The authors have stated explicitly that there are no conflicts of interest in connection with this article.To the Editor,We read the paper by Ohta et al. with great interest.However, in this paper, the patient selection criteria are very vague: “the presence of initial findings suggestive of vitamin B1 deficiency, assessed by general or internal medicine physicians on the day of admission based on physicians’ charts.” As no selection criteria are provided, the symptoms upon which the suspicion of thiamine deficiency is based are unclear.We have experienced a number of cases of thiamine deficiency in cancer hospitals.


Philosophers’ Imprint, 19(3). Philosophy and Phenomenological Research, 85(2). I argue for the unexceptionality of evidence about what rationality requires. Specifically, I argue that, as for other topics, one’s total evidence can sometimes support false beliefs about this. Despite being prima facie innocuous, a number of philosophers have recently denied this. Some have argued that the facts about what rationality requires are highly dependent on the agent’s situation and change depending on what that situation is like (Bradley,  Impossibility Thesis: Evidential situations requiring false belief about what rationality requires are impossible.A natural thought is that evidence about what rationality requires behaves in roughly the same way as evidence about other topics. If so, then your evidence can sometimes support false beliefs about what rationality requires. A number of philosophers have recently denied this. Some because they have thought that the facts about what rationality requires change depending on the agent’s evidential situation Bradley . Others not believe P. Are you required to believe P, or not? On the one hand, you seem to be required to not believe P, because this is what your evidence about what rationality requires supports. On the other hand, you seem to be required to believe P, since this is what the true requirements in fact require. You are in a bind—you appear to be required to both believe and not believe P. The Impossibility Thesis offers a way out—it rules out situations in which this inconsistency arises. However, it creates more problems than it solves. I argue that we should prefer the following Simple View of evidential support.Simple View: Evidential situations supporting false belief are possible about all topics.One reason to like the Impossibility Thesis is that it rules out an awkward evidential situation that threatens the consistency of our theories of epistemic rationality. Suppose that rationality requires you to believe P. Then, suppose that you have some evidence supporting the false belief that, in fact, you are required to Following others in this debate, I understand requirements of rationality as necessary conditions of having rational epistemic attitudes, without taking much of a stand on what these are.Section Logic 101. Suppose that it is a requirement of rationality to avoid contradictory belief. Suppose also that you are taking your first introductory course in logic. Unfortunately, your instructor is an overzealous advocate of dialetheism and believes that rationality sometimes requires contradictory belief, for example in response to instances of the Liar Paradox.Evidence can support false belief about all kinds of subject matters. The Simple View says that what rationality requires is a subject matter like any other, and so something that evidence can support false belief about. Studying philosophy is a good way to get evidence for false belief about what rationality requires. Consider:A natural explanation of this situation is that your evidence supports a false belief about what rationality requires. Your instructor counts as an expert in this domain, which makes his testimony evidence. The philosophical arguments you study also give you evidence. You lack the capacity to refute any of this evidence. However, none of this changes the facts about what rationality requires—rationality still requires that you avoid contradictory belief.Similar cases are possible for any other putative requirement of rationality. Epistemologists are in the business of making arguments for and against various requirements of rationality: requirements to believe/not believe lottery propositions,This simple explanation is ruled out by the Impossibility Thesis. As I argue in the following sections, neither of the ways of defending the Impossibility Thesis offers an adequate alternative explanation of situations like Logic 101.Perspectivism holds that what rationality requires depends on the agent’s evidential situation. For example, Bradley  avoids tdoes defeat the perceptual epistemic rule, but it is less clear that other, non-evidential, fundamental rules can be defeated so easily. Rational requirements are more fundamental than instrumental rules like ‘believe P if you have a perception that P’, and they need not be evidential.9Bradley focuses on epistemic rules such as ‘believe P if you have a perception that P’ and ‘believe P if you have testimony that P’. He argues that evidence that one of these rules does not apply in a particular case defeats that rule—it no longer applies in that case. Instead, what each agent is required to believe in a situation is specified by a complicated and indefeasible ‘Uber-rule’. However, an important shared feature of the rules that Bradley discusses is that they gain respectability from the more fundamental epistemic rule ‘believe what the evidence supports’. If this is right, then it is not surprising that evidence against the reliability of one’s perceptions can defeat the rule. The rule only ever had instrumental force—it was a good way of complying with the fundamental rule to believe what the evidence supports. Receiving evidence that trusting perception is not a way to believe what the evidence supports There are two options for interpreting how Bradley should respond to this point.If this is the position, then it is not as good an explanation as the Simple View of cases such as Logic 101. There are three key reasons for this. First, it amounts to an error theory about the more familiar rational requirements—requirements such as the Law of Non-Contradiction were never universally applicable requirements applying to all rational agents, as we might have thought they were. Logic 101 is not a case in which you are misled by evidence for a false view, but rather a case in which, in fact, you are required to believe a contradiction. On this view, requirements of rationality are flimsier than we might have thought. They are also not as good guides as we might have hoped. Bradley accepts this (2019: 14), but it is nevertheless a cost that must be justified by robust theoretical gains.12,Second, this view faces pressure to restrict what can count as rationally required in such a way that avoids collapsing into triviality, where any attitude at all can potentially be rationally required. It is unclear whether this can be done while maintaining commitment to the view that your evidential situation determines what rationality requires. This is particularly true if one shares Bradley’s view that something’s seeming to be evidence is sufficient for it to be evidence—for example, he expresses incredulity at the view that “what seems like evidence doesn’t even count as evidence” (2019: 6). However, what our evidence is and what our evidence seems to be are very different matters. Something may appear to be evidence from your perspective, even if it is not13Third, these awkward theoretical features are introduced unnecessarily. Bradley is right to point out that there is something positively evaluable about agents who have evidence that a rule does not apply and do not follow that rule. However, it does not follow from this that the rule ceases to apply to the agent in that case. Agents can break rules and be excused or even justified. Excuse and justification are positive evaluations in the sense that they remove blame that would have been deserved had the rule been broken without excuse or justification.Objectivism defends the Impossibility Thesis by making a claim about which evidential situations are possible. Specifically, it rules out the possibility of situations in which the total evidence supports false belief about what rationality requires.Assets: All agents, in all possible situations, have evidence that overwhelmingly supports the truth about what rationality requires.One way that some philosophers have attempted to rule out these situations is by appeal to the agent’s already having strong evidence supporting the truth about what rationality requires. For example, Smithies claims that “one always has a special kind of epistemic access to facts about which propositions one has justification to believe” : 273, anSmithies and Titelbaum take Assets to support the Impossibility Thesis, because they take it to rule out situations in which the agent has total evidence that supports a false belief about what rationality requires.One possible motivation for thinking that Assets is true is that it is a consequence of Bayesian commitments to logical omniscience. Logical omniscience says that for all agents in all situations, their evidence—whatever it is—supports all logical truths to credence 1. Some have thought that our a priori evidence is identical to that of the Bayesian ideally rational agent . One might think that because the question of what rationality requires is a priori, so long as agents have the ability to reflect, then they can easily acquire evidence for the truth about what rationality requires. While there are physical limitations on, for example, where I can transport my body to gather empirical evidence, there are no such limitations on what I can think about.15: 276. IndefeasibilitySufficiency: All agents, in all situations, have evidence for the truth about what rationality requires that is sufficient to rule out the possibility that their evidential situation requires false beliefs about what rationality requires.Even if true, Assets cannot establish the Impossibility Thesis alone. Assets says that we always have evidence that overwhelmingly supports the truth about what rationality requires. To get from this to the Impossibility Thesis, it must also be true that: this evidence is not defeasible by other features of the agent’s situation, and this evidence plays a meaningful role in determining what the agent ought to believe. In other words, Assets needs help from the following two claims:If all three of Assets, Indefeasibility, and Sufficiency were true, then the Impossibility Thesis would be true, because our total evidence would always require believing the truth about what rationality requires. All three claims are contentious. We have already seen some reasons to worry about Assets. As the following subsections outline, Indefeasibility and Sufficiency are also dubious. Even worse, this strategy rules out the simple explanation of cases of apparent misleading evidence about what rationality requires.indefeasible.22To get from the Assets claim to the Impossibility Thesis, our putative evidence for the truth about what rationality requires would need to be indefeasible. However, cases such as Logic 101 look like cases in which if there ever were any Assets, they are defeated. So, if we do have evidence that overwhelmingly supports true beliefs about what rationality requires, it is not clear that this evidence is For example, suppose, as some have thought, that we have default justification for basic laws of logic such as the Law of Non-Contradiction.According to standard accounts of defeat, justification can be defeated by both undermining and rebutting evidence, regardless of the strength of the justification Brown . So, notIf P is justified a priori, then it is justified independent of any experience.If justification is independent of experience, then it cannot be defeated by experience.The misleading evidence in the cases is derived from experience.So, misleading evidence cannot defeat our propositional justification for the truth about what rationality requires (indefeasibility).Indefeasibility does not simply fall out of the apriority of the Assets, as some have thought. A priori justification is, by definition, justified independent of experience. Some have taken this to imply that any misleading evidence one could acquire for a priori claims must be, in some sense, experiential. For example, Smithies makes the claim that “a priori justification for beliefs about logic has its source in logical facts, rather than psychological facts about experience, reasoning, or understanding.” (2015: 2270). Since misleading evidence about logic cannot be a ‘logical fact’, its source must, on this view, be a ‘psychological fact about experience, reasoning, or understanding’.However, both premises 2 and 3 are contentious. While you may have been justified in believing that rationality prohibits contradictions before you took Logic 101,27For premise 3 to be true, we need to assume a contentious understanding of ‘experience’, on which thinking through philosophical arguments is a (non-a priori) experience. On this contentious view, propositions rather than processes are a priori. Episodes of thinking that stray from logical and philosophical truth are not a priori, but rather experiential.Not only this, but this way of drawing the distinction means taking a disjunctive view of abstract thinking—particular instances of abstract thinking will have very different epistemic statuses depending on their truth value. When abstract reasoning results in truth, one is a priori justified. When it goes wrong, the reasoning was not a priori at all, merely empirical or experiential. Sturgeon argues against disjunctivism about visual experience : 182. HeMoreover, these points may turn out to be irrelevant. Theories of what rationality requires may not, in fact, be justified a priori. Anti-exceptionalist accounts of logic deny that logical theories are not justified a priori.31More importantly, Indefeasibility leaves us without a good explanation for cases in which our putative Assets appear to be defeated. Some have attempted to explain cases of apparent defeat using a distinction between defeat and some weaker defeat-like status. They have argued that although our Assets cannot be defeated, they can be ‘disabled’ Smithies , or the 32Views that make use of disabling typically recognise only two ways that a priori propositional justification can be disabled. Either the agent makes an error due to cognitive incapacity akin to Chomskian performance limitations or the agent has empirical evidence that she currently lacks the cognitive capacity to reason successfully. Limitation in cognitive processing capacity is an example of genuine cognitive incapacity. Some logical truths are too complicated to be deduced by ordinary human agents, even though they are entailed by their evidence, and propositions they already believe.well. Had you exercised them less well, you may not have followed the arguments, and so likely would have avoided acquiring evidence for the false belief about rationality. A possible line of response to this is that in Logic 101, your faculties are limited in one sense—your capacities for abstract philosophical reasoning are those of a beginner. Not only this, but you are being taught incorrect rules about how to reason rationally. There are, perhaps, many other good things to say about your reasoning capacities, but they are limited in some important respects. However, this response does not easily transfer to cases involving non-logical requirements of rationality, such as requirements to conciliate (or not) in response to peer disagreement. When you have false beliefs are about these, it is much less plausible that the mere fact that you endorse a false view compromises your general capacities for reasoning. If it did, this point would overgeneralise. It would also apply to experienced philosophers who hold false views about what rationality requires, but their reasoning capacities are clearly not limited.35However, this cannot explain cases like Logic 101. Here, your evidential situation supports the false belief about rationality because you are exercising your cognitive faculties really had (disabled) propositional justification for the truth, then the claim that an agent has propositional justification for P would be irrefutable, and ultimately trivial.One might reply that ideal agents would nevertheless have no false beliefs about what rationality requires. However, using the ideal to define what counts as a cognitive limitation risks generating a theory that is irrefutable. If every false belief about what rationality requires were an instance of limited capacity in which the agent evidence of cognitive incapacity explain the cases. This is the remaining option on offer for how Assets could be disabled, as in the following example:Coffee. Suppose that I work out my proof of T after having coffee with my friend Jocko. Palms sweaty with the excitement of logical progress, I check my work several times, and decide that the proof is good. But then a trusted colleague walks in and tells me that Jocko has been surreptitiously slipping a reason-distorting drug into people’s coffee—a drug whose effects include a strong propensity to reasoning errors in 99% of those who have been dosed (1% of the population happen to be immune). He tells me that those who have been impaired do not notice any difficulties with their own cognition—they just make mistakes; indeed, the only change most of them notice is unusually sweaty palms.  weight to evidence gathered using those capacities. Dealing properly with the evidence gathered from my capacities prevents me from forming justified beliefs based on my propositional justification for the logical facts. This is true for any other piece of logical reasoning I might undertake while I have evidence that I am under the influence of the drug. In contrast, in Logic 101, your evidence directly concerns what rationality requires. Responding appropriately to it means weighing it in your deliberation about the truth of what rationality requires, against other evidence that you have about this topic. This is quite different. This suggests that Logic 101 is not a case of disabling via evidence of cognitive capacity.So, Logic 101 does not seem to be a case of disabling. However, we might think there is another explanation of it available to Objectivists, namely that your evidence does not support the false belief about what rationality requires because the very process of weighing up what your evidence supports relies upon classical reasoning principles, and by extension the Law of Non-Contradiction. One might worry that this dependence undermines the evidence that you seem to have for the false belief that rationality sometimes requires contradictory belief.themselves count as part of our evidence. This is uncontroversial in other domains – compare our beliefs about the physics of middle sized objects. I believe that if I drop my coffee mug, it will fall to the ground. My evidence for this consists in past observations of dropping mugs, and some elementary and incomplete understanding of gravitational laws. However, its truth is underpinned by various more complicated principles of physics that I have never considered and would not understand if I did. These more complicated principles of physics do not form part of my evidence, but we might think that my reasoning about the physics of middle-sized objects implicates them in an analogous way that your reasoning in Logic 101 implicates principles of classical logic. However, my evidence about whether mugs will fall does not support the complicated principles of physics, and I do not have evidence that supports believing these principles—at least not unless I learn much more about physics. So, even if it is true that ordinary reasoning about evidence is underpinned by principles of classical logic, these principles are not part of your evidence in Logic 101, at least in the absence of further arguments, evidence, and understanding of logic. This is true even assuming that you exhibit tendencies to avoid contradictory belief. Once you have fully absorbed the arguments you study for dialetheism, you have evidence that this tendency is only a tendency and not supported by any requirement of rationality.It is worth pointing out that this explanation, even if correct, would only rule out cases of misleading evidence about requirements of rationality that are sufficiently close to principles of logic. It will not apply to evidence about requirements to conciliate in response to peer disagreement, or avoid believing lottery propositions. There is also some distance between the claim that one’s evidence supports or relies upon some logical principle, and the claim that it supports or relies on a requirement of rationality based on that logical principle. We cannot assume a straightforward relationship between the truths of logic and truths about what rationality requires. For example, while (P & (P → Q)) → Q) is a logical truth, it is not a demand of rationality that if one believes P, and believes that , then one also believes Q. Furthermore, even if there were a straightforward relationship between these, it is not clear that this would tip the evidential scales in favour of believing the true rational requirements, because it is not clear that the logical or rational principles we depend upon to acquire evidence So, Indefeasibility does not seem to be true. Moreover, the explanations of cases like Logic 101 that are compatible with Assets are inadequate. We should thus prefer the Simple View - evidential situations supporting false belief are possible about all topics, including what rationality requires. However, even if we did not prefer the Simple View, there is a further problem with Assets—it needs Sufficiency, and Sufficiency is false.Sufficiency: All agents, in all situations, have evidence for the truth about what rationality requires that is sufficient to rule out the possibility that their evidential situation requires false beliefs about what rationality requires.In addition to Indefeasibility, defending the Impossibility Thesis via Assets requires Sufficiency.strength of evidence. It says that the evidence postulated by Assets is maximally strong, such that it cannot be defeated. Sufficiency is a claim about how evidence, whatever its strength, can be used in deliberation. It says that the evidence postulated by Assets rules out other beliefs. The claims may appear similar because when indefeasible evidence for P does bear on what S ought to believe, it will typically rule out beliefs that not-P. However, Indefeasibility does not by itself imply the ability to rule out beliefs to the contrary. That the evidence bears on what S ought to believe is a claim strictly independent of Indefeasibility. Without Sufficiency, even if it were true that all agents have evidence supporting the truth about what rationality requires, and that this evidence was indefeasible, further argument would be required to establish how and why our having this evidence rules out the possibility of one’s total evidence supporting false beliefs about rationality, as the Impossibility Thesis claims.Sufficiency is importantly distinct from Indefeasibility. Indefeasibility is a claim about the For evidence to be able to rule out the possibility of one’s situation requiring false belief about what rationality requires, it would need to play a role in determining what the agent ought to believe. However, it does not seem that it does.48 An agent can reflect on her situation and come to recognize facts about what that situation rationally requires. Not only can this reflection justify her in believing those facts; the resulting justification is also empirically indefeasible (2015: 276).How is the justificatory map arranged such that one is never all-things-considered justified in both an attitude A and the belief that A is rationally forbidden in one’s current situation? The most obvious answer is that every agent possesses a priori, propositional justification for true beliefs about the requirements of rationality in her current situation.Smithies qualifies Assets as follows:[Assets] is not a doxastic thesis about which of one’s beliefs are justified, but is rather an epistemic thesis about which propositions one has justification to believe. Justified belief requires not only having justification to believe a proposition, but also using it in believing that proposition on the basis of one’s justification to believe it. By contrast, having justification to believe a proposition does not require using it in forming a justified belief. As we shall see, this distinction between epistemic and doxastic versions of the accessibility thesis is crucial for avoiding the problems of over-intellectualization and vicious regress (2012: 276).Restricting Assets to a claim about propositional justification can seem to remove some of the pressure to accommodate or explain cases like Logic 101. The idea is that in such cases, the agent is propositionally justified in the truth in spite of their misleading evidential situation. This can then be used to deny the claim that agents in such cases would be rational to believe falsely about rationality, and so avoid the problem of inconsistent requirements. However, this claim is silent on what agents are doxastically justified in believing, and this silence threatens the legitimacy of the restriction.The problem is that the justification that Assets provide is propositional, but not necessarily doxastic. Assets say that justification for the requirements of rationality is available to all rational agents in all situations, but this implies only that were they to reflect properly, then they would be rational to believe the truth about what rationality requires. This is a point on which Objectivist theories that postulate Assets agree. Here is Titelbaum again:P is propositionally justified for S.S believes P.S believes P on the basis of her propositional justification.There are three options for what you are doxastically justified in believing in Logic 101. Perhaps the most natural option is that you are doxastically justified in believing falsely about what rationality requires. This, after all, is what your evidence appears to support. However, if this is right, then it is less plausible that you are propositionally justified in believing the truth about what rationality requires. Traditionally, P is propositionally justified for S if and only if S has justifiers J sufficient to justify P.On this view, doxastic justification implies propositional justification. So, if you are doxastically justified in believing some false claim about what rationality requires, P, and you are propositionally justified in believing the true claim about rationality, not-P, then it is not true that the evidence postulated in Assets rules out evidential situations that require false beliefs about what rationality requires.This seems to be the situation in Logic 101. The proposition that rationality sometimes requires contradictory belief is supported by justifiers that you have—the evidence you acquire by taking Logic 101, albeit not to a maximal degree. So, in Logic 101, you meet all of (a)–(c). You believe it, you believe it on the basis of justifiers (i.e. evidence), and your justifiers support the truth of the claim. So, given the ‘orthodox view’ of the relationship between doxastic and propositional justification, you are both doxastically and propositionally justified in the false belief about what rationality requires. On this first option, Sufficiency is false. A second option is that you are doxastically justified in believing the truth about what rationality requires. This option is compatible with Sufficiency, but implausible. Even assuming you have propositional justification for the truth, you also have apparent defeaters for the true propositions, meaning that you lack a sound deliberative route from that justification to justified beliefs based on that justification.A third option, also compatible with Sufficiency and also implausible, is that you are doxastically justified in suspending judgement on the matter of what rationality requires. Suspension would be appropriate if it was clear to you that you had roughly equal amounts of evidence on either side—but it is not. You have what seems like strong evidence for a false belief about what is required, and propositional justification that you are unable to make use of. This does not add up to roughly equal evidence on either side.A final option, perhaps, is that you cannot be doxastically justified in believing anything in the neighbourhood of what rationality requires. However, this is just a restatement of the Impossibility Thesis. So, the only plausible option seems to be the first one, but if this is right then Sufficiency is false.Even worse, regardless of which option is correct, the propositional justification that Assets says we have plays very little role in determining what you should believe, given your situation. This suggests that Sufficiency is false—Assets cannot rule out evidential situations that require false beliefs in what rationality requires - they are inert. We can illustrate the inertia of Assets by comparing agents in good and bad epistemic situations, but who, according to Assets, have the same propositional justification. Consider a student, Anita, who takes Logic 101, and receives the same misleading evidence as you. Anita studies well, follows the arguments where they lead, and, like you, acquires the false belief that rationality requires contradictory belief. Compare another student, Bertha, who takes a different class. Bertha is luckier— her class teaches the true theory of what rationality requires. She also studies well and follows the arguments where they lead. She acquires true beliefs about what rationality requires.Assets says that both Anita and Bertha have the same propositional justification about what rationality requires. If this is true, it does not seem to make much of a difference to their epistemic situations. Their propositional justification, in so far as they have it, is epistemically inert. Anita’s alleged propositional justification for the truth about what rationality requires is of no help to her deliberation—she lacks a viable deliberative route to doxastically justified belief in this true view. To reason her way out of her situation and towards the truth, Anita would need to rationally dismiss the misleading evidence for the false view. It is not reasonable to expect her to do this. Anita’s indefeasible propositional justification plays no role in determining what she should believe, given her evidence. It is inert. Nor is Bertha’s propositional justification of any help to her. Bertha studies the true theory of what rationality requires. Assets says that Bertha also has indefeasible propositional justification for the truth about what rationality requires. She also believes the truth about what rationality requires. However, it is unlikely that her belief is based on the evidence that Assets postulates. This evidence, we are told, supports the truth about what rationality requires to a maximal degree, in a way that is immune from the arguments of dialetheists. It is not clear that this would be comprehensible by a beginner. So, even though Bertha believes what is supported by her propositional justification, it is not clear that she does so on the basis of it. So, Assets would seem to be inert even in the good cases—even here they are irrelevant to what the agent ought to believe given her evidential situation.Rejecting the Impossibility Thesis incurs the burden of tackling head on the problem of inconsistent requirements . In this section, I briefly mention three avenues for resolving the inconsistency without resorting to the Impossibility Thesis.One option is to simply accept the inconsistency as an epistemic dilemma.Alternatively, we might see the possibility of situations such as Logic 101 as support for denying that rationality requires enkratic coherence (see Lasonen-Aarnio The Simple View says that cases of apparently misleading evidence about what rationality requires are just ordinary cases of one’s evidence supporting a false belief - evidence can support false belief about any topic, including what rationality requires. The Impossibility Thesis avoids the problem of inconsistent requirements, but comes with its own problems. I argued here that we should reject the Impossibility Thesis. I argued that the routes for defending it face serious difficulties, and neither can offer as satisfactory an explanation of cases like Logic 101 as the Simple View. While his means that the problem of inconsistent requirements remains, there are reasons to think it is manageable.


Sir,—The paper by Laaksonen et al. (Acta Orthopaedica shows practice variation and possible overtreatment for a common orthopedic presentation: completely displaced (including overriding) distal radius fractures in children. While there is a considerable “grey zone” in treating these fractures, based on factors such as the age and sex of the child, and the proximity of the fracture to the wrist, there should be better agreement than has been demonstrated in this paper, and what many of us see in clinical practice.n et al.  in this We believe that the results presented by Laaksonen and colleagues raise several important questions about orthopedic practice, variation therein, and the management of these fractures in particular. Questions such as: “What is the cause of the variation?,” “Is better quality evidence for benefit and harms needed for the different treatments?,” and “How can the practice be brought in line with what is best for the patient?”Practice variation occurs where clear guidance is not provided by high-quality evidence. Sometimes practice variation occurs even when high-quality evidence exists .The findings of Laaksonen and colleagues should provide a stimulus for low-risk-of-bias comparative studies in this area so that practice can be narrowed to a range that targets the best patient outcomes with the least harm and cost. One such trial was planned and a protocol published (Adrian et al. We call on surgeons to question their current practice, to participate in planned trials, and to be open to new evidence as it is presented. Evidence-based surgery differs from non-evidence-based surgery in that the former necessitates that judgments are consistent with underlying evidence, while the latter does not.1 and Ian A HARRIS 21 Department of Clinical Sciences Lund, Orthopaedics, Lund University, Sweden.2 South Western Sydney Clinical School, University of New South Wales, and Department of Orthopaedic Surgery, Liverpool Hospital, Liverpool, NSW, Australia.Email:stefan.lohmander@med.lu.seEmail:iaharris1@gmail.comL Stefan LOHMANDER


The aim of this study is to discuss the available methodological resources and best‐practice guidelines for the development and completion of scoping reviews relevant to nursing and midwifery policy, practice, and research.Discussion Paper.Scoping reviews that exemplify best practice are explored with reference to the recently updated JBI scoping review guide (2020) and the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses Scoping Review extension (PRISMA‐ScR).Scoping reviews are an increasingly common form of evidence synthesis. They are used to address broad research questions and to map evidence from a variety of sources. Scoping reviews are a useful form of evidence synthesis for those in nursing and midwifery and present opportunities for researchers to review a broad array of evidence and resources. However, scoping reviews still need to be conducted with rigour and transparency.This study provides guidance and advice for researchers and clinicians who are preparing to undertake an evidence synthesis and are considering a scoping review methodology in the field of nursing and midwifery.With the increasing popularity of scoping reviews, criticism of the rigour, transparency, and appropriateness of the methodology have been raised across multiple academic and clinical disciplines, including nursing and midwifery. This discussion paper provides a unique contribution by discussing each component of a scoping review, including: developing research questions and objectives; protocol development; developing eligibility criteria and the planned search approach; searching and selecting the evidence; extracting and analysing evidence; presenting results; and summarizing the evidence specifically for the fields of nursing and midwifery. Considerations for when to select this methodology and how to prepare a review for publication are also discussed. This approach is applied to the disciplines of nursing and midwifery to assist nursing and/or midwifery students, clinicians, researchers, and academics. Foundational concepts and evidence can be mapped, allowing for examination of practice, policy, and research and gaps in evidence and policy can be identified. The results of scoping reviews can provide indications for where further research may be required and inform the development of these research endeavours , co‐authors for the study selection and extraction process, software to support the process (such as SUMARI and/or reference management software)  and Web of Science (webofknowledge.com), but not currently through PROSPERO. Examples of protocol templates can be found on JBI SUMARI  and question(s). The PCC framework used for developing the research objective(s) and question(s) will also inform inclusion and exclusion criteria and consequently the literature search strategy , may be relevant. The PsycInfo database can also be useful for questions which combine nursing/midwifery practice with mental health, psychological, and social science concepts, for example, identifying the range of tools to measure behavioural aspects of the nurse–patient relationship . Searching for grey literature can be difficult as it is not necessarily organized or indexed like peer‐reviewed articles in academic databases. Balancing the sensitivity and specificity of the search with resource limitations, particularly time restrictions, is challenging. If a grey literature search is conducted, it is necessary to determine and justify the extent of the search in the protocol and finalized scoping review.Aromataris and Riitano  provide Developing and implementing the search strategy should occur in three stages and in collaboration with a research librarian. These stages include:Initial search: Search for articles relating to the review topic in at least two relevant databases and identify words and phrases found in the title, abstract, and index of papers that would likely be included in the review to inform your final search strategySecond search: Using the identified search terms, formally conduct a search in the selected databases, and grey literature locations. Document these searches for inclusion in the final PRISMA flow chart  all the identified studies from the initial search (consider time restrictions), (b) studies included from full‐text review, or (c) studies included in the review. It can also be useful to scan the reference list of related reviews identified in the search. To evaluate the search strategy, the Peer Review of Electronic Search Strategies (PRESS) is a checklist developed by librarians is a useful tool  and objective(s) been addressed? If all elements of the review question and objectives have been met in the results section, the discussion section can focus on the extent of evidence available and place this evidence into context. Given the nature of scoping reviews, there are circumstances where review questions and objectives may not be addressed due to insufficient literature. If this is the case, the discussion section provides an opportunity to discuss gaps in knowledge, new hypotheses, and considerations for future research.Firstly, has the review question been addressed accurately? The discussion section provides an opportunity to demonstrate the alignment of review results with review questions and objectives. When conducting a scoping review, the information that is located can highlight new avenues of inquiry. It can be tempting to discuss these tangential subjects in the discussion section, without re‐focussing on the primary aim of the review. Ensuring alignment among the review question, objectives, results, and discussion will strengthen the integrity of the review.Secondly, has the paper been adequately situated within the context of the relevant field of literature, practice and/or policy? A good discussion section will highlight the contribution the review has made to the relevant field through reflecting on what has preceded the review and projecting the potential implications for future investigation and planning. The purpose of many scoping reviews is to describe the nature and diversity of available evidence  guidance and the PRISMA‐ScR  , provides a useful decision‐making tool. Further resources include the JBI Scoping Review Working Group  and their newly updated JBI reviewer's manual . Another resource, which includes video presentations for the individual steps of a scoping review, is the UniSA Scoping Review website . Further information and resources about the PRISMA‐ScR can be found here: https://knowledgetranslation.net/portfolios/the‐prisma‐scr2/ (accessed 12 December 2020).Resources are available to assist with planning and developing scoping reviews. If there are challenges with deciding what type of review to undertake, the website: 5Scoping reviews are a valuable form of evidence synthesis. The scoping review approach to evidence synthesis is increasingly being adopted by nurses and midwives who are seeking to map evidence and describe relevant literature. As the methodology for undertaking a scoping review advances and becomes more refined, it is important for nurses and midwives to be using the most current and appropriate guidelines, particularly if wanting to publish results or to use the results to inform future research. This study provides an overview of best practice and current guidelines for nursing and midwifery students, academics, and clinicians who are considering undertaking a scoping review. Examples and advice are offered to assist with the appropriate adoption of this methodology and the distribution of results to the broader community.6The scoping review methodology presents nursing and midwifery academics and clinicians with a valuable and adaptable opportunity to synthesize evidence. This approach to evidence synthesis has certainly grown in popularity in these professions and will no doubt continue to be used in the future. As this type of review continues to be adopted, it is vital that they are conducted rigorously and in accordance with the latest methodological recommendations. The process for how to perform a scoping review from inception to publication has been outlined in this study with a goal of facilitating conceptual clarity for nursing and midwifery academics, clinicians, and policymakers who are undertaking a scoping review.https://publons.com/publon/10.1111/jan.14743.The peer review history for this article is available at


Electronic health literacy skills and competences are important for empowering people to have an active role in making appropriate health care decisions. The aims of this cross-sectional study were to (1) examine the frequency of use of the Internet for seeking online information about chronic pain, (2) determine the level of eHealth literacy skills in the study sample, (3) identify the factors most closely associated with higher levels of eHealth literacy, and (4) examine self-efficacy as a potential mediator of the association between eHealth literacy and measures of pain and function in a sample of adults with chronic pain. One-hundred and sixty-one adults with chronic pain completed measures assessing internet use, eHealth literacy, pain interference, anxiety, depression, and pain-related self-efficacy. Results indicated that 70% of the participants are active users of the Internet for seeking information related to their health. The level of eHealth literacy skills was not statistically significantly associated with participants’ age or pain interference but was significantly negatively associated with both anxiety and depression. In addition, the findings showed that self-efficacy fully explained the relationship between eHealth literacy and depression and partially explained the relationship between eHealth literacy and anxiety. Self-efficacy should be considered as a treatment target in eHealth literacy interventions, due to its role in explaining the potential benefits of eHealth literacy.  Chronic pain is a common health condition worldwide, associated with financial, physical, and emotional burdens ,2,3,4. ISpecific knowledge domains addressed by pain education include information about the possible causes of pain, treatment options, effective self-management strategies, and prognosis . PatientElectronic health literacy  is a relatively new construct that extends the study of the traditional health literacy to encompass health literacy as it relates to the Internet. Specifically, eHealth literacy has been defined by Norman and Skinner as “…the ability to seek, find, understand, and appraise health information from electronic sources and apply the knowledge gained to addressing or solving a health problem”  (p. 2). Although a considerable amount of research has examined traditional health literacy in individuals with chronic pain ,21,22,23Research has also shown that having higher levels of eHealth literacy skills is associated with better health outcomes, as indicated by greater medication adherence, higher levels of quality of life and psychosocial well-being, and the adoption of adaptive health behaviors ,30,31,32Given these considerations, the current study had four primary aims. First, we sought to examine the frequency of use of the Internet for seeking online information about chronic pain. Second, we wanted to better understand the association between eHealth literacy skills related to pain  and measures of pain and function in a sample of adults with chronic pain. Third, we sought to identify the factors most closely associated with higher levels of eHealth literacy in the study sample. Finally, we sought to test pain-related self-efficacy as a possible mediator of the associations between eHealth literacy and measures of function. On the basis of the research published to date, we hypothesized that seeking health information online would be common in a substantial subset of participants . We alsStudy participants were recruited from the general population through sending an invitation via associations of patients or groups of patients with chronic pain in social networks. For individuals to be considered as potential participants, they had to (1) be aged 18 years old or older, (2) report having a pain problem of at least three months’ duration, (3) be able to understand Spanish, and (4) have access to an electronic device connected to the Internet to be able to respond to the online survey. 2 = 0.15; α = 0.05 at 2-tailed; power = 0.95; two predictors).Sample size estimation was calculated using G*Power . The reshttps://www.limesurvey.org/es/, accessed on 24 November 2021) that included all the variables and instruments of interest for this study. The survey was made available during the months of November 2019 through January 2020. A short description of the study, which included a link for contacting research staff if a potential participant was interested in participating, was shared via social networks mainly through the profiles of associations of patients. We also encouraged individuals to share the study information through their own social networks to reach a wider audience. Once individuals clicked on the survey link, they could read additional details about the objectives and procedures of the study. The description of the study provided information about the study’s aims. Specifically, participants were told that the study aimed to examine what people do, feel, or think when they have pain. They were also informed that participation in this study was anonymous and voluntary. After providing their consent to participate, they were able to respond to the survey questions. Participants did not receive any compensation for completing the survey. As no follow-up was planned, we decided to collect responses anonymously. On average, participants spent 17 min to respond to the survey. Participants were requested to respond to each question in the survey. Responses from 18 individuals were excluded from the planned analyses due to their failing to respond to all questions (the completion rate in this study was 90%). A cross-sectional study was conducted to address the objectives of the study. We created an online survey using the LimeSurvey program . Health Literacy in an Electronic Context: We used a modified version of the 8-item eHealth Literacy Scale (eHEALS)  to assesAnxiety and depression symptom severity: The Hospital Anxiety and Depression Scale (HADS) originally developed by Zigmond and Snaith  was usedPain self-efficacy: We used the 10-item Pain Self-Efficacy Questionnaire (PSEQ)  to measuPain interference with daily activities: The Pain Interference Scale of the Brief Pain Inventory (BPI)  was usedhttps://www.processmacro.org, accessed on 24 November 2021). For bootstrap, 5000 samples were computed. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) for Windows Version 23.We first computed the means and standard deviations (continuous variables), as well as number and percentages  of the demographic and study variables to describe the sample and address the first two study aims . We computed a Pearson correlation coefficient between participants’ age and scores on the eHEALS to test the hypotheses that these variables would be positively associated. We then computed Pearson correlation coefficients between the eHealth literacy score and the measures of pain interference, depression, and anxiety symptoms to test the third hypothesis, with the plan to only proceed with a formal mediational analysis if these associations were statistically significant . Finally, we performed mediation analyses to test the hypothesized mediating role of pain-related self-efficacy between eHealth literacy (independent variable) and criterion variables using the PROCESS macro version 3.4 for SPSS developed by Hayes , ranging in age from 24 to 68 years, with a mean age of 44.63 years (SD = 9.55). The most common pain sites were the lower back , the shoulder, and the upper limbs . Participants consisted of a sample of 161 adults with chronic pain problems who were recruited from the general population. The overwhelming majority of participants were women (One-hundred and thirteen (70%) participants in our study reported that they used the Internet “almost always” or “always” for seeking information related to their chronic pain problem , almost never = 3 (2%), sometimes = 44 (27%), almost always = 38 (24%), and always = 75 (46%) of the total participants). The mean score on the eHEALS scale for the whole sample was 29.53  and 42 (26%) of the participants had scores below 26, indicating low levels of eHealth literacy skills . p = 0.845). In addition, the measure of eHealth literacy was not statistically significantly associated with pain interference . However, it was significantly associated with both anxiety and depression . We therefore proceeded to evaluate the extent to which general self-efficacy mediated the association between eHealth literacy and anxiety and depression symptoms. No statistically significant association was found between participants’ age and the total score of the eHEALS scale . The data indicated a direct and negative association between pain-related self-efficacy and depressive symptoms . Moreover, the relationship between eHealth literacy and depression is fully explained by self-efficacy . Bootstrapping using confidence intervals not including zero confirmed that the mediating role of pain self-efficacy was statistically significant .p < 0.001). Unlike the previous models, the effect of eHealth literacy on the criterion variable  was found to be statistically significant . In this case, the findings support partial mediation for pain-related self-efficacy on the relationship between the level of eHealth literacy and anxiety, that is, the indirect effect  explains part of the relationship between eHealth literacy and anxiety. Bootstrapping method with confidence intervals not including zero value confirms that the mediating role of pain self-efficacy was statistically significant .A summary of the results of the mediational analyses when examining the role of pain-related self-efficacy as a mediator on the relation between eHealth literacy and anxiety symptoms is shown in This study contributes knowledge about the eHealth literacy in individuals with chronic pain, as well as on the association between chronic pain-related eHealth literacy and function in these individuals. Consistent with the study hypothesis, a large number of participants in this sample (70%) were active in seeking information related to their chronic pain condition on the Internet. This percentage is considerably higher than that found by other authors  and it mMost of the participants in this study (74%) obtained a score of 26 or higher on the eHEALS scale, indicating high levels of eHealth literacy skills on the basis of the cutoff used in previous studies ,39. In oThe results did not support the hypothesis that participants’ age would be negatively associated with higher eHealth literacy skills. Although the majority of previous studies have found that being younger is associated with a higher level of eHealth literacy ,26,27,28On the other hand, the hypothesis that eHealth literacy would be associated with health outcomes was partially supported. eHealth literacy was significantly and negatively associated with anxiety and depression but was not significantly associated with pain interference. Finally, the data partially supported the hypothesis that self-efficacy mediated the association between eHealth literacy and patient function . As noted in the Introduction section, self-efficacy has been identified as a mediator of the association between eHealth literacy and health status both in clinical and community samples e.g., ,58). Mor. Mor58])The results of this study have important research and clinical implications. First, this study contributes to a better understanding of the relationship between eHealth literacy and psychological health status in a sample of adults with chronic pain, a target population in which these associations have not yet been thoroughly explored. In addition, the direct associations found between measures of electronic literacy and psychological function highlight the potential importance of having adequate electronic literacy skills. This finding is consistent with the results of others that show that higher electronic literacy skills are more likely to be associated with better health-related outcomes . With respect to the clinical implications of the study findings, eHealth literacy encompasses a set of abilities that can be learned. The current studies suggest that eHealth literacy interventions should emphasize increases in self-efficacy as a component of intervention, in order to maximize the potential benefits of the intervention on psychological function. Research to evaluate the efficacy of treatments that could enhance eHealth literacy, as well as to enhance self-efficacy for using the Internet in adaptive ways to better manage pain, is warranted. Although some eHealth literacy interventions have demonstrated benefits for patients and community samples, more efforts are required to develop these interventions in ways that are informed by eHealth literacy conceptual models . This isA number of limitations of this study should be considered when interpreting the results. First, the sample was composed by adults with chronic pain problems recruited from patients’ associations with an active role in social networks who were responding to an online survey. Thus, the extent to which they generalize to other adults with chronic pain who would not be interested or willing to participate in a study such as this one, or to adults with chronic pain seen in clinics and health care centers , is not known. Future studies should be conducted with other samples of individuals with chronic pain to help determine the generalizability of the findings. Second, and also related to the characteristics of the sample, almost all the participants in our study were females. As a result, we were not able to examine gender-related differences in the variables studied. Research is needed to study eHealth literacy in more balanced samples and looking at other variables that could help explain the quality of the experience in using the Internet for health-related purposes among individuals with chronic pain. For example, it would have been interesting to include some additional measures examining the attitudes and experience in the use of electronic sources for seeking health-related information, beyond the frequency in which they do that. Further research needs also to examine the role of the eHealth literacy skills as moderators between sociodemographic variables  and health care habits and outcomes. This study provides new information about eHealth literacy and its association with psychosocial variables in individuals with chronic pain. The data showed that seeking information about health is a common practice. However, contrary to what was hypothesized, participants’ age was not significantly associated with eHealth literacy. In addition, the findings of this study showed the potential role of this literacy on emotional symptoms and the role of self-efficacy as a mediator between eHealth literacy and function in adults with chronic pain. Future efforts should be focused on the development and assessment of effective educational programs which enhance electronic health literacy in individuals with chronic health conditions as well as for the population from the community. Further studies examining the association between eHealth literacy and function and its mediators are also warranted.


There is a growing interest in the named “acidic sterolbiome” and in the genetic potential of the gut microbiome (GM) to modify bile acid (BA) structure. Indeed, the qualitative composition of BAs in feces correlates with the bowel microorganisms and their collective genetic material. GM is responsible for the production of BA metabolites, such as secondary and oxo-BAs. The specific BA profiles, as microbiome-host co-metabolic products, could be useful to investigate the GM-host interaction in animals under physiological conditions, as well as in specific diseases. In this context, we developed and validated an ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry method for the simultaneous analysis of up to 21 oxo-BAs and their 9 metabolic precursors. Chromatographic separation was achieved in 7 min with adequate analytical performance in terms of selectivity, sensitivity (LOQ from 0.05 to 0.1 µg/mL), accuracy (bias% < 5%), precision (CV% < 5%) and matrix effect (ME% < 10%). A fast solvent extraction protocol has been fine-tuned, achieving recoveries > 90%. In parallel, the gut microbiota assessment in farming animals was evaluated by 16S rRNA next-generation sequencing, and the correlation with the BA composition was performed by multivariate analysis, allowing to reconstruct species-specific associations between the BA profile and specific GM components. They are synthetized by the liver and once conjugated with glycine/taurine secreted in bile into the proximal intestine. A major portion of conjugated BAs, is selectively and efficiently actively absorbed in the terminal ileum; the unconjugated fraction is passively absorbed along the entire intestinal tract. All the absorbed BAs reach the liver via the portal vein, where they are taken up by the liver and partial spillover in the systemic blood take place. Therefore, BAs are recycled in the enterohepatic circulation, where they exert their physiological function in bile to facilitate lipid absorption via mixed micelles thanks to their detergency. The dynamic of their recycling is driven by the gallbladder (if present) and intestinal motility2. In addition, as already demonstrated, they are ligands of nuclear receptors, such as farnesoid X receptor (FXR), largely expressed in the liver and intestine, which is involved in several biological pathways, including BA own synthesis. The finding of their “hormone activity” at the end of the twentieth century has elicited an increasing interest in the BAs family, especially for their potential use as drugs in several hepato-biliary diseases. For example, semisynthetic Obeticholic acid is recognized as the most potent (FXR) agonist and is used as a drug to treat primary biliary cholangitis, and it is undergoing development for deployment in further liver diseases and related disorders2, such as nonalcoholic steatohepatitis (NASH).Bile acids (BAs) are the end products of cholesterol catabolism in more than 650 vertebrate species3. BA chemical structures vary in three main ways: (a) structure of the side chain, which determines the BA class; (b) stereochemistry of the A/B juncture [cis (5b) or trans (5a)]; and (c) hydroxylation pattern  of the nucleus and/or side chain.No other class of small molecules in vertebrates exhibits the variety of chemical structures shown by BAs, and the identification of new bile alcohols and BA derivatives continues to date4. For example, human FXR has a curved binding pocket best suited for the bent steroid ring structure typical of evolutionarily more recent BAs. Several isoforms of FXR have been determined in mice and are differenty expressed in liver, intestine, kidney, adrenal gland, stomach, fat, and heart, where they differentially regulate gene expression in numerous tissues in vivo5.This huge chemical diversity is also related to the fact that the ligand binding pocket of the BA receptors has changed across species during the evolution process of vertebrates6. Several papers have reported the BAs composition in numerous animals, specifically in bile and blood samples7. The study of BA variations across vertebrates, has highlighted a clear evidence of the evolutionary transition from C27 ancestral bile alcohols to C24 BAs, with an intermediate phase in some animal classes where C27 BAs are present.Each vertebrate therefore presents a different pattern of cholesterol metabolites, as the result of evolutionary changesThe complexity of the BAs family, named the “acidic steroid profile”, is also due to the interaction with the intestinal microbiome and the resulting microbiome-host co-metabolic network with species-specific declinations. Indeed, it is well known that a small percentage of BAs not absorbed by the intestine across the ileal-caecum valve, enters the colon, where in the presence of the bacterial microflora is extensively metabolized. Bacteria bile salt Hydrolase (BSH) is the main metabolic pathway in gut which is responsible for the cleavage of the amide bond with glycine and /or taurine to produce unconjugated BAs. Metabolites are partially reabsorbed by the colon, and the remaining metabolites are excreted in the stools. The amount of excreted BAs is balanced by the BAs of nova synthesis from cholesterol in the liver by a feedback mechanism, thus ensuring the steady-state BA pool size and acting as a way to control cholesterol excretion.9. In particular, different strains of bacteria can metabolize steroid compounds during their intestinal transit, leading to the formation of a number of BAs derivatives that can therefore have different activities towards receptors as the major transcriptional regulator of BAs synthesis FXR8. Indeed, the bacteriostatic effect of BAs on the colon content is known, but the specific BAs structure action is not yet well defined, and the bacterial specificity is still poorly understood.In this process, since different host species possess a species-specific pattern of gut microbial components with an interspecies gut microbiome diversity matching the host phylogenetic distance, the species-specific gut microbiome structural and functional profile combines with the host genome in the definition of the “acidic sterolbiome” of a peculiar vertebrate host9. The cholic acid (CA) structure and one of its potential metabolic derivatives, (5β)-3,7,12-trioxocholan-24-oic acid, are reported in Fig. Among gut microbiome metabolites, keto (or oxo) BAs produced by steroid dehydrogenase enzyme expressed by microbiota, mainly 3,7 and 12 Hydroxysteroid dehydrogenases (HSD), have been onlyanalyzed in human faeces10.There are low concentrations of these species in human blood due to the reduction step of the oxo groups by liver after their hepatic uptake. On the other hand, high levels of these molecules in the entire intestinal tract have been determined. Moreover, high fecal concentrations of several oxo bile acids (oxo-BAs) have been highlighted by HPLC–ESI–MS methods, specifically oxo-derivatives of the secondary BA deoxycholic acid (DCA). Indeed, such hixi-derivatives cover a significant fraction of fecal BAs, approximately 30% of the total pool11. In addition, considering their nature as microbiome-host co-metabolic products, more detailed knowledge of the fecal acidic steroid profile could represent a valuable tool to study gut microbiota-host interactions in vertebrates under physiological and pathological conditions12, as well as to establish fingerprinting biomarkers useful for feces characterization . In fact, thanks to their high stability and specificity, BAs and oxo-BA metabolites are promising biomarkers for the identification of human fecal inputs, giving a significant contribution to the growing field of bioarchaeology10.These oxo BA derivatives have never been properly evaluated in either human or animal feces. The lack of information about oxo-BAs in vertebrate feces hampers the evaluation of their physiological role, for example their activity as endogenous ligands for receptors or as bacteriostatic agents14. Here, we aimed to investigate the BAs profile mainly in livestock animal feces to develop new tools potentially applicable to future studies on farm animal welfare, based on the evidence that BAs are powerful species-specific markers related to microbiota and disease conditions in these species. For example, the identification of negative effects of intensive farming and specific feeds, as well as displacement of physiological condition in animals, could be better achieved with higher capillarity. Indeed, several papers suggest controversial or poor specific effects on the BA profile by diet in vertebrates8, but oxo-BAs and their strict interaction with the host gut microbiota have been actually overlooked so far.High resolution mass spectrometry is definitely the most powerful and comprehensive technique to investigate metabolic profiles due to its extreme selectivity. The coupling of mass spectrometry with chromatographic techniques of the latest generation has allowed in recent years to study and identify new metabolic pathways, and their products, on molecules of endogenous and exogenous origin15. The analysis of BAs in biological liquids requires the use of highly selective analytical techniques, due to the complexity of either their profile and the sample matrix. Indeed, due to the multiplicity of the BAs structures  method for the analysis of 30 compounds, 21 oxo-BAs and their metabolic precursors, in 10 vertebrates. This UPLC method permits faster identification and quantification with high selectivity thanks to the use of new-generation high-resolution mass spectrometry (HR-MS). The UPLC-MS method was fully validated to achieve high analytical performances in terms of reproducibility, accuracy, selectivity, and sensitivity in fecal samples.16, which is reflected in the overall bulk composition of the stool samples. Furthermore, a systematic evaluation of the MS/MS spectra was carried out to identify characteristic fragmentation patterns. Specific fragments and their different relative abundances can in fact be diriment in the discrimination of isobaric compounds with similar chromatographic behaviour.The extraction procedure and percentage BAs recovery were evaluated for each animal. This step was necessary on the basis of the great variability of diets (herbivores and omnivores) and digestive systemsAdditionally, the present study deals with gut microbiota assessment in farming animals wvia 16S rRNA next-generation sequencing, which successfully allowed to reconstruct species-specific associations between the BA profile and specific gut microbiome components.Methyl alcohol, isopropyl alcohol, and acetonitrile (ACN), hypergrade for LC–MS (Lichrosolv), were purchased from Sigma-Aldrich . Acetic acid (98% pure), and ammonium hydroxide (98% pure) were purchased from Fluka . Water of HPLC–MS grade (Millipore) was produced by Milli-Q Synthesis A 10 . Authentic chemical standards of the BAs and deuterated BAs  were purchased from Sigma-Aldrich . Commercially available standards of the oxo-BAs were purchased from Steraloids .9. The purity of the synthetized compounds was determined by NMR and HPLC coupled with high-resolution mass spectrometry. All the standards used in this study were > 95% pure , and acetonitrile (ACN), all of MS grade, were purchased from Merck . Acetic acid, formic acid, and ammonium hydroxide  were purchased from Fluka . Milli-Q Synthesis A 10 depurative system  was used to produce water of high purity grade. Other solvents were all analytical grade.9. These stock solutions were further diluted in isopropanol to obtain working solutions, stored at 4 °C and used for 4 weeks at most.Stock solutions of each analyte and IS were prepared in isopropanol at a concentration of 1 mg/mL and stored at − 20 °C as the procedure already reportedFecal samples were collected from 9 different mammalian vertebrates and one nonmammalian vertebrate: pig, wild boar, dog, rabbit, mouse, chicken, horse, donkey and cow. Ten healthy adult subjects, 5 males and 5 females, were selected for all investigated animals, except for wild boar samples that were collected without sex checks. The farm animals were under a regular diet from antibiotic-free and not intensively exploited livestock. The study was carried out in accordance with ARRVE guidelines. Ethical review and approval was not required for the animal study because faces were collected from hosted animals in farms without touching nor disturbing them in any way.Fecal samples were collected also from 10 healthy human subjects , 5 males and 5 females, from 18 and 60 years old, with no colorectal disorders, digestive, biliary and other gastrohepatic/hepatogastric disorders. Informed consent was obtained from all subjects and/or their legal guardian(s), and all methods were carried out in accordance with the Declaration of Helsinki guidelines and regulations.15. Four different procedures were tested for oxo-BAs in animals: isopropanol, acetonitrile, methanol extractions and sodium hydroxide 0.1 M.The fecal extraction has been optimized according to the different procedures for the most common BAs reported in the literature9. Aliquots of wet fecal sample homogenate (1 g) were extracted with 3 mL of isopropyl alcohol. The mixture was stirred for 30 min at 37 °C and then centrifuged at 600 g for 10 min. The supernatant was then diluted 1:10 (v/v) with 40% isopropanol in 15 mM ammonium acetate at pH 8.00, filtered, transferred to an autosampler vial, and 1 μL injected into the UPLC-QTOF-MS system. The results obtained from the analysis are expressed as μg/g of wet feces.Samples were obtained fresh and kept on ice until storage at − 80°. According to our results for oxo-BA extraction, sample preparation was performed following the selected conditions described below, already reported in our paper with minor modification9. Liquid chromatography was performed using an ACQUITY UPLC  Waters BEH C18 column  kept at 37 °C throughout the analyses. The mobile phase was composed of 15 mM ammonium acetate buffer at pH 8.00 (phase A) and methanol 99.92% pure (phase B). Final separation was achieved at a 0.3 mL/min flow rate under gradient elution conditions: 40% B for 0.1 min, 40–55% B from 0.1 to 0.4 min, 55% from 0.4 to 1 min, 55–65% B from 1 to 2.1 min, 65–80% B from 2.1 to 3.3 min, and 90% B from 3.3 to 7 min. Re-equilibration at 40% B between analyses was achieved in 4 min for a total run time of 11 min. The injected sample volume was 1 μL. The autosampler temperature was kept at 10 °C to preserve the sample from precipitation.The choice of the chromatographic and mass spectrometry conditions were based on our previous HPLC–MS/MS method for BAsMass spectrometry analysis was performed using a Qtof system  with an ESI source. High-resolution multiple reaction monitoring acquisition mode (HR-MRM) was used for quantification.The major fragment for quantification and the qualifier ion fragments for identification Table  were det2) 400 °C and 800 L/h, respectively; and collision energy of 40 eV were the same for all compounds. The software UNIFI was used to control the instrument and acquire and analyse data.The detection parameters of the ESI source were as follows: capillary voltage 2.5 kV; sample cone 40 V; source offset 80 V; source temperature 100 °C; declustering potential 80 eV; flow rate of cone gas 50 L/h; temperatures and flow rate of desolvation gas , obtained by diluting the working solution with mobile phase and analysed in duplicate. Internal standard (IS) concentrations were kept constant at 1 µg/mL for LCA-D4, CA-D4 and DCA-D4. According to the similar chemical structures and to obtain the best available IS for each analyte, CA-D4 was used to quantify CA, MCA and oxo-CAs; DCA-D4 was used to quantify secondary BAs and their oxo metabolites; LCA-D4 was used to quantify LCA and 3-OXO LCA.The limit of detection  and limit of quantification  of the method for each single analyte were experimentally determined by injecting stepwise dilutions of the standard solution. Accuracy (bias%) and precision (CV%) were determined intra- and inter daily through triplicate analysis over three different days (n = 9) by analysing mixed standard solutions at low (0.2 μg/mL), medium (2 μg/mL), and high (15 μg/mL) concentration levels.p > 0.05).The matrix effect was evaluated by comparing standard calibration curve slopes and matrix-matched calibration curve slopes with a t-test  was added to 1 g of feces and incubated for 1 h at 60 °C before the addition of 4 mL of water. The sample was homogenized and centrifuged at 600 g for 10 min. The supernatant was collected and extracted using a 500 mg SPE cartridge C18. Preconditioning was carried out with 5 mL methanol and 5 mL water. The cartridge was loaded and rinsed successively with 10 mL water to discard salts and hydrophilic metabolites. BAs were then eluted with 5 mL methanol. The methanol fraction was collected and dried under a nitrogen stream. The residue was dissolved in 150 µL methanol, and 1 µL was injected into the mass spectrometer.Recovery was assessed for each extraction protocol in each animal by comparing the areas of standard fecal extracts (2 µg/mL) fortified before extraction with those fortified after extraction. The results are obtained from 3 different experiment at least.According to our results, the recovery for the best extraction protocol  was also evaluated for each analyte in human fecal sample at three concentration levels .17. Briefly, the homogenization step was performed three times using the FastPrep instrument  at 5.5 movements per sec for 1 min. Afterward, samples were heated at 95 °C for 15 min. Then, at the end of the purification step, DNA was quantified by using a NanoDrop ND-1000  and stored at − 20 °C until PCR amplification and library preparation. After the isolation of microbial DNA from fecal samples, the amplification of the V3–V4 hypervariable region of the 16S rRNA gene was carried out using the 341F and 785R primers18 with added Illumina adapter overhang sequences, as previously reported19. PCRs were purified by using Agencourt AMPure XP magnetic beads , and Nextera Technology was used to prepare indexed libraries by limited-cycle PCR. After a further clean-up step as described above, libraries were normalized to 4 nM and pooled. Sequencing was performed on an Illumina MiSeq platform using a 2 × 250 bp paired end protocol, according to the manufacturer's instructions .Total bacterial DNA was extracted from individual fecal samples obtained from the following animals: pig, wild boar, dog, rabbit, chicken, horse, donkey, using the DNeasy Blood & Tissue Kit  with a modified protocol as previously shownwet feces) ± standard deviation (SD) and as a percentage with respect to the total quantity of BAs.Data analysis for BAs quantification was performed using GraphPad Prism 5.0 software . The quantitative data are presented as the mean  according to a 99% similarity threshold using UCLUST22. Taxonomy was assigned using the RDP classifier against the SILVA database23. OTUs were filtered for their presence in at least 5 samples.Raw microbial sequences were processed using a pipeline combining PANDASEQAlpha rarefactions were analyzed by using Faith's phylogenetic diversity (PD whole tree), OTU species count (observed species) and Chao1 index metrics for microbial richness.https://www.r-project.org/). Beta diversity was estimated by computing unweighted UniFrac distance, which was used as input for principal coordinates analysis (PCoA) to explore inter-sample variability in relation to the different animal species. The contribution of covariates to the ordination space was found by using the function ‘envfit’ of the R package vegan. A permutation test with pseudo-F ratios (function ‘adonis’ in the vegan package) was used to determine the significance of separation on the PCoA plot. The Kruskal–Wallis test followed by the post hoc Wilcoxon rank-sum test were used to assess significant differences in taxon/BA relative abundance between animal groups. The hierarchical cluster analysis was computed on the matrices of relative abundances  with the function ‘hclust’, using as distance the Pearson correlation coefficient, and Ward’s linkage method (ward. D2 function). The optimal number of clusters was defined with the elbow method using the function ‘fviz_nbclust’ in the factoextra package. Correlation analysis was performed using the ‘rcorr’ function in R with the Pearson method, which allowed us to find any correlation (positive or negative) between BAs and bacterial genera. Univariate linear regressions were computed with the function ‘lm’ in R. P-values were adjusted using the false discovery rate (FDR) function in R, and a p value ≤ 0.05 was considered statistically significant.All gut microbiota statistical analyses were performed using the R project  was experienced working in HR-MRM mode monitoring the transition [M–H]−  > [M–H]−, pseudo-MRM, while the other product ions were used for the qualitative analysis of the samples  is one of the major fragments for CA, along with the fragment ion [C19H29O2]− (289.2 m/z) , from the cleavages of ring-B, from C5–C6 to C9–C10 [C16H27O2]—(251.20 m/z), and from C6–C7 to C9–C10 cleavage [C14H21O]—(195.17 m/z). The loss of water for MCAs represents a common fragmentation pathway for these BAs, although neutral loss of hydrogen is also abserved in alpha MCA. Few product ions were detected in the spectra of BAs without a 12-hydroxyl group , CDCA (373.3 m/z), HDCA (373.3 m/z) and LCA (357.3 m/z). On the other hand, the low intensity of the dehydrated product of LCA , was the major fragment for DCA together with the dehydrogenated form [M–H–H2O–CO–H2]− (343.3 m/z) and the di-dehydrated form [M–H–H2O–CO–H2O]—(327.3 m/z). According with the described results, it has been recently highlighted25 that the dissociation mechanisms of BAs with a 12 hydroxy group involve the rotation of the carboxylate side chain and the subsequent rearrangements accompanied by proton transfer between 12-hydroxyl and 24-carboxyl groups.The product ion, [M–H–H19H29O2]− (m/z 289.2) is one of the highest fragments for 3-oxo-CA, 7-oxo-CA and also for 3-oxo-UCA  with its dehydrated form (m/z 341.24)  is the most abundant for 3-oxo-CA  as the main fragment. In contrast, the DCA oxo metabolite at position 12 shows a low signal of this product ion  of DCA and the oxo-DCAs Fig. F–H with 2O–CO]− 43.3 m/z 2O]− (m/z 371) and its dehydrated form (m/z 353) are only slightly detectable for secondary BAs without 12-hydroxyl groups, such as 3-oxo-CDCA, 7-oxo-CDCA, 6-oxo-HDCA, 3-oxo-HDCA, and 3-oxo-UDCA  was detectable only for 3,12-dioxo-DCA  and the dehydrated form (309 m/z) were specific for 3,6-dioxo-HDCA  and the fragment [C21H33O2–CO2]− (273 m/z) were specific for 3,7-dioxo-CDCA  ≥ 0.995 for all the BA molecules. LOD values ranged from 5 to 10 ng/mL, while LOQ values ranged from 15 to 30 ng/mL for all compounds. The results are shown in Supporting Material , expressed as CV% and bias%, respectively, were < 10% for all compounds at three different concentration levels  in the same concentration range.The matrix-matched calibration curve slopes did not differ significantly from those in neat solution (The best results among extraction protocols were obtained with both isopropanol and sodium hydroxide extraction (Rec > 90%) for oxo BAs without significant differences among the different animal fecal samples. On the other hand, the SPE step was a longer sample handling that introduced more variability related to different analyte recoveries. For this reason, isopropanol was used for sample extraction, with the advantage of a low impact of pre-analytical errors The results are reported in the supporting material Table .26. Indeed, the abundance percentage was also used to better compare BAs profiles , SD and relative abundances (%) of oxo-BAs and their precursors are reported in Tables The method has been applied to evaluate the quali-quantitative composition of BAs in the feces of investigated animals and humans. The total content was reported as μg/g ± standard deviation (SD) of wet feces for each animal. The high variability of data is explained considering several factors that could affect the amount of BAs in feces: intestinal motility, daily variation, diet, age of the animal and genetic factorsles Fig. . Concentsus scrofa domesticus) are reported along with wild boar (sus scrofa) as belonging to the same family Suidae  BAs profile presents a relative complexity, with several analytes detected . Oxo metabolites were detected at very low percentages with respect to the other species, and as expected, the most abundant, although at low levels, was the 12-dehydrogenated derivative 12-OXO-DCA (4%).The dog  feces is CDCA (80%), a primary BA, in agreement with previous studies29 . The oxidated derivative of DCA represents the main fraction of the oxo-BAs, with a relative abundance of 23% for 3,12-dioxo-DCA. Oxo-derivatives of CDCA have been detected at concentrations below the LOQ.Rabbits (Oryctolagus cuniculus) also present a complex BAs profile in terms of oxo-BAs Table E, Fig. 4Equus africanus asinus) are reported along with horses , since they belong belonging to the same family (Equidae). For both species, the absolute BA concentrations were very low  profile is quite different from the others, considering the presence of the named muricholic acids (MCAs).+.The main quantified oxo BAs are the oxidated derivatives of DCA and MCA, covering more than 10% of the total pool Table I, Fig. 49. In particular, the main oxo derivatives are the oxo derivatives of DCA, covering approximately 35% of the total content of BAs in feces . After the sequencing process of the V3-V4 hypervariable region of the 16S rRNA gene and bioinformatics pipeline, a total of 429 868 high-quality reads were obtained . Sequences were clustered into 2 828 operational taxonomic units (OTUs) at 99% identity.p < 0.05). Specifically, horses, donkeys and pigs showed higher diversity values in the three metrics used  compared to rabbits, chickens and dogs, which showed lower values, as assessed by the Wilcoxon rank-sum test across groups (p < 0.05)  based on unweighted UniFrac distance was carried out. As expected, each animal clustered apart from the others Fig. A, highliSpecificities of the gut microbiota composition at the phylum, family and genus levels are shown in the Supplementary material Fig. . At the p ≤ 0.001).Next, we sought the Pearson correlation coefficients between microbiome genera and BAs in the comprehensive dataset from all farming animals. Only genera with a relative abundance ≥ 1.5% in at least two samples were considered. In Fig. Peptoclostridium, Blautia, Collinsella, [Ruminococcus] gnavus group, Megamonas, Fecalibacterium and Catenibacterium, was positively correlated with DCA, CA and 12-oxo-CA. Members of cluster 2 foresee two groups of microorganisms, the first including Enterococcus, Romboutsia, Turicibacter and Escherichia Shigella and correlating positively with 7-oxo-CDCA, 3-oxo-CDCA and 3,7-dioxo-CDCA, and a second group of microorganisms  correlating with a different set of BAs, such as UDCA, HDCA, 3,6-dioxo-HDCA, 3-oxo-UDCA and 3(o)6-oxo-HDCA. Finally, cluster 3, encompassing members of the Ruminococcaceae NK4A214 group, Ruminococcaceae V9D2013 group, Ruminococcus 2, Ruminococcaceae UCG-014 and Candidatus Saccharimonas, was positively correlated with 12-oxo DCA, 3,12-dioxo-DCA and 12β,3-oxo-DCA, while cluster 4 included a heterogeneous group of gut components showing a sparse pattern of positive and negative correlations with different BAs. Interestingly, microbial clusters 1–3 were dominated by genera that have been previously shown to characterize the gut microbiome from different farming animals . In particular, cluster 1 was dominated by microorganisms characterizing the dog’s gut microbiome, while the first and second groups of cluster 2 and cluster 3 included microorganisms characteristic of the chicken, pig and rabbit gut microbiomes, respectively. To strengthen these observations, for the different farming animals, we calculated one to one linear regression between the abundance of the specific gut microbiome components and the load of each BA in the different subjects. Figure This approach allowed the identification of 4 bacterial clusters showing a characteristic correlation profile with BAs. In particular, cluster 1, including microorganisms such as ls Figs. , S6 (Kru2 of 0.8) were obtained in rabbit for members of the family Ruminococcaceae  and the BAs 3,12-dioxo-DCA and 12β,3-oxo DCA. Tendencies toward positive associations have been observed for swine, where the family Prevotellaceae NK3B31 associates with UDCA and HDCA, and for dogs, where both the Ruminococcus gnavus group and Blautia are associated with DCA abundance.In particular, strong positive associations  was optimized, which allowed us to quantify BAs and oxo-BAs present in different concentration orders of magnitude and in different fecal matrices. Especially the variable water content among the samples did not affect the recovery reproducibility for oxo-BAs and BAs.In this scenario, we have developed a comprehensive LC–MS method for the analysis of 28 BAs, including 21 oxo-BAsThis method allowed us to determine the same BAs with the addition of MBAs in a short time with high selectivity due to the high-resolution MS. The analysis time was shortened up to 5 times compared to previous methods for oxo-BAs, maintaining, if not improving, the resolution of the chromatographic peaks and the selectivity by using high resolution.25, has been also confirmed in oxo-BA spectra.Careful evaluation of the oxo-BA fragmentation patterns improved the specificity of these analytical methods, considering the similar chromatographic behaviour of the isobaric compounds analysed. Specific product ions and different ion abundances are useful tools to better discriminate among oxo bile acids with the same molecular weight and close retention time in columns. The key role of the 12-hydroxy group on the fragmentation patterns, supposed to be correlated to proton transfer between 12-hydroxyl and 24-carboxyl groupsTo our knowledge, this is the first time that an almost complete profile of BAs and their metabolites produced by GM has been characterized in fecal samples by UPLC-HR-MS. In addition, the method was applied for the first time to the characterization of the "acidic steroid prolife" of 10 vertebrates, including humans. We focused our investigation on animals that have lived with humans for millennia for the purpose of breeding, work or companionship. Among the animals analyzed, we had 9 mammals, both herbivorous and omnivorous, and a bird.3 but also highlighted for the first time that the composition of the fecal oxo-BA GM metabolites differs for each species, even if belonging to the same family.Our study not only confirmed the peculiar profile of fecal BAs for each animalThe profile of fecal BAs and oxo-BAs was then correlated to the composition of the intestinal microbiota to shed light on a possible microbiome-host co-metabolic network with species-specific declinations.35, such as the Ruminococcus gnavus group, Ruminococcus 2, Collinsella, Fecalibacterium and Roseburia, including taxa possessing dehydroxylases and dehydrogenases able to metabolize various BAs. A complete list of bacteria possessing dehydroxylases and dehydrogenases able to metabolize various BAs is reported in the supplementary material , not only for excretion but also for their role as a potential ligand (agonist) to the FXR receptor, and in general for a better understanding of pathological events involving the liver and starting and connected with the intestine in the so-called gut-liver axis.36. Future developments may include a broader class of compounds found especially in other mammals and vertebrates, for example, to investigate pathologic conditions affecting the intestinal tract of animals.Minor BA fecal constituents at trace levels could be potentially present such as glycine/taurine-conjugated BAs, glucuronides, sulfate BAs or other metabolites under physiological and pathological conditionsThe analytical method reported was developed and fully validated for the analysis of up to 30 compounds in feces, including 21 oxo-BAs. Careful evaluation was performed to determine the best extraction procedure for these compounds in fecal samples from different animals. The fecal oxo-BA profile was determined and reported for several farming animals for the first time in the literature. This analytical method is a powerful tool to better understand the key role of acid steroid compounds largely present in the intestinal tract of vertebrates. According to this purpose, the gut microbiota in farming animals was evaluated, allowing us to reconstruct species-specific associations between the BAs profile and specific GM components.Supplementary Information.