, further research is required in order to identify a complete set of robust indicators in this specific context. Finally, feasibility constraints should be taken carefully into account as they can affect the reliability of the evaluation. For example, the assessor may decide to collect the individual indicators during either morning or evening milking, choosing the moment when the light is higher, as a scarce illumination can negatively affect the results. Furthermore, it may be advisable to collect FHAT according to the farm routine, for example, when the farmer gathers the flock before entering the barn for the evening milking.Specific research should be conducted on daily activities and biorhythms of farmed goats to select the best moment of observation for evaluating the synchrony during feeding and, in particular, during resting. This research highlighted the lack of animal-based indicators to assess the effect of prolonged thirst in semi-extensive conditions; hence, specific research is needed to fill in this gap.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the animal study because according to the National Italian Law (D.L. 26/2014), no specific ethical approval was required, as no pain, suffering, distress or prolonged damage equivalent to or greater than that caused by the insertion of a needle was applied. Written informed consent was obtained from the owners for the participation of their animals in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.MG collected data and organized the database. MB and MG performed the statistical analysis. MB wrote the first draft of the manuscript. MR and SM wrote sections of the manuscript. LB supervised the work. All the authors contributed to conception and design of the study, manuscript revision, read, and approved the submitted version.The authors received funds for open access publication fees by Open APC Project provided by University of Milan.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The original IPDAS checklist, based on evidence syntheses, focusedon components of PtDAs known to support informed, values-based reasoning andengagement with health care professionals. In this article, we present IPDASEvidence Update 2.0. The 13 articles that make up this update provide the latestevidence on 11 core IPDAS domains: development process,14 At a minimum, PtDAs describe the health condition or problem; makeexplicit the decision; provide information on options, benefits, and harms;and help patients clarify which benefits and harms matter most.15 Optional features in PtDAs are probabilities of outcomes of options,narratives describing patients’ experiences with making decisions, andguidance in the process of decision making. They are designed to be used asadjuncts to counseling and are often used to facilitate shared decisionmaking between patients and their clinician. A systematic review of 105randomized controlled trials demonstrated that compared with usual care,patients exposed to PtDAs have improved knowledge, more realisticexpectations, less decisional conflict and participate more actively inmaking decisions.16 Given that few have been used in clinical practice after trials were completed,17 there is increasing research focused on the process used for theirdevelopment, evaluation, and implementation.PtDAs are evidence-informed resources to guide patients in the process ofmaking quality decisions.18 For example, there were decreased hysterectomies and fewer herniateddisc surgical procedures when patients were aware of nonsurgical options toaddress the condition. The effect on uptake of options was judged to bepositive when PtDAs were unbiased and the change addressed variations inclinical practice.20 Concurrently, there was concern that PtDAsdeveloped without a guiding set of standards could be used to present biasedinformation.Evidence was emerging in 2003 that PtDAs can affect the uptake of options.21 The collaboration has been an entirely volunteer organization with noformal affiliation with a professional society, and members have produced aseries of evidence-based IPDAS resources , and comments on thefeasibility from those trained in using the IPDAS instrument, the expertcommittee proposed 6 criteria for qualifying to be defined as a PtDA, 6criteria for certifying PtDAs, plus 4 for screening PtDAs (to minimize riskof bias), and others were described as quality criteria. In 2013, IPDASmembers published the updated theoretical and empirical evidence on the 12original broad domains, plus 1 extra team published evidence onimplementation of PtDAs.23 In addition tosupporting the IPDAS criteria, this update provided more detailed guidanceon developing PtDAs and discussed ways of describing the quality of theevidence used to inform PtDAs  and the need todisclosure actual or potential conflict of interest, particularly forfunding received from commercial for-profit entities used to develop orexclusively distribute PtDAs. The 2013 evidence update did not includechanges to the IPDAS criteria at that time.Given the number of IPDAS items and some challenges with applying the items,IPDAS proposed a minimum set of standards for defining and certifying PtDAs.24 The certification program is noteworthy because it provides aheightened level of legal protection to clinicians who use certified PtDAswith their patients.26 This programtypically announces a call for PtDAs based on specific conditions , and certified PtDAs areannounced on their website. Concurrently, the IPDAS criteria are: being usedby the Norwegian Health Department for reviewing PtDAs approved for thenational platform,27 being used for the International A to Z Inventory at the OttawaHospital Research Institute,28 formally approved in the Netherlands by national stakeholders,29 and they were proposed for national standards for certification ofPtDAs by the National Quality Forum.20 The standards are also available in Japanese, Spanish, and Chinese(IPDAS website).21In 2016, Washington State Health Care Authority launched the first program tocertify PtDAs based on the IPDAS criteria.3032 Based on theIPDAS quality dimensions and other reporting guidelines, the 26-item SUNDAEChecklist is meant to promote greater transparency and completeness ofintervention studies that evaluate PtDAs.In 2018, the IPDAS reporting guidelines workgroup published the Standards forUniversal reporting of patient Decision Aid Evaluations (SUNDAE)Checklist.33 and the wealth of new research about their use and effectiveness, theIPDAS Steering Committee identified the need for another evidence updatewith a specific focus on identifying recommendations for changes to theIPDAS criteria.Given the increased use of IPDAS, the rapidly growing number of clinicalpractice guidelines recommending PtDAs,In fall 2018, the IPDAS Steering Committee identified 11 team leads for each ofthe 12 original broad domains with 2 changes: 1) balanced information wasmerged with the presentation of information on options, benefits, and harmsand 2) the delivery of PtDAs on the internet was merged with theimplementation of PtDAs. Senior researchers were chosen based on theirinvolvement in previous evidence updates and their research in the area ofthe specific domain. They were encouraged to identify co-leads from anothercountry. Volunteers for each of the domains were recruited through the IPDASlistserv and at the 2018 Society for Medical Decision Making Shared DecisionMaking special interest group meeting in Montreal, Canada. Concurrently, weasked for other topics that should be included in this update.Domain teams were tasked with drafting a proposal for the process they plannedto use for updating the theoretical and empirical evidence published sincethe 2013 update and making recommendations of changes to the original IPDAScriteria. Teams were given examples from the 2013 update and asked to createan update of publishable quality. The proposals were reviewed by members ofthe IPDAS Steering Committee in spring 2019 based on the following criteria:1) names and affiliations of working group leaders and members withrepresentation from 2 or more countries, 2) proposal based on previous IPDASwork including definitions and original criteria, 3) proposed methods aim tosynthesize the best available theoretical and empirical evidence, 4)indication in the proposal that 1 outcome of Update 2.0 is verifying and/orrevising the original criteria with justification for changes, 5) timelineaims to have work completed, and 6) completed disclosures of interest.The IPDAS Steering Committee gave careful attention to how potential conflictsof interest would be disclosed among the team leads and members. At theoutset of the update, members were asked to declare direct interests wherethere was an opportunity for financial gains  for themselves, aspouse, or dependent children. Other reportable debts, outside positions,agreements or arrangements, and gifts or travel were also disclosed.Finally, indirect interests where there was an opportunity for benefit for athird party closely associated with the member were also disclosed.Declarations of interest were regathered at the time of release of theupdates from all members.113Two of the broad domains published 2 articles: communicating probabilitiesabout outcomes4 and health literacy.10 Other topicssuggested for this update were theories and mechanisms, training in shareddecision making, application of shared decision making in support of chronicconditions, whether or not to provide probabilities, and targeting specificdisadvantaged populations. Given IPDAS’s mandate is focused on PtDAs, weexcluded suggestions more broadly focused on shared decision making andasked teams to report on updated theories and mechanisms. The other 2suggestions were assumed by the communicating probabilities team and thehealth literacy team.The update involved 105 unique participants from 11 countries. While the IPDASSteering Committee did not determine any conflicts that rose to the point ofdisqualifying a member from participating in the update, promotingtransparency is an ongoing priority of the collaboration. The articles inthe IPDAS Update 2.0 series reflect the 11 broad domains.IPDAS is commonly used to inform the development and evaluation of PtDAs.Evidence continues to support the minimal criteria. Although a few newcriteria were proposed by authors of articles in this update, the IPDASSteering Committee will engage in a broader consensus process before changeswill be made to the IPDAS criteria. Ongoing updates such as these are avital part of maintaining the IPDAS criteria as responsive to changes inemerging evidence and relevant to PtDA development, content, evaluation, andimplementation.