The aim of physiology is to elucidate the factors that maintain homeostasis, and therapeutics aim to correct abnormal factor functions. The homeostasis paradigm does not formally recognize influences outside its controlled experimental frames and it is variable in its modeling of neural contributions. The paradigm of allostatic orchestration (PAO) extends the principle of allostasis (“stability through change”) as originally put forth by Peter Sterling. The PAO originates from an evolutionary perspective and recognizes that biological set points change in anticipation of changing environments. The brain is the organ of central command, orchestrating cross-system operations to support optimal behavior at the level of the whole organism. Alternative views of blood pressure regulation and posttraumatic stress disorder (PTSD) illustrate differences between the paradigms. For the PAO, complexities of top-down neural effects and environmental context are foundational (not to be “factored out”), and anticipatory regulation is the principle of their interface. The allostatic state represents the integrated totality of brain-body interactions. Health itself is an allostatic state of optimal anticipatory oscillation, hypothesized to relate to the state of criticality, a mathematical point of poise between phases, on the border between order and disorder (or the “edge of chaos”). Diseases are allostatic states of impaired anticipatory oscillations, demonstrated as rigidifications of set points across the brain and body (disease comorbidity). Conciliation of the paradigms is possible, with “reactive homeostasis” resolved as an illusion stemming from the anticipation of environmental monotony. Considerations are presented with respect to implications of the two paradigms for brain-focused intervention or advancement; the hypothesis that the state of criticality is a vehicle for evolutionary processes; concordance with a philosophy of freedom based on ethical individualism as well as self-creativity, non-obsolescence, empowerment, and citizenship; and concluding reflections on the science and ethics of the placebo, and the potential for virtuous cycles of brain-Anthropocene interactions.There are two main paradigms for brain-related science, with different implications for brain-focused intervention or advancement. The paradigm of homeostasis , originating from laboratory-based experimental physiology pioneered by Claude Bernard, shows that living systems tend to maintain system functionality in the direction of  The human brain is the most precious resource on our planet. Excitement about the brain takes many forms, and actions on behalf of the brain go in many different directions. It may be valuable to articulate and clarify whether there are particular approaches to the brain that favor the recognition of particular categories of data, and whether they lead to different goals and strategies for intervention. Such an exercise may help visualize alternative trajectories for brain-related advancement at the level of “broad brush strokes.”This essay contends that there are two main paradigms Kuhn,  for braiallostasis , to point toward his original ideas and to extend them.Over the course of three decades, a series of brain-centric, evolution-inspired insights have been articulated with increasing refinement, as principles of The essay reviews the origins and principles of homeostasis and allostasis and illustrates their differences by considering how they approach the phenomena of blood pressure regulation and posttraumatic stress disorder (PTSD). It contends that the Sterling principle of allostasis is sufficiently different that it warrants characterization as a new paradigm—the PAO—which is articulated as a set of propositions, and it also contends that the two paradigms can be conciliated. Selected considerations are presented pertaining to their different interventional implications; a falsifiable model regarding the vehicle or mechanism of evolutionary processes; the alignment of the PAO with a philosophy of freedom; and the proposed utility of allostatic planning and action in the context of the Anthropocene epoch.milieu interieur :Potentially, it may have been his studies of sugar mobilization in the setting of insulin-induced hypoglycemia, that may have spurred his attention to the organizing principle of In an open system such as our bodies represent, compounded of unstable material and subjected continually to disturbing conditions, constancy is in itself evidence that agencies are acting, or ready to act, to maintain this constancy.If a state remains steady it does so because any tendency towards change is automatically met by increased effectiveness of the factor or factors which resist the change.Any factor which operates to maintain a steady state by action in one direction does not also act at the same point in the opposite direction.Homeostatic agents, antagonistic in one region of the body, may be cooperative in another region.The regulating system which determines a homeostatic state may comprise a number of cooperating factors brought into action at the same time or successively.When a factor is known which can shift a homeostatic state in one direction, it is reasonable to look for automatic control of that factor or for an opposite factor or factors having an opposite effect.factors which move system parameters in one direction or another.In concordance with these postulates, laboratory physiological research from the early twentieth century and beyond has made tremendous progress in the understanding of normal level of blood pressure.Blood pressure regulation is a phenomenon exhaustively studied under the homeostatic paradigm. The main actors for blood pressure maintenance include the heart, which acts as a pump; the vessels, which constrict and relax as they transport blood throughout the body; and the kidneys, which manage blood volume by filtering fluid and conserving sodium to preserve osmotic pressures. Exhaustive search for the factors of blood pressure regulation and their sites of production or action has led to recognition that the adrenal glands, lungs, and liver also play roles. The main “equation” renders blood pressure to be a function of cardiac output and total vascular resistance, analogous to Ohm’s law in physics where the voltage in an electrical circuit is calculated as the product of current and resistance. Cardiac output is itself a product of heart rate and stroke volume, with stroke volume, in turn, being influenced by blood volume (“preload”), the pressure against which the heart must contract (“afterload”), and the strength of each contraction (contractility). If sensors in the carotid artery or kidney detect a decrease in blood pressure, there will be a cascade of factor actions that alter volume, vascular resistance, and cardiac output, to increase it. If they detect a rise in blood pressure, there will be cascades in the opposite direction. Life itself—through the capacity for homeostasis—is given the credit for checks and balances that preserve the When the system fails to maintain its normal state—for example hypertension, the state of persistent blood pressure elevation—then causes are sought in the various factors which would otherwise return the blood pressure to “normal.” Yet though there are certain well-characterized abnormalities of homeostatic mechanisms, that are correctable causes of hypertension—for example renal artery stenosis or endocrine tumors—many anomalies arise when explaining blood pressure regulation as the steady state of local tissue factors. Ninty-five percentage of individuals with hypertension are deemed to have a type that has no identifiable local factor as the cause . Thus, studies which aim to explore physical changes in PTSD are themselves conducted in some defiance of reductive explanation based on physical factors alone. Nonetheless, stimuli can be carefully designed with the intention of producing controlled forms of trauma within a laboratory setting, and the brain can be taken to be the organ of behavior . By refining such frameworks for experimentation, specific physical factors for PTSD have been sought in cellular failures to extinguish associative conditioning between environmental cues and fear learning mechanisms , the naturalist Alfred Russel Wallace wrote a letter to Charles Darwin in which he proposed that species may change through the pressures of scarcity generated by nature itself, which would lead to the differential reproductive success of some variants over others. Subsequently, Darwin  publisheallostasis, beginning with a personal appreciation for the influence of environmental context on biological regulation. In the 1960s, Sterling was working as both a neuroscientist and a social activist, and while canvassing in African-American communities he noticed that many individuals had limps or facial droops indicative of a history of stroke, as a consequence of the high prevalence of hypertension. Over the next two decades, Sterling integrated his naturalistic observation, which was being confirmed in epidemiological studies of the links between social disruption and chronic disease, with other growing bodies of knowledge. There were electron microscopic findings of the ubiquity of nerve fibers on blood vessels, endocrine cells, and elsewhere, suggesting that “the brain has close access to essentially every somatic cell,” and also primate studies showing that demands for sustained attention caused elevations of blood pressure and release of catabolic hormones.The neuroscientist Peter Sterling has given the following account (2004) of the origins of allostasis (1988), which states that “an organism must vary all the parameters of its internal milieu and match them appropriately to environmental demands.” If an individual is exposed to an environmental condition presenting potential danger and high need for vigilance, the brain orchestrates systems on an anticipatory basis, toward a physiological arousal state that is a match for those demands  homeostatic schematics of blood pressure regulation do highlight the brainstem centers of the autonomic nervous system; direct neural control and afferent signaling between the brain, heart and blood vessels; and neural ganglia at the kidneys. Nonetheless, the role of neocortical influences on the brainstem and autonomic signaling has yet to be understood in depth, and the full significance of these pathways for neural influence has yet to be appreciated across physiological subspecialties, clinical medicine or public health.For example, there is now an expansive corpus of studies that have provided an increasingly granular appreciation for the environmental factors associated with blood pressure, including an early report from Bevan et al. . In grouThe brain-centric allostatic perspective in conjunction with the polyvagal theory Porges,  can alsoToo, the Sterling principle of allostasis may shed light on the controversy around the role of salt consumption as a factor in hypertension. The allostasis perspective is consistent with the idea that dietary salt may be not a culprit but rather an “innocent bystander” , Sterling articulated allostasis as the following set of principles: (1) organisms are designed for efficiency; (2) efficiency requires reciprocal trade-offs (managed by the brain); (3) efficiency requires predicting (by the brain) what will be needed; (4) prediction requires each sensor to adapt its sensitivity to the expected range of input; (5) prediction requires each effector to adapt its output to the expected range of demand; and (6) predictive regulation depends on behavior whose neural mechanisms also adapt.An objective of the present essay, addressed in greater depth in Section “Conciliation of the Two Paradigms,” is to help resolve questions and occasional debate as to whether the concepts or principles of allostasis merit the cognitive costs that come with the introduction of new language. Post-Sterling, many have used the allostasis concept to refer to the general phenomenon that biological set points become temporarily altered under the condition of acute environmental stress . Yet Cannon himself recognized that systems do not maintain absolute constancy in their set points, and he was careful to note that he had chosen the prefix “homeo” (similar) rather than “homo” (same) when coining the term homeostasis (1929). It seems likely that the answer to the question, whether or not allostasis is simply homeostasis in “new clothes,” lies mainly in Sterling’s heightened appreciation for the brain. Sterling’s recognition that the brain is necessary as a dedicated organ for higher-order processing, for management of trade-offs or anticipatory orchestration among actors, is in some respects the same conclusion reached by Bernard more than 100 years earlier.between the brain and all organ systems , which can replace the idea that health is a capacity for reactive (or corrective) feedback. Optimal anticipatory oscillation builds on appreciation of the brain as a “prediction machine”  is the rule among individuals with chronic diseases  that is most conducive to the generation of rapid and context-sensitive variations in its activity. A given brain activity pattern can only be characterized as “optimal” for a given environmental condition; it is the pluripotent capacity to express a range of activity patterns, that should be the most optimal pattern of all. An “empty” state of latent potentiality may have great import for anticipatory regulation, when external circumstances entail unpredictable and last-moment changes  are themselves secondary phenomena, which invoke the need for investigation of primary causes. Tendency to maintain the state of criticality  is hypothesized to explain the likelihood to express optimal anticipatory oscillations.As noted in Section “Allostasis—From Principles to Paradigm,” the current presentation aims to help resolve the question of whether allostasis (to include the PAO) is genuinely different from homeostasis. Attention to this question entails empirical, analytical, inferential, interventional, and ethical considerations, some of which are further explored in Section “Selected Considerations and Summary.” Clarification of terminology shared by the two paradigms, as put forth by different authors, is presented in replace the concept of homeostasis, which is itself flawed. In this view, homeostasis erroneously perceives that biological parameters maintain a categorically constant set point (they do not), and erroneously explains that these set points are maintained through corrective or reactive feedback (which is inconsistent with the ubiquity of anticipatory regulatory behaviors demonstrated throughout nature). It is the brain that acts as a data-integrating prediction-machine, to orchestrate operational set points and anticipatory behaviors in ways that support whole-organism health (with implications for species evolution). In the main, critics of Sterling’s original allostasis concept have focused on his point against biological constancy, and dismissed it as a mischaracterization of the thinking of Bernard and Cannon, who both recognized variability of biological functions. Consequently, there have been doubts as to whether the allostasis concept adds meaning beyond homeostasis  of physiological research post-Cannon. Though Cannon made many statements which can be selectively highlighted to give the appearance that Sterling has misunderstood him (and Bernard’s appreciation for the brain has been unrecognized by many), a bird’s-eye view of homeostatic science makes clear that it is generally designed for the isolation of local factors, in hopes for discovery of “silver bullets”—with sparse attention to complex environments which will favor anticipatory variations in functional set points, or to the integrative and top-down role of the brain. The only way to validate the existence and efficacy of such bullets is to “factor out” the contributions of top-down neural regulation  and environmental variations. Sterling can be forgiven for using Bernard and Cannon as straw-men in order to make a set of crucial points that have still yet to reach biomedical researchers as a community. There are vacuities in research which proceeds without a view informed by evolutionary processes , and that processes which support homeostasis can simply be labeled as “homeostatic mechanisms”  arises because data collection and causal inference in biology are substantially still driven by a methodology that systematically limits or ignores parameter alteration or variability that may be decisively influenced by factors from complex environments, as well as neural complexity including top-down efficacy for regulation of whole-body physiology . Adequate updating of the classical view of homeostasis would entail the need for three formal revisions. Again, there would be a need for formal recognition of anticipatory behaviors . Second, there is a need for formal attention to top-down neural effects and their complexity . Third, there is a need for an expansion in formal modeling of the complexity of the natural environment.Nonetheless and conceivably, if the sciences which explore homeostasis—the collective methodologies essential to the maintenance and utility of a Kuhnian paradigm—were sufficiently broadened or revised, there would be no need to burden biological researchers with the additional concept of allostasis. appears to be a reactive (or “corrective”) process may be parsimoniously understood as another form of anticipatory behavior, if the complex environment has been “controlled” to appear monotonous and unchanging, and especially if neural regulation is relatively limited in its own complexity or criticality. An additional implication of conciliation is to offer lines of exploratory investigation for any given phenomenon that appears to be homeostatic. The PAO predicts that relative constancy in any biological parameter will yield to findings of increased variability if there are systematic alterations in either the complexity of neural dynamics , environmental complexity, or both. A straightforward illustration is to consider a subject who provides continuous daytime blood pressure readings for 10 consecutive days. He or she may show maintenance of an average  blood pressure in the vicinity of 120/80 for each day, irrespective of occasional changes from being seated to standing, or whether they read the morning newspaper or receive an animated neighbor as a guest. Consistency of the mean value at roughly 120/80 for each of those 10 days can be taken as empirical evidence for homeostasis. However if 10 days of readings were then to be collected from the same subject after they had begun a new role associated with high job strain , whereas while the PAO would also be sensitive to genetic substrates, it would search for differences in environmental and top-down neural regulatory influences.Thus, the PAO begins with two critical complexities that are foundations, not after-thoughts, and that are themselves interfaced through anticipatory regulation; and it avoids the possible introduction of “kludges” to rescue homeostasis. The PAO’s potential to resolve “reactive vs. predictive homeostasis” may be one illustration of its economy, in that it identifies “reactive homeostasis” as a kind of illusion. What Following is a more provocative example of how homeostatic phenomena can be viewed as the special case of allostatic orchestrations that arise when a product of neural and environmental complexities is minimized. The constancy of core body temperature in homeotherms, despite major changes in seasonal weather, seems to give evidence for the McEwen-Wingfield formulation that homeostasis is the rule for a few fundamental parameters. Yet perhaps the narrow range is only a demonstration of relatively limited critical capacity for top-down neural regulation of temperature. Many mammal species reduce their core temperatures markedly during winter , at a time when their foraging opportunities are limited. Interestingly the evolutionary origins of hibernation are not genetically defined ; or “Alteration in pre-prandial glucose allostasis through a comprehensive lifestyle intervention” (attention to anticipatory regulation and whole-person behavior); or “Allostatic intervention for critical brain dynamics in diabetics results in tighter glucose control without increasing episodes of hypoglycemia” . At this juncture it may also be helpful to identify socio-cultural and ethical currents as well as a key lacuna of scientific understanding—the physics of consciousness itself—which have likely contributed to the value and durability of the homeostatic paradigm, and may be presumed to associate with any application of the PAO . Theological dictums have influenced progress in biology  no less than they have in physics. Separately yet relatedly, physicians and healers judiciously rely on the biological value of their patients’ subjective confidence. Bernard and Cannon formalized an experimental method explicitly designed to ascertain material causes and effects in living systems, without adducing idiosyncratic or sometimes exploitative ideas and practices of theologians, vitalists, physicians, or faith healers. These two giants of physiology are in a lineage that extends the legacy of the American polymath Benjamin Franklin one century and more before them, with respect to Franklin’s participation in a commission that falsified the “magnetism” claims of Franz Mesmer. It seems possible that Cannon’s own relative neglect of the brain’s top-down influences may have been partly motivated by his own principled ethical secularism . Ethical secularism remains alive and well, easily recognizable in the commitments to liberal political philosophy dating back to the Western Enlightenment  which are central to modern biomedical ethics. Stated otherwise, modern biomedicine at its best may shun the benefits of the placebo response not out of antipathy to human self-empowerment, rather because it greater disdains the sense of charlatanism  that can sometimes be associated with its efficacious induction, and the distaste is exacerbated by the lack of a hold on the essential biology of subjective intentionality. The objectivity of the laboratory has not only identified the fine mediators of biological operations, it has also been constitutive for the needful expunging of “sin” in particular as an etiological construct in the understanding of health and disease.Nonetheless and with appreciation for the weight of the aforementioned considerations, including the cost of progress and the work still undone, the thrust of the PAO is that the present need for advancements in neuroscience, healthcare, and beyond, points us to cease clinging to an era where the significance of subjectivity, or consciousness itself, is ignored in neural research or excluded with scrupulosity from the healthcare toolkit. To be clear, the PAO makes no call for a return to vitalistic reasoning, disease as “punishment,” or the methods of Mesmer. As the PAO admits consciousness and all its qualities, the “old” scientific conundrums and ethical dilemmas will not disappear; they will require advanced forms of conceptualization and engagement.Innovation is needed in the field of brain functional enhancement or advancement, that begins and ends with a homeostatic perspective. For example, a neuroprosthetic is a miracle if it permits recovery of homeostatic capacities for an individual with gross brain damage. Nonetheless even the most successful homeostatic interventions may entail systematic under-recognition of unintended consequences, because such effects may not be easily visible in the controlled context of early laboratory investigations. In contrast, the PAO perspective may be well suited to inform brain advancement objectives on larger scales, with heterogeneous populations, for objectives that may be less “controlling” yet potentially more ambitious. To clarify the differences entailed by homeostatic and PAO approaches to brain advancement, this section begins by considering how homeostasis and the PAO are associated with different approaches to “brain normality” and therapeutics in general.or time.Homeostasis begins with the (often unstated) presumption that there do exist “truly normal” forms of biological phenomena including brain functionality; that “true and discrete abnormalities” can be identified; and that the goal of therapeutics including brain-focused interventions is to “restore the state of normality.” While scientists acknowledge that the parameter estimates associated with these assumptions may be frail, there is little argument about the biological validity of the constructs themselves , and indeed without them, it is not clear how knowledge can be gained, or interventions can be devised. In contrast, the PAO definition of health as an active state of anticipatory behaviors with respect to the changing environment is not consistent with the identification of a single biological state that is objectively “normal.” An “average” specimen may be the one that is obliterated, in the next new environment; it may be the highly “unusual” individual that becomes the “next new normal” Taleb, , 2012. Mper se, to include its association with whole-organism behaviors in context, that is central to the Sterling principle of allostasis or the PAO. For example, many pharmacological strategies that are designed to clamp set points for neurotransmission, even if they “target allostatic load,” are still likely to entail the complications and limitations associated with homeostatic therapeutics , or to support the brain to move toward a pluripotent and flexible capacity for meeting the needs of changing situations. Allostatic intervention may still include the goal of “improvement” for a given output parameter, yet strictly speaking, such a change should be modeled as a secondary outcome (given its contingency on assumptions about the “better” which may or may not be durable). Whereas a homeostatic medication for sleep promotion may also cause lethargy that impairs attention, in contrast an allostatic intervention should support a state of “wholeness” that can be expressed as relaxation or concentration, depending on the contextual need. Moreover, robust allostatic interventions should have effects on bodily facets of the allostatic state, not only on cognitions, emotions or behaviors.natural selection are tautological—we know that some variants are more fit because they have survived; what permits some variants to survive, is being more fit. The PAO theorizes that the critical state of the brain , demonstrated behaviorally as optimality of anticipatory oscillation  variation, “field-tested” over repeated reproductive cycles that “judge” its fitness for a given environment  to have been desirable, it does not necessarily follow that struggle should be the guiding principle for producing new outcomes in the future. Consequences of the naturalistic fallacy are shown in darker chapters of modern history, post-Origin. Repressive movements such as eugenics, along with ideologies that informed twentieth-century fascism, for example, were products of misconceived justifications based on “survival of the fittest.”One error perpetuated by some evolutionists is the naturalistic fallacy Moore, . For preallostatic neuro-education can present many advantages for learners, especially by better preparing them for the rapidly changing environment of the twenty-first century ” there are anomalies that block the advance of understanding or practice with respect to PTSD, and many of them can be attributed to competition between foundational definitions held by different societal stakeholders including afflicted individuals and their loved ones, clinicians and healthcare advocates, military culture-bearers, clinical researchers, laboratory scientists, epidemiologists, and others. Whether these definitions are constructed upon, or against, the homeostatic paradigm (and either implicitly or explicitly), in either case, the persistence of homeostatic conceptualizations as our dominant “mental furniture” does not leave researchers or anyone else with much room for innovation at a fundamental level. Until there is a viable and attractive framework for biological research other than homeostasis, in some ways the limitations in scientific and societal progress around PTSD are predictable. If it has merit, the PAO should help to honor and integrate the most valuable elements of different perspectives that pertain to traumatic stress-related phenomenology, in a way that goes beyond the political brokering of ontological consensus, toward the construction of genuine and “needle-moving” solutions.healthy strategies for their prevention or management.In the first place, a strong application of the PAO points us to dispense with the notion that the clinical construct of PTSD is a tenable way to distinguish between “healthy” and “diseased” individuals. As discussed in Section “Implications of the Two Paradigms for Intervention on the Brain,” it is problematic to define “normal” biology in any absolute sense, including for the state of the brain, and a visceral consciousness of this truth is appreciated greatly in, for example, communities of special operations military service members. To these individuals on mission, who have been trained to maintain extraordinary levels of alertness and sleep deprivation, it is counter-contextual to state that certain “symptom criteria”—including hypervigilance and sleep disturbance, core features of the clinical PTSD definition—can be used as indicators of their “abnormality.” Yet in a different setting, neither is it acceptable to suggest that these traits should have no negative impact on a service member’s ability to interact with civilians in a non-combat environment, or that service members should not receive tailored forms of healthcare or support from the governing agencies that trained them to develop their characteristics. If the “PTSD” concept must persist in order to serve civilian life, then perhaps it is a label that should simply be carried in a graded form by everyone, anywhere  more endemic to modern life than homeostatic thinking might point us to consider Selye, , the PAOmoonshot to prevent and eliminate the negative effects of stress, for example, are likely to be unacceptably fragile or absent. Not only is the homeostatic evidence for subtle yet chronic stress effects inadequate for the kinds of conceptualizations entailed by the PAO, also the scientific appreciation for neural complexity is still only elementary (including the girders or limits of free will), and there is a need for advanced allostatic interventional strategy. Yet we should not be lulled into complacency excused by the lack of sufficient data collected from existing paradigms. Homo sapiens currently seem to be on path for exacerbations (or compounding) of stress effects that are already impairing our lives in joy and functionality  management and prevention, including allostatic interventional strategies described in Section “Implications of the Two Paradigms for Intervention on the Brain.” Given the complexities alluded to in PTS(D) ontology, pathogenesis, and comorbidity, it is unsurprising that there is no consensus regarding what to do about PTS(D) on a population basis, whether it can be prevented and if so then how, and whether efforts toward such challenges are worth the resources they might require. Though studies have been conducted on these questions (e.g., Cohen et al.,  Harari, . For reaThe reflections below aim to further exemplify the potential usefulness of the PAO with respect to the science of consciousness, as it pertains to the “placebo phenomenon,” and the science of human interactions with the global environment, in the present temporal context. Other implications of ongoing development and application of the two paradigms and their interaction with one another are beyond the scope of this essay.There are data to indicate that effects from placebo interventions in clinical trials are increasing over time (e.g., Rief et al., Homo sapiens are now the single largest influence on the natural environment as a complex whole, then it is no small matter to state that the essential function of the brain is to generate optimal anticipatory behaviors with respect to that self-same environment. The PAO predicts that if human brains can be successfully supported to be in allostatic states of criticality, with increased sensitivity to the environment and human impacts upon it, then they should demonstrate behaviors that anticipate the value of re-creating those environments so as to be recursively supportive of their allostatic criticality, the theorized bedrock of health itself. Allostatic agendas for brain advancement—to include both an allostatic perspective on the brain, as well as allostatic interventions—have the potential to generate virtuous cycles that leverage the inextricability of humans and their environments.Geological data suggest that the earth is now in an Anthropocene epoch, characterized by human factor influences more than any other and that in recent decades we are experiencing a “Great Acceleration” (Lewis and Maslin, allostasis, here expanded upon as the PAO, which posits that neurally directed anticipatory behavior—not corrective feedback—is the general principle of biological regulation. The PAO’s construct of the allostatic state serves to represent the integrated totality of brain-body interactions, and optimal anticipatory oscillation, potentially a function of the mathematically-defined state of criticality, is intended to spur the scientific imagination toward advancements in models, data, and interventions for positive health. As an evolutionary approach to brain advancement and in its alignment with a philosophy of freedom, the PAO aims to inspire new forms of allostatic thinking, research and action, including new attention toward currently under-attended classes of biological phenomena.In the late nineteenth century, Claude Bernard laid the foundation for the homeostatic paradigm by showing that biological systems tend to maintain constancy, and Walter Cannon later used this idea to lead a long prolific run. As Bernard was concluding that the brain has a privileged position in its relationship to other organs, Charles Darwin brought forth a context-sensitive, evolutionary perspective to life on earth as a whole. Beginning 100 years later, Peter Sterling and Joseph Eyer re-realized the importance of environmental context; and they re-realized, with Bernard, that the brain is the upstream regulator of other organs. To synthesize those insights, Sterling and Eyer introduced the principle of SL is responsible for the ideas presented in this manuscript, and was the sole writer.The author was formerly an employee at Brain State Technologies LLC, a company that has developed an allostatic neurotechnology, and he retains stock options with the company.


It was the main goal of this study to investigate performance on the mental rotation test (MRT) in Brazilian and German adolescents. Mental rotation is the ability to mentally transform a three-dimensional stimulus in mind and relates to science education. 60 German and 60 Brazilian adolescents  completed the Mental Rotation Test, a physical activity and media use questionnaire and a Number Connection Test. The result showed no difference between Brazilian and German adolescents in the cognitive processing speed measurement. German adolescents are more active and show a less media use compared to the Brazilian adolescents. Furthermore, German adolescents demonstrate a better MRT performance than Brazilian ones, as well as boys show a better performance than girls do. A multiple regression analysis indicated that the MRT performance could be predicted by nationality, sex, and cognitive processing speed. Since cognitive processing speed did not differ between Brazilian and German adolescents, the worse MRT performance of the Brazilian adolescents could be explained by different educational systems. Further studies have to follow which investigate the reasons for the different nations in more detail. Mental rotation is defined as the ability of mental transform of how an object appears if it is rotated from its original position . This spDifferences in spatial abilities between males and females favoring males are clearly established findings, with MRT performance producing the largest effects  and beinStudies that investigated the effects of long-term physical activity on MRT performance found better MRT performance for students of sports sciences compared to students of educational sciences . Also moThere are only a few studies investigating cultural differences regarding Mental Rotation Test performance: Omani students show a worse MRT performance compared to German students , and chiWith regard to cross-cultural cognitive differences between German and Brazilian children, German children scored higher than Brazilian children in intellectual and emotional autonomy in a self-rated survey by school teachers and students , and theThus, the objective of this study is to investigate whether there are performance differences in solving the MRT between Brazilian and German boys and girls and the contributing factors of cognitive processing speed, physical activity, and media usage. According to the literature presented above, we hypothesize (1) that the MRT performance should be better in German than Brazilian adolescents and (2) that the sex differences should be smaller in the Brazilian than German sample. Furthermore, we explore the possible role of nationality, age, sex, cognitive processing speed, sports practice, and media usage in predicting the MRT performance.M = 13.42 years, SD = 1.80), half German , half Brazilian  participated in the current study. Regarding age and sex, the Brazilian children were matched to the German sample.Around 120 children aged between 11 and 17 years . The study was conducted according to the guidelines of the declaration of Helsinki.Demographic data of the participants concerning sex, age, and time spent practicing sport were recorded with a self-generated questionnaire. Hours of practice and use of electronic devices were registered by the questionnaire used in the health behavior in school-aged children (HBSC) study protocol  includinThe number connection test (ZVT)  was admiThe mental rotation test (MRT-A) test is a speeded mental rotation test and appropriate to use for children and adolescents older than 10 years of age . The tesThe pupils were tested in each country in group sessions, in the following order: (1) demographic questionnaire; (2) physical activity and sedentary behavior questionnaires; (3) cognitive processing speed test; (4) mental rotation test.To investigate if there are differences in the physical activity (hours per week) and media behavior (hours per week), two analyses of variance with the dependent variables (1) “hours of sports practice per week” and (2) “hours of media behavior per week” were conducted, with the independent factors “nationality” (German and Brazilian) and “sex” . For the media behavior, the use of electronic devices, TV, video and computer games, chatting, emailing etc., were meant.Two univariate analyses of variance were conducted for the dependent variables (1) “cognitive processing speed,” and (2) “number of correctly solved items in the MRT,” with the independent factors “nationality” (German and Brazilian) and “sex” . Furthermore, a multiple regression on MRT performance with the predictors’ nationality, age, sex, cognitive processing speed, hours of sports practice per week, and hours of media used per week was conducted to investigate the relevant predictors for MRT performance.F = 38.92, p < 0.001, partial η2 = 0.25 , as well as in use of electronic devices likewise for “nationality” F = 33.22, p < 0.001, partial η2 = 0.22 . Both analyses revealed neither an effect for sex, physical activity: F = 2.92, p = 0.09, partial η2 = 0.02, media behavior: F = 0.39, p = 0.53, partial η2 < 0.01, nor an interaction for “nationality” and “sex,” physical activity F = 1.65, p = 0.2, partial η2 = 0.01; media behavior: F = 0.57, p = 0.45, partial η2 < 0.01.The analysis regarding physical activity revealed a significant main effect for the factor “nationality,” F = 1.085, p = 0.300, partial η2 = 0.009 nor for the factor “nationality,” F = 0.73, p = 0.394, partial η2 < 0.006 and neither a significant interaction between both factors, F = 0.17, p = 0.681, partial η2 = 0.001. The ZVT values for the German girls  and boys  did not differ. There was also no difference between the Brazilian girls  and boys .A univariate analysis of variance with the dependent variable “cognitive processing speed” showed no significant differences for the factor “sex,” F = 7.38, p = 0.008, partial η2 = 0.06. Adolescents from Brazil  performed significantly worse than the ones from Germany . There was also a significant effect of the factor “sex,” F = 12.61, p = 0.001, partial η2 = 0.01, but no significant interaction between “nationality” and “sex,” F = 0.503, p = 0.480, partial η2 = 0.004. Boys  showed a better performance than girls , see In the analysis of the standard performance measurement with the MRT, our results show that there was a significant effect of the factor “nationality,” 2R = 19.7%) was explained by the predictors “cognitive processing speed,” “nationality,” and “sex,” F = 5.951, p < 0.001, see The multiple regression with including all variables results on MRT performance with the predictors nationality, age, sex, cognitive processing speed, hours of sports practice per week, and hours of media used per week showed that 23.8% of the variance . According to this, the quasi-experimental study was conducted according to the guidelines of the declaration of Helsinki.PJ designed the study, analyzed the data, and wrote the first draft of the paper. SH helped to design the study, organized the data acquisition, and discussed the first draft of the paper. FP and SM acquired the data and discussed the first draft of the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


One of the most common complications following autologous cranioplasty is bone flap resorption (BFR). Severe BFR can lead to revision surgery with implantation of synthetic bone flap and also necessarily lead to higher hospital expenses. This study aims to perform a meta-analysis to summarize available evidence regarding risk factors of BFR requiring a second surgery in patients with autologous cranioplasty.Cohort, case-control, and cross-sectional studies that report the incidence and risk factors of BFR among patients with autologous cranioplasty, published in English, will be considered for selection. Three databases from inception to May 2020 will be searched. The process of data selection, quality assessment, and data extraction will be assessed by 2 authors independently. The study quality will be assessed by Newcastle-Ottawa Scale (NOS) and Agency for Healthcare Research and Quality checklist.The statistical analysis of this meta-analysis will be calculated by Review manager version 5.3.The results of this systematic review and meta-analysis will be disseminated through academic conferences and expected to publish in a peer-reviewed journalThis study will offer high-quality evidence about risk factors for BFR after autologous cranioplasty.INPLASY202050063. The reported prevalence of BFR with autologous cranioplasty was varied significantly, up to 50% in previous studies.,12 BFR can lead to weakening, loosening and significant disintegration of the implanted autologous bone, which eventually results in loss of the bone coverage. Revision surgery with replacement of synthetic material is necessary in severe cases of BFR and second surgery could be associated with higher expenses and poor clinical outcomes.,15 It would be reasonable to identify high-risk group that might suffer BFR and take preventive measures or choose alloplastic material cranioplasty for those patients.Despite the advantages and simple technique, cranioplasty can carry high rates of complication.,16–18 Other potential risk factors, such as bone flap size, preservation of bone flap, and time interval between DC and cranioplasty,17,19,20 need to be assessed in a systematic approach. Therefore, we will undertake a systematic review and meta-analysis of studies presented data on risk factors for BFR requiring a second surgery after autologous cranioplasty.The risk factors of BFR, however, remains unclear, and the data is not comprehensive and no systematic review with respect to the prevalence rates and risk factors of BFR has been implemented. Previous reports have found that younger age, bone flap fragmentations, and hydrocephalus shunt implantation to be associated with higher incidence of BFR.22.1https://inplasy.com/inplasy-2020-5-0063/) and the study registration number is INPLASY202050063. It is reported to be in line with the meta-analysis of observational studies in epidemiology (MOOSE) guidelines and the Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocol.,22 If adjustments are needed throughout the study, we will update the details in the final version.This systematic review protocol has been registered on the INPLASY website  and cross-sectional studies will also be included. The language of literature will be limited in English, but there will be no restriction on publication data. Case report, letters, conference abstracts, reviews, non-clinical research, technical note will be excluded.2.3.2In study group, any patients should be diagnosed with BFR requiring a second surgery after autologous cranioplasty with no restrictions on ethnicity, sex, or nation. People in control group should be patients without BFR or those patients with BFR but not requiring a revision surgery with implantation of synthetic materials. The diagnosis of BFR will be based on valid clinical and radiographic findings.2.3.3The outcomes should be explicitly reported as the followings:1.Incidence of BFR;2.Study reported at least 1 risk factor for BFR requiring a second surgery;3.Study reported findings in terms of risk estimate  or provided sufficient data to calculate. Those studies that risk estimates cannot be directly extracted or obtained will be excluded.2.4A systematic and comprehensive search will be carried out in 3 databases, including PubMed, Embase, Cochrane Library database, from the inception to May 1, 2020. Detailed search strategy of PubMed will be ((((cranioplasty[Title/Abstract]) OR post-cranioplasty[Title/Abstract])) AND (((((autologous[Title/Abstract]) OR bone[Title/Abstract]) OR autologous bone[Title/Abstract]) OR autogenous[Title/Abstract]) OR autograft[Title/Abstract])) AND ((((resorption[Title/Abstract]) OR necrosis[Title/Abstract]) OR bone resorption[Title/Abstract]) OR bone necrosis) The search strategies for other electronic databases will be modified appropriately.2.5Two independent members in our group will select all of the studies and import them into Endnote version X8 software to manage. First, the duplicated studies will be removed. Then, the 2 authors will independently screen all potentially qualified studies by reading titles and abstracts. Studies will be excluded if they do not meet inclusion criteria. Finally, the 2 authors will screen the full text and determine the final qualified articles that in line with the inclusion and exclusion criteria. The 2 reviewers will crosscheck the included studies, disagreements in the process will be resolved after mutual discussion. If no agreement is reached, the third author will individually evaluate the matter. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses Flowchart Fig.  will be 2.6For eligible studies, data were independently extracted into Microsoft Excel by 2 authors. The following information were collected: name of the first author, study period, preservation of the bone flap, where the study was conducted, study design, sample size, mean (median) age of participants, the rate of bone flap resorption. We will extract the risk factors if they reach statistical significance of 5% in univariate and multivariate analyses. The risk estimates with a 95% confidence interval (CI) were extracted for each risk factor or the absolute number of case and control for each risk factor were available. If data is missing, we will email the corresponding author to ask for assistance.2.7 which contains 3 categories: 4 items for patients selection, 1 item for study comparability and 3 items for outcomes assessment. The score is classified into 3 scales: 7-9 defined as good, 5-6 is fair quality, and 0-4 is poor quality.All included studies will be cohort, case-control and cross-sectional studies. The quality of included studies will be independently assessed by 2 authors and possible discrepancies will be adjusted by a third author. Quality assessment will be conducted according to guidance of Newcastle-Ottawa Scale (NOS),2.82.8.1Meta-analysis will be performed using Review manager version 5.3. The outcome measures for the meta-analysis will be risk factors associated with BFR requiring revision surgery. If relevant risk factors are reported in 2 or more studies, the pooled odds ratio with 95% confidence intervals will be calculated. If the risk factors could not be included in this meta or reported only once, the results will be presented separately or in discussion part.2.8.2I2 index. When P value <.10 or the value of I2 < 50%, studies will not be considered heterogeneous and a fixed effect model will be adopted in the meta-analysis; otherwise, a random effects model will be applied. In the case of heterogeneity, the quantitative synthesis is not appropriate, the results will be presented in tables or charts.Heterogeneity of the studies will be assessed using Cochrane Q test and 2.8.3If the results of meta analyses are heterogenous, we will perform a subgroup analysis based on several aspects, such as race, study country, study year, different bone flap preservation, and study quality.2.8.4The sensitivity analysis is used to evaluate the robustness and stability of conclusions. It will be conducted by removing low-level quality study one by one and then merges the data to probe the sample size, study quality, and missing data on results of the study.3BFR is one of the most common complication following autologous cranioplasty, which could lead to prolonged hospital stay and neurological deterioration and economic burden. Recently, there has been a growing number of studies about identification of risk factors and potential strategies for lowering BFR rates, avoiding a second surgery. The results are different and no studies have summarized the existing evidence. Therefore, the purpose of this systematic review and meta-analysis is to summarize the evidence from previous researches and investigate potential risk factors for BFR. For those patients with risk factors, prevention strategies or implantation with alloplastic material are necessary.There are strengths in this study. This is the first meta-analysis to summarize findings about risk factors for BFR, which could provide clear evidence for clinical work and improve clinical outcomes. However, there may be some limitations in our meta-analysis. First, we only search 3 international database that may lead to selection bias. Second, the included types of studies are varied, for example RCTs, case control studies and cohort studies, this may cause substantial heterogeneity. Third, the methods of bone flap preservation are different, this may also be a source of heterogeneity.In conclusion, this study will help to identify risk factors for BFR after autologous cranioplasty. We hope this systematic review and meta-analysis can provide a high evidence for predictions for BFR and guide future clinical works.Conceptualization: Jingguo Yang, Junwen Guan.Data curation: Jingguo Yang, Yikai Yuan, Yicheng Zhou.Methodology: Tong Sun.Project administration: Xuepei Li.Writing – original draft: Jingguo Yang.Writing – review & editing: Junwen Guan.


The Kessler Foundation Neglect Assessment Process (KF-NAP) is an assessment tool for unilateral spatial neglect (USN), which is the scoring method for the Catherine Bergego Scale (CBS) based on detailed instructions. This study is aimed at determining the reliability and validity of the Japanese version of the KF-NAP (KF-NAP-J), evaluating the improvement of neglect assessment with KF-NAP-J, and comparing it with the original CBS for subacute stroke patients. We assessed subacute stroke patients admitted to our intensive rehabilitation hospital. Two KF-NAP-trained occupational therapists (OTs) assessed 22 patients. Before implementing the KF-NAP at the hospital, two other OTs assessed the other 23 patients using the CBS. We evaluated the interrater reliability of the KF-NAP and CBS using intraclass correlation coefficients (ICC) for the total scores, weighted kappa statistics for each subscale, and internal consistency using Cronbach's alpha. We assessed the validity of the KF-NAP against the Behavioral Inattention Test (BIT) and Functional Independence Measure (FIM) using Spearman's correlation coefficient. The reliability of both the KF-NAP and CBS was excellent. The weighted kappa results demonstrated that each subscale was in better agreement with the KF-NAP than with the CBS. In the KF-NAP, all eight subscales in which weighted kappa could be calculated were in significant agreement, and two were almost in perfect agreement. The KF-NAP moderately correlated with the subscales of BIT and FIM representing USN and activities of daily living. The USN detection rates of KF-NAP and BIT in the KF-NAP group were 63.6% and 22.7%, respectively. These results suggest that the KF-NAP, as well as the CBS, is useful to assess USN, which strongly impacts the rehabilitation outcomes in subacute stroke patients. Unilateral spatial neglect (USN) is defined as a failure to report, respond, or orient to novel or meaningful stimuli presented to the side opposite a brain lesion, when this failure cannot be attributed to either sensory or motor defects . This diChen et al. proposed the Kessler Foundation Neglect Assessment Process (KF-NAP) , 14, whiOur hospital is a rehabilitation hospital in Japan that provides intensive rehabilitation for subacute stroke patients hospitalized within 2 months after onset. In this phase, most patients are in the process of functional recovery, and improvement of USN strongly affects rehabilitation outcomes . Therefok = 2) assessment, 95% confidence interval (CI), and CI width 0.4; we calculated the minimum sample size as n = 20. We determined that 20 or more participants were required.We recruited participants from patients with cerebral stroke who were admitted to Saiseikai Higashi-Kanagawa Rehabilitation Hospital from July 2018 to June 2019, before implementing KF-NAP-J at our hospital, for the CBS group, and from July 2019 to August 2020 for the KF-NAP group. A chief occupational therapist, who did not participate in this study, randomly assigned patients for screening. We included participants admitted within 2 months after the onset of stroke and who could follow the examiner's instructions. We excluded participants whose behavior could not be assessed with the CBS or the KF-NAP-J due to severe aphasia, severe cognitive impairment, severe deafness, or blindness. For the CBS group, we screened 25 stroke patients, and 23 patients fulfilled the inclusion criteria. For the KF-NAP group, of the 39 patients who were screened, 26 fulfilled the inclusion criteria. Four patients were excluded: three had severe aphasia, and one was severely deaf. Therefore, we eventually recruited 23 participants in the CBS group and 22 in the KF-NAP group. We calculated the sample size according to the study by Doros . We assuExperiments were performed according to the Declaration of Helsinki and were approved by the Saiseikai Higashi-Kanawa Rehabilitation Hospital Research Ethics Committee . All participants provided written informed consent.The KF-NAP was translated into Japanese (forward translation) by a professional life science translator. The comparison and panel discussion led to the primary Japanese version. The translators translated the Japanese form back to English (backward translation), and this was checked and verified by members of the Kessler Foundation who developed the KF-NAP. Finally, the committee comprised two physiatrists and two occupational therapists, who compared the original English form and the obtained form to produce the final Japanese version (KF-NAP-J).The CBS  was baseThe KF-NAP , 14 was The BIT is a standard test used to assess USN and consists of conventional and behavioral tests. The conventional test consists of six subscales , with a score ranging between 0 and 146; a higher score indicates a better spatial awareness. In the original version, the cut-off score is 129 , whereasThe functional independence measure (FIM), which was developed to ensure uniformity in assessing the ADLs, includes motor and cognitive subscales and is subdivided into 18 items . For eacThe Mini-Mental State Examination (MMSE) is a simple examination used to evaluate the decline in cognitive function  and is conducted using a verbal questionnaire . It is cTwo OTs (who had not participated in translating the KF-NAP) assessed participants using the CBS (CBS group). Two other OTs who were members of the KF-NAP-J translation team and trained in KF-NAP assessment assessed participants using the KF-NAP-J (KF-NAP group). The raters of both the CBS and the KF-NAP-J possessed similar clinical experience and skills. The ADLs in both groups were assessed using the FIM. The USN of the KF-NAP group was assessed using the conventional BIT.t-test , and FIM) and chi-squared test . The level of statistical significance for the variables was set at 0.05.The two groups were compared by performing the unpaired Interrater reliability was calculated employing intraclass correlation coefficients (ICC) for total scores and weighted kappa coefficients for each subscale. Internal consistency was calculated using Cronbach's alpha. The level of statistical significance for the variables was set at 0.05.The KF-NAP was evaluated for convergence and discriminative validity of Rater 1. Convergence validity was obtained using the Spearman correlation coefficient of the KF-NAP against conventional BIT and FIM. The level of statistical significance for the variables was set at 0.05.In the KF-NAP group, we calculated and compared the percentage of patients detected with USN symptoms by KF-NAP and the percentage of patients who were below the cut-off point with the BIT conventional test by Rater 1. In addition, we calculated the detection rates of the injured side in the participants assessed using the KF-NAP and BIT.Data analysis was performed using IBM SPSS Statistics for Mac, Version 23 .P > 0.05). The CBS group had more patients with left hemisphere damage and lower MMSE scores than the KF-NAP group. Four participants in the CBS group could not participate in MMSE assessment due to motor aphasia. However, CBS assessment in these participants could be performed because they could understand and follow the instructions.The demographic data of all participants are presented in The ICC and Cronbach's alpha of the two groups are shown in The weighted kappa coefficients of each subscale of the KF-NAP and CBS are shown in The order of the CBS was changed to correspond with that of KF-NAP.The correlation between the KF-NAP and conventional BIT is shown in The correlation between the KF-NAP and FIM is shown in 1The correlation between the KF-NAP and FIM is shown in The USN detection rates of KF-NAP and BIT in the KF-NAP group are shown in We evaluated USN using the KF-NAP in subacute stroke patients during rehabilitation in Japan. In the Japanese healthcare system, stroke patients are transferred to a rehabilitation hospital within 60 days, and the time between stroke onset and admission to rehabilitation hospitals is generally 3 to 5 weeks. Subsequently, patients receive intensive rehabilitation treatment for several months.In this study, both interrater reliability and internal consistency were slightly better in the KF-NAP group than in the CBS group. Each subscale was generally in better agreement in the KF-NAP group than in the CBS group. Previous studies reported that both CBS , 10 and In this study, weighted kappa coefficients of two subscales (auditory attention and navigation) could not be calculated because one rater assigned the same score (zero) on the subscales for all the participants. However, the other rater also scored zero for all except two participants on the auditory attention (scored 1) and except for one (scored 1) on the navigation. Therefore, even these two subscales were in good agreement between the raters. Although most subscales of KF-NAP were in moderate or better agreement, only the personal belonging subscale remained fair even with KF-NAP, possibly due to the assessment method and the space constraints in the Japanese hospital room setting. We assessed the personal belonging subscale from 3 to 6 based on the patients' daily use of personal belongings placed in the room and assessed their behavior. It is difficult to evenly position personal belongings around a patient on the left and right because of limited personal space in the hospital rooms in Japan. Therefore, it is challenging to appropriately arrange the room for the assessment. The interrater reliability of the subscales was less consistent than in previous reports both in the KF-NAP and the CBS. The possible reasons are as follows: (1) the participants were in the subacute intensive rehabilitation ward, and most of the patients experience improvement in their function and abilities day by day, and the symptoms of USN fluctuated. Therefore, we expect that the scores would be significantly different between raters compared with those of patients during the acute phase, as reported in previous studies. (2) Most participants with USN had mild to moderate symptoms. Potentially, in these patients, USN symptoms would have been impacted by intensive rehabilitation more rapidly than in patients with more severe USN, and (3) the raters were not adequately trained, which could be improved by thorough education.In our study, KF-NAP-J was correlated with the line cancellation test, star cancellation test, and figure and shape copying of BIT. Azouvi et al.  reportedKF-NAP-J was also correlated with motor and total FIM but not with cognitive FIM. Chen et al.  showed tThe USN detection rate of the KF-NAP was higher than that of the BIT. Subacute patients receiving rehabilitation treatment may learn to compensate for USN symptoms in the visual search task. In addition, paper-and-pencil tests tend to improve when the tests are repeated regardless of improvement of USN . TherefoIn patients with left hemisphere damage, the USN detection rate of KF-NAP was considerably higher than that of BIT. No USN patients were detected using BIT, whereas 60% were diagnosed with USN by KF-NAP. It is difficult to evaluate USN using paper-and-pencil tests if the patients have aphasia and/or severe dominant hand paresis. However, right-sided USN of patients with left hemisphere damage equally predicts poor functional outcomes as left-sided USN , 4. TherUSN is a strong negative predictor of poor rehabilitation outcomes , 6, 24, One of the main limitations of this study is the small sample size. There were only 22 participants in the KF-NAP group and 25 participants in the CBS group. Therefore, we could not match the lesion side and distribution of severity between the two groups. Future studies with a large number and a wide spectrum of participants are required to ascertain the reliability and validity of the method. In addition, we assigned the participants and the raters to either the CBS group or the KF-NAP group. Ideally, to effectively compare the reliability between the CBS and KF-NAP is to assess the same participants by two KF-NAP trained raters and two other raters trained with the original CBS but not KF-NAP trained. However, it is also difficult to retain blindness and independence among raters if many raters assess the same participants in a small hospital. Therefore, we initially separated the raters for the two groups, and two raters assessed participants in the CBS group, whereas the other two raters were involved in translating and KF-NAP training. Owing to these limitations in the study design, we cannot confirm the validity and reliability based on the results of this study only. Despite these limitations, the investigators believe that the KF-NAP-J is useful for assessing CBS in stroke patients undergoing subacute rehabilitation.In this study, the KF-NAP exhibited good interrater reliability and correlated with the subscales of BIT and FIM, which represent USN and ADLs in subacute stroke patients. Unilateral spatial neglect strongly impacts rehabilitation training and its outcomes, therefore requiring accurate assessment of USN. In addition, behavioral testing is even more important in patients with left hemisphere damage because it facilitates assessment even if the patients experience aphasia and paralysis of the right hand. These results suggest that KF-NAP and CBS are useful in assessing USN in subacute stroke patients.


Ovis aries) from Qinghai-Tibetan Plateau. Bacteroidetes and Spirochaetae were enriched in sub-adult sheep, while Firmicutes and Tenericutes were more abundant in young individuals. The sub-adult individuals had higher alpha diversity values than those in young sheep. Metabolomics analysis showed that the content of essential amino acids and related gene functional pathways in rumen were different between the lambs and sub-adult population. L-Leucine that participates in valine, leucine and isoleucine biosynthesis was more abundant in the lambs, while phenylethylamine that takes part in phenylalanine metabolism was more enriched in the sub-adults. Both rumen microbial community structures and metabolite profiles were impacted by age, but rumen SCFA concentration was relatively stable between different age stages. Some specific microbes  were positively associated with L-Leucine but negatively correlated with phenylethylamine, implying that rumen microbes may play different roles for metabolite production at different ages. Mantel test analysis showed that rumen microbiota was significantly correlated with metabolomics and SCFA profiles. Our results indicates the close relationship between microbial composition and metabolites, and also reveal different nutritional requirement for different ages in ruminants, thus having important significance for regulating animal nutrition and metabolism by microbiome intervention.The rumen microbiota plays an important role in animal functional attributes. These microbes are indispensable for the normal physiological development of the rumen, and may also convert the plant polysaccharides from grass into available milk and meat, making it highly valuable to humans. Exploring the microbial composition and metabolites of rumen across developmental stages is important for understanding ruminant nutrition and metabolism. However, relatively few reports have investigated the microbiome and metabolites across developmental stages in ruminants. Using 16S rRNA gene sequnecing, metabolomics and high-performance liquid chromatography techniques, we compared the rumen microbiota, metabolites and short chain fatty acids (SCFAs) between lambs and sub-adult Tibetan sheep ( Ruminants have a close relationship with their symbiotic microorganisms in the digestive tract. These microbes include bacteria, archaea, fungi, viruses, and protozoa , which pRuminococcus and Fibrobacter) in the bovine had a low abundance in the newborn (1–3 days) individuals, while had higher abundance in the adult sheep and maintained a relatively constant level , which can extract many metabolites from samples and uncover the metabolic phenotypes in humans , animalsOvis aries) are one of the most widely distributed and numerous livestock on the Qinghai-Tibetan Plateau, as their population size reaches more than 50 million  and 6-month-old Tibetan sheep  in the same cohort. Three portions (~5 g per portion) of the contents from the anterior, middle and posterior parts of the rumen were taken and mixed well-before sample collection. These Tibetan sheep were all males. The sub-adult individuals were grazed on pasture  on the Qinghai-Tibet Plateau, and supplied commercial feed#8876  at dusk. The main nutritional components of this commercial feed include crude protein ≥ 16%, crude fat ≥ 3%, crude fiber ≤ 8.0%, and crude ash ≤ 9.0%. The daily feed intake of sub-adult individuals was 4.75 ± 0.32 kg. The lamb individuals were mainly fed milk, and also ate a small amount of the grass (~0.72 ± 0.0.07 kg) and commercial feed (~0.54 ± 0.0.12 kg) mentioned above. Drinking water was freely available to these Tibetan sheep. The body weight of lamb and sub-adult groups was 15.75 ± 5.90 and 26.35 ± 4.01 kg, respectively. After collection, the ruminal contents were immediately divided into three parts on ice for the following microbiome, metabolome and short chain fatty acid (SCFA) analysis, and were temporarily kept in −20°C portable refrigerator in field. Finally, all samples were transferred to our lab within 24 h and stored at −40°C refrigerator.Rumen contents were collected between February and July from the Haibei Demonstration Zone of Plateau Modern Ecological Animal Husbandry . After the sheep were anesthetized using diethylether and dissected, we obtained a total of 12 rumen samples from 1-month-old  was applied to extract total microbial DNA from the ruminal contents according to the product manual. Thereafter, Qubit 2.0 Fluorometer  was used to detect the concentration of DNA samples. The V3-V4 regions of 16S rRNA gene were amplified using the primer pairs 341F(CCTACGGRRBGCASCAGKVRVGAAT) and 806R (GGACTACNVGGGTWTCTAATCC) . PCR amphttp://qiime.org/scripts/index.html) from our Microbiome and Bioinformatics Platform in School of Public Health, Lanzhou University. The detailed analysis methods and procedures were described in our previous reports with slight modification , were calculated using QIIME Pipeline. Unweighted UniFrac distance matrice is based on the absence or presence of OTUs, while weighted UniFrac is dependent on the relative abundance of OTUs . Princip2O (4°C) were added into the tubes. The mixture was thoroughly vortex-mixed for 60 s. Thereafter, 1,000 ul of methanol (pre-cooled at −20°C) was added into the samples, and the mixed liquids were shaken for 30 s. Thereafter, we further placed the tubes into an ultrasound machine at room temperature for 10 min, and then stew for 30 min on the ice. The samples were centrifuged for 10 min at 14, 000 rpm 4°C, and then 1.2 mL supernatant was transferred into a new centrifuge tube. Samples were further blow-dried by vacuum concentration. Thereafter, samples were dissolved using 400 μl methanol aqueous solution , and underwent 0.22 μm membrane for filtration. For the quality control (QC) samples, 20 μL of prepared samples were extracted and mixed. These QC samples were used to monitor deviations of the analytical results from these pool mixtures. Finally, samples were ready for LC - MS  detection. More detailed methods for LC-MS procedures have been described in the previous report . Then the metabolomic data underwent peaks identification, filtration and alignment using the XCMS package in R (v3.3.2). In order to compare data of different magnitude, peak area was normalized for further statistical analysis .Short chain fatty acid (SCFA) profiles of ruminal content samples were measured using an Agilent 1100 series high-performance liquid chromatography (HPLC) system . We measured the concentration of acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and hexanoate using an Alltech IOA-2000 organic acid column. The detailed procedures for measuring SCFAs have been described previously .U-tests were used. Permutational multivariate analysis of variance (PERMANOVA) analysis based on the unweighted and weighted UniFrac distance matrices was used to evaluate whether the community structures were significantly distinct between groups using the procedure “Adonis” in the R “vegan” package. In order to identify significant taxonomic differences of rumen microbiota between groups, we used LEfSe analysis at phylum, genus and OTU level. This method considers both biological and statistical significance of data. Linear discriminant analysis (LDA) effect size (LDA > 2) was applied to assess the magnitude scale of the effect of differentially abundant taxa. This analysis was performed at Galaxy module of LEfSe .To test the differences of the alpha diversity indices (phylogenetic diversity and observed OTUs) of ruminal microbiomes, Mann–Whitney P < 0.05) between the groups ES and SS. The identification of these compounds was based on their MS/MS spectra, online databases and literature. Subsequently, the related metabolic pathways of the above different metabolites were analyzed based on Hypergeometric test using the online software MetaboAnalyst . The impacting factor plots of KEEG (Kyoto Encyclopedia of Genes and Genomes) based on those different metabolites were visualized.For metabolomic data, the detailed analysis methods were described in Li et al. . Brieflypost-hoc test in SPSS13.0 was used to analyze the differences of SCFA concentration of sheep ruminal contents between young and sub-adult groups. The spearman correlation analysis between rumen bacterial genera and SCFAs, differential metabolites was also performed using SPSS13.0. Heatmap2 was used to visualize the correlation results. In addition, mantel test was used to detect the relationship between rumen microbiota and metabolic profile or SCFAs using the “mantel” procedure in the R vegan package.One-way analysis of variance (one-way ANOVA) with Tukey's PRJEB36249 (http://www.ebi.ac.uk/ena/data/view/PRJEB36249).All the original 16S rRNA gene data in this study were submitted to the European Nucleotide Archive, and were available by accession NO. Ruminococcaceae_UCG-005 (10.22%), Bacteroides (8.18%), Rikenellaceae_RC9_gut_group (7.58%), two unknown genus from Lachnospiraceae (7.42%) and Ruminococcaceae_UCG-010 (5.77%). The rumen microbial composition of each sample was visualized in We obtained 698,333 original paired-end reads across all samples  from MiSeq sequencer. A total of 800 unique OTUs were detected in the Tibetan sheep rumen across all samples using VSEARCH clustering. By taxonomic classification, we found that the rumen microbiota was dominated by Firmicutes (mean relative abundance = 56.57%), Bacteroidetes (39.46%), Proteobacteria (1.88%) and Verrucomicrobia (1.24%), followed by other rare phyla (mean relative abundance <1%), such as Spirochaetae, Actinobacteria, Tenericutes and Saccharibacteria. At genus level, five most abundant genera were Clostridium_sensu_stricto_1, Turicibacter and Ruminococcaceae_UCG_010. The SS (or sub-adult) group was enriched by 22 genera, including Escherichia_Shigella, Prevotellaceae_UCG_001, Succinivibrio and Treponema_2. At OTU level, a total of 27 OTUs were more abundant in the ES group, these OTUs were mainly affiliated with Clostridium_sensu_stricto_1, Ruminococcaceae, Barnesiella, Akkermansia and Eubacterium. In contrast, 26 OTUs showed a higher abundance in the SS group, these OTUs mainly belonged to Lachnospiraceae, Succinivibrio, Rikenellaceae_RC9_gut_group, Ruminococcaceae, Eubacterium and Bacteroidales. In addition, we compared the alpha diversity values between the ES and SS groups. The rumen microbiota of sub-adult sheep had higher phylogenetic diversity and observed OTUs .Using LefSe analysis, we compared the significant different bacteria taxa between developmental stages of Tibetan sheep at phylum, genus and OTU level . We founMetabolic biomarker can reveal the significantly different metabolites between young and sub-adult Tibetan sheep. A total of 32 biomarkers were identified between the two groups . Among tAccording to the PCA or PLS-DA plots, we found that the young sheep had significantly different metabolomic profiles with sub-adult individuals regardless of ion mode -Hexenal, 3-Hydroxy-3-Methylglutaric acid, glycochenodeoxycholic acid, 5,6-Dihydrouracil, 9(S)-HpOTrE, barceloneic acid A, galactitol, and L-Leucine, while this genus showed negative correlations with 16 metabolites .Some rumen microbes were associated with metabolites . For exar = 0.296, P = 0.028; negative ion mode, r = 0.343, P = 0.019). In addition, SCFA profiles also showed significant associations with rumen microbiota .Mantel test was also used to detect the relationship between rumen microbiota (using weighted UniFrac distance) and metabolomic or SCFA profiles. We found that rumen microbial communities were significantly correlated with metabolomic profiles (positive ion mode, Most of previous reports on ruminants only focused on rumen microbiome, but lacked functional and metabolic information. Here, we investigated the relationship among age, rumen microbiome, metabolomics and SCFA profiles. Our data indicate that age significantly influence the rumen microbiome and metabolomic profiles, but had no impacts on SCFA concentrations. In addition, we found that rumen microbial communities were significantly correlated with metabolomic profiles and SCFAs. These results have important significance for understanding nutrition and metabolism of ruminants on the Qinghai Tibetan Plateau.The rumen microbial diversity in the sub-adult Tibetan sheep was more diverse than that in the young individuals. This finding is consistent with those of previous studies , 26, whiIn addition, the community structures of rumen microbiota were also significantly divergent between the young and sub-adult Tibetan sheep. These results were consistent with those in previous studies , 26, whiTreponema, Ruminococcus and several unknown genera from Ruminococcaceae and Spirochaetaceae were enriched in the sub-adult Tibetan sheep. Some members of these bacterial taxa were involved in the degradation of plant cellulose, hemicellulose, and chitin (Barnesiella, Akkermansia and Eubacterium were enriched in the young sheep. Some members of Barnesiella have also been isolated from human feces (Barnesiella intestinihominis may activate anti-cancer immune response of spleen T cell, and thus inhibit tumor growth and improve chemotherapy effect (Eubacterium, especially Eubacterium hallii, may increase fecal butyrate concentrations and modify bile acid metabolism. More importantly, it can improve the insulin sensitivity in diabetic mice, thus contributing potential benefits to animal health (Akkermansia is mucin-degrading bacteria, and may protect hosts against pathogen invasion. The abundance of this genus in gut microbiome is positively correlated with health and metabolic ability of hosts (Our data showed that Bacteroidetes were more enriched in the sub-adult individuals, while Firmicutes were more abundant in the young sheep. This finding was partly consistent with that in Jami et al. , who foud chitin . At OTU an feces , and arey effect . Eubactel health . Akkermaof hosts , 35, thuOur data showed that the metabolomics profiles were significantly different between the young and sub-adult Tibetan sheep based on PCA or PLS-DA analysis, indicating that age is likely to influence the metabolic profiles of animals. These results were consistent with those in Li et al. , who alsSeveral organic acid were more abundant in the young sheep, such as barceloneic acid A, terephthalic acid, 3-Hydroxy-3-Methylglutaric acid, fumaric acid, and glycochenodeoxycholic acid. These organic acids probably play important roles in host development and metabolism. For example, it has been reported that terephthalic acid was tightly correlated with the ruminal development of lambs . Glycochin vivo. For instance, acetate can been transported to peripheral tissues, and then to liver for participating in cholesterol synthesis (Staphylococcus aureus internalization into bovine mammary epithelial cells and regulate antimicrobial peptide gene expression (SCFAs are the end metabolic products of plant cellulose and other dietary polysaccharides via rumen microbial fermentation. These metabolites contributes to energy and nutrition of ruminants. For example, butyrate can supply ~60–70% energy for normal epithelial cells . Besidesynthesis . Propionynthesis . In addipression , 49. OurOur results showed that rumen microbiota structure was associated with metabolic profiles. This result was congruent with that in goat , fish 5, human , gorillaIn conclusion, we found that both rumen microbial community structures and metabolite profiles of Tibetan sheep were distinct at different ages, but rumen SCFA concentration was relatively stable between the two age stages. Notably, metabolomics analysis showed that the nutritional requirement of essential amino acids in rumen were different between the lambs and sub-adult Tibetan sheep. We found L-Leucine that participates in valine, leucine and isoleucine biosynthesis was more abundant in the lambs, while phenylethylamine that takes part in phenylalanine metabolism was enriched in the sub-adults. In addition, rumen microbita was associated with metabolomics and SCFAs, indicating the close relationship between microbial composition and metabolites. These results have important significance for regulating animal nutrition and health by microbiome intervention.PRJEB36249.All the original 16S rRNA gene data in this study were submitted to the European Nucleotide Archive, and were available by accession no. Animal ethics approval for the present project was obtained from the Animal Ethics Committee of Lanzhou University.HL and JQ designed this study. JQ carried out the animal experiments and sample collection. HL was responsible for the molecular experiments, data analysis, and wrote the original manuscript. All authors revised and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.