Magnesium, an essential mineral for human health, plays a pivotal role in the cardiovascular system. Epidemiological studies in the general population have found an association between lower dietary magnesium intake and an elevated risk of cardiovascular events. In addition, magnesium supplementation was shown to improve blood pressure control, insulin sensitivity, and endothelial function. The relationship between magnesium and cardiovascular prognosis among patients with chronic kidney disease (CKD) has been increasingly investigated as it is becoming evident that magnesium can inhibit vascular calcification, a prominent risk of cardiovascular events, which commonly occurs in CKD patients. Cohort studies in patients receiving dialysis have shown a lower serum magnesium level as a significant risk for cardiovascular mortality. Interestingly, the cardiovascular mortality risk associated with hyperphosphatemia is alleviated among those with high serum magnesium levels, consistent with in vitro evidence that magnesium inhibits high-phosphate induced calcification of vascular smooth muscle cells. Furthermore, a harmful effect of high phosphate on the progression of CKD is also attenuated among those with high serum magnesium levels. The potential usefulness of magnesium as a remedy for phosphate toxicity should be further explored by future intervention studies. Magnesium is an essential mineral for human health and acts as a co-factor for more than 500 enzymatic reactions in the body. Dietary magnesium intake in developed countries has decreased over the past decades owing to the increased consumption of low-magnesium diets, such as processed foods and fast foods; as a result, more than half of the US population do not meet the estimated average requirement of dietary intake of magnesium . Meta-anDespite this evidence implicating magnesium as a protective mineral for the cardiovascular system, this divalent cation has received little attention in the field of chronic kidney disease (CKD). However, the low magnesium status may also be unfavorable for CKD patients since it is associated with inflammation, atherosclerosis, and intradialytic hypotension among patients undergoing hemodialysis ,14. MoreVascular calcification is one of the most important aspects of the mineral and bone disorders of CKD. In particular, coronary artery calcification strongly predicts an incidence of cardiac events and mortality in patients with CKD ,21,22,23Spiegel et al. have reported that magnesium supplementation for hemodialysis patients may be useful to suppress the progression of coronary artery calcification in a small-scale uncontrolled trial . RecentlAnimal studies, both in CKD and non-CKD models, suggested that magnesium inhibits vascular calcification in vivo although it has long been unclear whether magnesium directly acts on calcification ,32. Rece50) in serum, a strong predictor of mortality in patients with kidney disease [The inhibitory effect of magnesium on vascular calcification may also be attributed from its function in the extracellular space. In the circulation, where calcium and phosphate are supersaturated, some kinds of proteins, such as Fetuin-A, interact with a calcium ion on the surface of the calcium phosphate cluster to form calciprotein particles (CPPs) that have lower cytotoxicity compared with a naked calcium-phosphate crystal . CPPs ar disease . In thisBased on the evidence discussed above, it is anticipated that magnesium is protective against cardiovascular risk of patients with CKD. To test this hypothesis, we examined the relationship between serum magnesium levels and cardiovascular mortality risk in a cohort of 142,555 patients undergoing hemodialysis with a one-year follow-up period . As expeGiven the in vitro evidence showing the potent protective effect of magnesium against phosphate-induced vascular calcification, it can be assumed that the influence of magnesium on the cardiovascular risk is particularly manifested among patients with high phosphate levels. In fact, there is a significant interaction between serum levels of magnesium and phosphate on the cardiovascular mortality risk of hemodialysis patients; the risk associated with hyperphosphatemia was aggravated in those with lower serum magnesium levels, whereas the risk was substantially attenuated in those with higher serum magnesium levels  Figure . This re2 [The counteracting action of magnesium against the phosphate-induced pathological condition, as evident in vascular calcification, might also be exerted to other target organs of phosphate toxicity. It has long been known that excess phosphate causes tubular injury and interstitial fibrosis in animal models of CKD ,61,62,632 . While s2 . Consist2 . This evThe magnesium content in the body is tightly regulated by an orchestrated interaction between the intestines, kidneys, and bones . Magnesi(1) Low Magnesium IntakeMagnesium is abundantly found in green leafy vegetables, seaweeds, beans, legumes, and nuts. Processed foods and fast foods are poor in magnesium. Although severe magnesium deficiency due to low dietary magnesium intake is uncommon, except for patients who are maintained on magnesium-deficient parenteral nutrition, mild deficiency is becoming prevalent as the consumption of processed foods and fast foods has increased, especially in developed countries . (2) Reduced Gastrointestinal AbsorptionIntestinal absorption of magnesium mainly occurs in the jejunum, ileum, and colon through both an active transcellular transportation and passive paracellular diffusion. When magnesium intake is low, the active transport pathway via TRPM6 becomes dominant. Loss-of-function mutations of TRPM6 is a cause of autosomal recessive familial hypomagnesemia with secondary hypocalcemia. Recently, it has become widely recognized that use of proton pump inhibitors induces hypomagnesemia even in hemodialysis patients ; the und(3) Enhanced Urinary ExcretionAbout 70% of magnesium in the plasma is filtered by the glomeruli. Filtered magnesium is reabsorbed in the proximal tubules (10%–25%), the loop of Henle (50%–70%), and distal convoluted tubules (~10%) where fine-tuning of the total magnesium reabsorption takes place by TRPM6. Fractional excretion of magnesium (FEMg) can be as low as 0.5%. On the other hand, FEMg of more than 4% in the presence of hypomagnesemia is considered as renal magnesium wasting. In addition to genetic disorders, there are many clinical factors that cause renal magnesium wasting. Among them, the most frequent cause is drug-induced hypomagnesemia , etc.). Insulin resistance is another important cause of magnesium loss from the kidney and it appears that a resultant magnesium deficiency aggravates the glucose metabolism . Since urinary magnesium excretion is mostly negligible among hemodialysis patients, serum magnesium levels of these patients are largely determined by dietary magnesium intake as well as dialysate magnesium concentrations. Under the same dialysate magnesium concentration, serum magnesium levels are well correlated with the dietary amount of magnesium . Luis etThere are several ways to elevate serum magnesium levels of patients receiving hemodialysis. While it would be unrealistic in clinical practice for dialysis patients to increase their dietary magnesium intake since they need to restrict potassium, magnesium supplementation can increase serum magnesium levels without affecting potassium levels. In this regard, magnesium-containing phosphate binders are useful and can correct the low magnesium–high phosphate status. It has been reported that the magnesium-containing phosphate binder suppresses vascular calcification more effectively than other types of binders in an animal model of CKD  as well Another expeditious and secure method to control magnesium levels is to alter dialysate magnesium concentrations. Notably, dialysate magnesium concentrations may affect intradialytic hemodynamics since magnesium directly influences cardiac contractility and vascular tone. It has been reported that the use of a low magnesium–low calcium dialysate increases the occurrence of intradialytic hypotension and decreases cardiac outputs . TherefoIn the current clinical practice of dialysis patients, the primary treatment option for hyperphosphatemia is to decrease the phosphate load by dietary management and the use of phosphate binders, as well as to remove phosphate by dialysis therapy. Despite the integrative approach by these therapeutic options, however, the problems related to hyperphosphatemia have not been fully resolved over the past decades because of poor adherence to the treatment, side-effects of the drugs, and nutritional disadvantages inherent to dietary restriction. We may have to recognize that the current strategy to reduce phosphate load is reaching a limit and develop an additional way to alleviate the harmful effects of phosphate. Although more compelling data are needed to justify the use of magnesium as a “remedy for phosphate toxicity” in clinical practice, it is worth testing whether the correction of low magnesium status improves the prognosis of patients with hyperphosphatemia. At the same time, it is important to clarify to what degree serum magnesium levels can be safely elevated since excessively high serum magnesium levels may also be harmful . Further


Innate personality traits are supposedly less evident in animals when confounded by learning acquired with experience through time. Learning might play a key role in the development and adoption of successful anti-predator strategies, and the related adaptation has the potential to make animals that are more experienced less vulnerable to predation. We carried out a study in a system involving a large herbivorous mammal, female elk, Cervus elaphus, and their primary predator, i.e., human hunters. Using fine-scale satellite telemetry relocations, we tested whether differences in behaviour depending on age were due solely to selection pressure imposed by human hunters, meaning that females that were more cautious were more likely to survive and become older. Or whether learning also was involved, meaning that females adjusted their behaviour as they aged. Our results indicated that both human selection and learning contributed to the adoption of more cautious behavioural strategies in older females. Whereas human selection of behavioural traits has been shown in our previous research, we here provide evidence of additive learning processes being responsible for shaping the behaviour of individuals in this population. Female elk are indeed almost invulnerable to human hunters when older than 9–10 y.o., confirming that experience contributes to their survival. Female elk monitored in our study showed individually changing behaviours and clear adaptation as they aged, such as reduced movement rates (decreased likelihood of encountering human hunters), and increased use of secure areas (forest and steeper terrain), especially when close to roads. We also found that elk adjusted behaviours depending on the type of threat (bow and arrow vs. rifle hunters). This fine-tuning by elk to avoid hunters, rather than just becoming more cautious during the hunting season, highlights the behavioural plasticity of this species. Selection on behavioural traits and/or behavioural shifts via learning are an important but often-ignored consequence of human exploitation of wild animals. Such information is a critical component of the effects of human exploitation of wildlife populations with implications for improving their management and conservation.In animal behaviour, there is a dichotomy between innate behaviours ( Animal behaviour is determined by the genetic makeup and the experience of the animal, resulting in a complex configuration of innate and learned behavioural patterns –3. BehavAdaptation to new environmental conditions  leads to changes in animal behaviour that can occur very rapidly and involve learning, and hence can be attributed to behavioural plasticity  learn to adjust their behaviour as they age under human hunting pressure. Female elk are ideal for studying learning in the wild because the hunting pressure on female elk is moderate to low compared to males [Here we investigated whether and how female elk  but without the learning process .(i) Are age-related behavioural differences in females driven only by selection by hunters , 23? If (ii) Are age-related behavioural differences in females driven both by human selection and learning? If learning with experience also is involved, then animals should adjust their behaviour as they age, with special regard to those behaviours adopted to avoid human hunters. This second set of models, therefore, includes the true age of the elk as a covariate, which is allowed to change over time.Our first hypothesis relies on the rationale supported by Ciuti et al.  that surOur data collection complied with relevant federal laws of Canada and provincial laws of Alberta. Procedures were reviewed and approved by the University of Alberta Animal Care and Use Committee ACUC–Biosciences , by all jurisdictions of the Alberta Government , and by Parks Canada .2 in south-west Alberta and south-east British Columbia, Canada. This is a diverse landscape, ranging from flat agricultural grasslands in the east, through the foothills to mixed conifer/deciduous forests and mountains in the west. The range of this elk population is an area under multiple jurisdictions administered by the provinces of Alberta and British Columbia. The elk winter range includes both private and provincial  land of Alberta, whereas elk migratory corridors and summer home ranges are on provincial lands in Alberta (and to a lesser extent in British Columbia). Cattle ranching constituted the dominant land-use on private land. Activities in the public land also included cattle grazing and natural gas extraction as well as extensive recreational use including camping, all-terrain vehicle (ATV) use, hunting, fishing, and hiking [Puma concolor) and wolves (Canis lupus) taking approximately 5% of the study animals , mostly during winter. Grizzly bears (Ursus arctos) and black bears (U. americanus) also pose a predation risk, especially to calves during spring. For an overview on predator distribution, see previous research conducted in the study area [The study was conducted over a six-year period in an area of 46,000 kmd hiking . Elk in d hiking . In thisudy area –29. HoweWe captured elk during 2007–2012 using net guns from a helicopter when soft snow cover was present on the ground to avoid elk injuries during captures. We equipped female elk with GPS radiocollars  and released them immediately after. Experienced personnel carried out all captures. A vestibular canine was taken using dental lifters during the capture to assess exact age through cementum analysis  ; forestsensu [We modelled variation of three response variables  as a function of time of the year (months: Sept.-Dec.), canopy cover  and terrain ruggedness (in those models where use of terrain ruggedness was not the response). We included a quadratic term for the use of terrain ruggedness and canopy cover when used as predictors to account for non-linear effects. We also included proxies of human activities: such as the time of day , distance to roads sensu ) compareWe modelled step-length  using a linear mixed model (LMM) as a function of environmental variables and human-activity-related variables, and the interaction between age and human-activity-related variables. Likewise, we modelled variation in the use of terrain ruggedness using LMM as a function of environmental variables (same as for step-length excluding ruggedness), human-activity variables, and the interaction between age and human-activity variables. Finally, to examine the use of cover by elk, we used logistic regression to model use of forest  as a function of environmental variables , human activity variables, and the interaction between age and the human-activity variables. Female home ranges were stable and overlapping across years, with over 90% of the home range (minimum convex polygon) of one year included in the next year’s home range. Each elk had the same habitat availability throughout the monitoring period. Thus, variation in the use of terrain ruggedness and forest across years can be assumed to be proportional to variation in selection, because availability was fairly constant through time. We were more interested in the actual use (and change in use over time) of a resource rather than its selection, because we expected that the use of a given resource would be more tied to mortality risk than its selection. A resource unit might be strongly selected by one animal, e.g., when 10% of relocations are located within open areas that were only 1% of the available resources, However, a resource might be weakly selected by a second animal when 50% of relocations were located within open areas that amounted to 49% of the available resources. The latter animal would appear to show weaker selection for open areas (use/availability) but spends more time in open areas than the former animal thereby exposing it to higher mortality risk  and reduced step length could be a result of migratory strategies combined with age and individual behaviour , 38, 39.i.e., true age). See i.e., behaviour changes due to learning). In contrast, if age at capture were used, then within-individual behavioural adjustments due to aging were not considered , and the model investigates the difference among individuals of different ages. In practice, when age at capture was included in the model structure, then we could use the model to detect selection . When true age was included in the model structure, the model did not allow us to exclude human selection but we could detect learning .To evaluate our alternative hypotheses, we compared models with different ways to account for elk age  when casensu .lme4 [We fitted the 5 alternative linear mixed-effect models using the Restricted Estimate of Maximum Likelihood (REML) method in R  using thlme4 . For thelme4 .a priori set of models that we fitted by Maximum Likelihood (ML). Each model differed only by the inclusion of true age and the two-way interactions between true age and human activity measures as fixed effects. This enabled us to verify whether elk behaved differently towards proxies of human activity as they grew older  is not Female elk in our study population adjusted their behaviour as they became older, adopting behaviours to avoid human hunters. Despite extensive wilderness in our study area compared to more heavily human dominated landscapes, effects of humans on behaviour of wildlife exceed those of natural predators in this landscape of fear . HuntingBy comparing alternative random structures in our mixed-effect models, we showed that individual elk adjusted their behaviour (movement rate and use of rugged terrain and forest) as they grew older, due to both learning and selection. Movement rate and the use of terrain ruggedness and forest were adjusted by older individuals depending on the ongoing hunting regime , proximity to a road, and as a function of the time of the day . Our study highlights elk behavioural plasticity to hunting pressure, confirming patterns in habitat selection previously documented by Proffitt and colleagues . Female Poecilia reticulata [Macropus eugenii [Innate responses by a species are expected to be performed in stereotypic fashion , whereasticulata  and wall eugenii . Learnin eugenii , 55–58.  eugenii , such asPomacentrus amboinensis, that highlights how information on predator identities can be passed to group members quickly without a dilution of information content [Rangifer tarandus habituated to humans [Gerbillus andersoni allenbyi do not appear to have that ability [Learning about mortality risks should be more efficient in gregarious species (such as for elk aggregations) due to opportunities for non-lethal exposure to mortality, e.g. –62. Lear content . Gregario humans —are ableo humans –17, wher ability .Capreolus capreolus. The consequence of inter-individual differences in learning can lead to the ability for an animal to learn and to adapt its behaviour as a function of its behavioural type, i.e., are shyer individuals more plastic? See Kareklas et al [i.e., random slope for age and random intercept for individual, thus allowing animals of different age to learn differently). Further investigations should identify personality traits from spatial behaviour [The perception of risk might differ according to the individual personality type, because animals might adjust their behaviour differently according to their behavioural type. See Bonnot et al.  for an eas et al  for an eas et al , we mighehaviour , and theehaviour  to betteAs introduced earlier, human hunting pressure on elk is much higher on males than females. This means that the likelihood for certain behavioural traits selected by humans  to occur is higher among older females compared to older males. On the one hand there is a strong selection by hunters on male behavioural traits  where”boThe likelihood that a female elk will be shot by a hunter decreases markedly with age , 23, witWe showed that learning can have a long-term effect on the behaviour of individuals in a wild population. We also showed that animals were able to differentiate between hunters using different equipment and tactics and to fine-tune their behaviours accordingly. This helps to explain why other indices of human activity such as roads can have vastly different effects on behaviour of large mammals inside and outside of national parks. For example, animals inside parks without hunting do not avoid roads like they do outside parks . ConsequS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.


Animal behavioural traits often covary with gene expression, pointing towards a genomic constraint on organismal responses to environmental cues. This pattern highlights a gap in our understanding of the time course of environmentally responsive gene expression, and moreover, how these dynamics are regulated. Advances in behavioural genomics explore how gene expression dynamics are correlated with behavioural traits that range from stable to highly labile. We consider the idea that certain genomic regulatory mechanisms may predict the timescale of an environmental effect on behaviour. This temporally minded approach could inform both organismal and evolutionary questions ranging from the remediation of early life social trauma to understanding the evolution of trait plasticity. Gene expression and behaviours are intimately related, and their interactions can play out over timescales from developmental to evolutionary. Here, the authors review how temporal aspects of gene expression mediate behavioural responses to the environment, a key question in behavioural genomics. Indeed, each external influence on behaviour has its own timescale, i.e., a timespan over which the input perturbs an organism’s internal processes with behavioural consequences , here we narrow our focus to gene expression dynamics and their regulation. Variation at other mechanistic levels has important temporal correlates, but gene expression dynamics are easy to measure, and they show plasticity across a very broad temporal range . Furthermore, gene expression measures allow for a range of analytical approaches, from a focus on the dynamics of a specific pathway to plasticity at the whole-genome scale, agnostic to gene identity. This range enables high-powered statistical approaches.Though there are many types of mechanisms that underpin behavioural variation  is a particularly popular method to infer that an environmental input causes a lasting behavioural change10, the relationship is complex and likely indirect25, and both phenotypes are to some degree independent of one another. Furthermore, the direction of causation between behaviour and gene expression change is unclear and under-investigated. Though these are general issues for linking many levels of physiological organization to behaviour, the problem is particularly acute for genomics research, where studies often show surprisingly large changes in gene expression with limited causal validation. These facts present important challenges to using genomic state to predict timescale of behavioural change, or vice versa. In the current review, we explore the use of gene expression analyses to predict timescale of behavioural changes, assuming at least some direct relationship between expression values and behavioural trait values. However, in Box It is important to note that although the co-occurrence of gene expression variation and behavioural variation suggests an interaction between these levels of organizationIn the following section, we highlight diverse cases in which behavioural change over different timescales, both within the lifespan of an organism and across generations, is correlated with variation in expression of similar sets of genes. These patterns, which are observed with increased regularity, provide a basis for the argument that mechanisms of behavioural variation with respect to time are non-independent, and that gene expression reflects the integration of information from ancestral environments  with parental, developmental, and current environment effects  using a single currency.26. Studies have examined gene expression signatures associated with behavioural variation across a range of time points, including over evolutionary time where sequence level variation underlies behavioural variation across distinct species and populations27, cases in which cues from the environment are transmitted from parents to offspring30, developmental time, during which cues shape both behaviour and morphology31 and current environmental time, in which acute interactions with conspecifics, predators, or pathogens influence behaviour32. The widespread co-occurrence of variation in behavioural and genomic states across different points in time and with different timescales of effect suggests gene expression is a relevant mechanistic level to examine how environmental inputs are integrated to give rise to behavioural phenotypes.One generalized finding from behavioural genomics is that even relatively ephemeral shifts in behavioural phenotype are accompanied by widespread changes in the expression of hundreds and even thousands of genes in tissues including, but not limited to, the brain33, have found overlap in gene expression associated with heritable variation and within-population plasticity in the same trait34. One of the earliest cases of gene overlap across ancestral and current environmental time for a behaviour involved the foraging gene, for which sequence level variation in the fruit fly (Drosophila melanogaster) influenced both gene expression and behavioural differences across individuals with different genotypes , while regulatory changes over the course of adulthood  gave rise to similar effects in the honey bee (Apis mellifera)35. In killifish, genes showing population level expression differences correlated with variation in salinity tolerance were also responsive to acute osmotic shock36. In sailfin mollies, there is substantial overlap between genes differentially expressed as a result of plastic vs. genetically fixed differences in male mating strategies37.A number of studies have found that patterns of differential gene expression associated with phenotypic or behavioural traits indeed involve similar sets of genes regardless of the timescale of effect, providing evidence that molecular mechanisms of behavioural change are non-independent. Many studies have identified mechanistic links across ancestral and more proximate timescales. For example, investigations of genetic accommodation, in which plasticity in a given trait or expressed gene product precedes evolved differences that reflect sequence level variation38. This could include cases in which developmental regulatory mechanisms are co-opted later in life to regulate plastic phenotypes, a phenomenon that has been demonstrated in the context of neural plasticity39. In the zebra finch, genes that are constitutively expressed during song learning periods are largely suppressed during adulthood, which is outside of the critical period for song learning40. However, these genes can become activated in response to a strong social stimulus, even during adulthood40. Box Fewer studies have evaluated mechanistic overlap for environmental inputs received at different time points within a single individual’s lifespan 41. A recent study of D. melanogaster genomic response to dietary stressors yielded similar results, with overlap in 108 genes differentially expressed as a result of experimental evolution vs. acute exposure to a specialized diet. Similar to the guppy example, over 90% of these overlapping genes were expressed in opposite directions across timescales 42. These studies, notable because they are experimental and not correlational, raise the possibility that patterns of gene expression evolution may be a function of evolutionary divergence time, where early divergence is characterized by non-adaptive plasticity (which is strongly selected against until it is purged), while divergence in later generations reflects the patterns expected if adaptive plasticity facilitates evolutionary change42. Also known as ‘counter-gradient selection’42, another explanation for this pattern is that evolutionary and induced gene expression effects combine to yield little net change in phenotype, suggesting a set-point  that is maintained by integrating evolutionary history with current environmental conditions. Further experimental tests of ‘early-non-adaptive, late-adaptive facilitation’ may be a useful empirical starting point for gaining a better understanding of molecular plasticity mechanisms and their evolution41. These patterns for molecular plasticity are particularly intriguing given positive relationships between trait plasticity and trait evolution for other phenotypes evaluated in experimental studies43.Despite considerable evidence for shared gene expression patterns across timescales, some studies have revealed interesting exceptions and complexity in the nature of transcriptional change. For example, in guppies, an evaluation of the brain transcriptional response to predator exposure during development reported that genes that evolved rapidly over evolutionary time were the same as those showing plasticity in response to predator exposure during development; however, the direction of plasticity induced by predator exposure was largely opposite in direction to their evolutionary trajectory. Thus, the transcripts that were revealed to show ‘non-adaptive’ patterns of plasticity during development were also the first to evolve changes in expression, presumably because non-adaptive plasticity would be subject to very strong negative selectionGene expression dynamics are a function of underlying regulatory mechanisms that could constrain or predict capacity for plasticity at both the gene expression and behavioural levels. In the next section, we review some major mechanisms of transcriptional regulation, including ones often invoked to predict stability in gene expression and behavioural state. We discuss the known temporal rules associated with these mechanisms in order to assess the state of knowledge in the field, but also to evaluate whether this type of information could provide a means to predict the time course of experience-dependent changes in behaviour.18, despite substantial differences among taxa in the prevalence and function of these modifications44. An extremely broad array of gene regulatory mechanisms entrains environmental inputs to influence mRNA levels and gene transcription. Determining whether these mechanisms have predictive value in terms of the timescale of effect, i.e., the relative stability of the effects on the transcriptome has been emphasized recently as a controversial yet critical area of research in a number of different plasticity contexts, from learning and memory to transgenerational inheritance of environmental effects48. Here we systematically review some of the major types of gene regulatory mechanisms and what is known about their temporal stability and reversibility. Although these mechanisms could differ across taxa in terms of their prevalence or function49 , here we cover broad taxa in general terms because there is a lack of evidence for taxon-specific differences in the temporal properties associated with these regulatory mechanisms.Many studies use the existence of particular gene regulatory mechanisms to infer the relative stability of an experience-induced behavioural change. One common example is the evaluation of epigenetic modifications , which are used across species from insects to mammals to infer stable shifts in gene expression and behaviour50. Experience-induced variation in gene expression can also be maintained by self-sustaining feedback loops in which protein concentrations modulate mRNA transcription46. Such mechanisms have been known to play a role in circadian rhythms51, hormone signalling more generally52, and energy metabolism and metabolic capacity53. Functional genomics studies have linked shifts in all three of these biological processes to variation in behavioural phenotypes57; thus it seems likely that mechanisms of autoregulatory transcriptional control could play an important role in modulating experience-induced behavioural change. However, because elucidating such mechanisms is difficult to do in a high throughput manner in naturally behaving organisms, there is limited knowledge of the temporal resilience of these regulatory mechanism, and their prevalence.A number of relatively understudied regulatory mechanisms may play critical roles in modulating gene expression and behaviour, especially over relatively short-term timescales. Temporary inductions in gene expression may be maintained passively for a period of time depending on the rate of mRNA decay47. These mechanisms have been associated with environmentally induced behavioural variation across generations58, during development49, and throughout adulthood45. When considering the timescale of changes in behaviour and gene expression, one simple heuristic is that stimuli that result in lasting behavioural shifts will be those that cause chromatin-based epigenetic changes. However, though it has been demonstrated that these types of changes can lead to stable shifts in gene expression that last for substantial portions of an organism’s life59, there is limited understanding of the reversibility of these mechanisms, both in terms of the time course and the relevant stimulus inputs60. For example, in mammals, contextual fear learning requires a combination of active DNA methylation and demethylation45 to tune gene expression patterns in neurons. In the honey bee, some of the most highly methylated genes show the greatest degree of context-dependent plasticity61. The assumption of DNA methylation persistence over time in particular is at least in part historical, since this mechanism was first appreciated in the context of stable maintenance of cell identity across cell divisions58. Interestingly, over some of the longest timescales for behavioural change, e.g., transgenerational inheritance of parental effects, organisms undergo known periods of epigenetic reprogramming that may essentially erase experience-based effects on gene expression46; thus, a simple relationship between the duration of an environmental effect and chromatin-based regulatory mechanisms may lead to spurious conclusions.Chromatin-based transcriptional regulatory mechanisms, e.g., DNA methylation and histone modifications, have received a large amount of attention in a variety of disciplines58. One feature that makes the study of these mechanisms and their temporal components so challenging is that the occurrence and the outcomes of chromatin modifications  are extremely varied across taxa64. In addition, several types of RNAs have been implicated in transcriptional regulation, independently and in conjunction with other epigenetic modifications. For example, small RNAs and long non-coding RNAs can modulate other regulatory mechanisms including chromatin modification68. Rapid and reversible shifts in behaviour can be difficult to study in any context, which may explain the limited application of chromatin-based analyses to short-term timescales. However, there are many experimental tools that can be used to manipulate and measure chromatin-based modifications towards a better understanding of their temporal effects on gene expression70.New research continues to alter the perspective on the time course and reversibility of epigenetic modifications, particularly in natural contexts that involve repeated and varied environmental inputs throughout life72)  may provide insights about plastic phenotypes and their evolution9. For example, animal personalities  can arise if environmentally responsive behavioural phenotypes are stabilized over time through their dependence on slower changing intrinsic state variables77. Genomic state, as an underlying intrinsic state variable, could thus impose a limit to plasticity in some cases. For instance, in the honey bee, chronic exposure to predator disturbances leads to baseline differences in both aggression and brain gene expression78, and this shift persists for several days after the threat is removed. One hypothesis to explain this phenomenon is that the behavioural response to the repetitive predator stimulus persists until the gene expression response decays, suggesting the behaviour is stabilized because the genomic state shifts slower than the external environment. The result is that behaviour−environment match at a given time point is limited by previous experiences that influence both behaviour and genomic state (a form of ‘plasticity-history limit’ to plasticity evolution)9.A plasticity limit is a type of constraint on the evolution of phenotypic plasticity9. Studies suggest this type of plasticity limit may have generalizable gene expression characteristics. For example, in Locusta migratoria, which can transition between a solitary and gregarious morph, increased density and the resulting heightened interaction between conspecifics initiates the transition to the gregarious morph79. Repeated stimuli are required to complete this physiological and morphological transition. The switch to the gregarious morph is relatively slow compared to the reverse despite the fact that this transition is accompanied by more robust changes in gene expression34. This robustness could be the result of repeated stimulus exposure over an extended timeframe, compared to the shift towards the solitary state, which is initiated by the absence of stimuli80. Thus, the degree of gene expression change is a function of the nature of the stimuli that cause the phenotypic shift, not the rate or degree of the phenotypic shift itself. In this example, there are limits to plasticity associated with a time lag in the phenotypic shift, which may result in fitness costs associated with environmental mismatch. The tolerance of these asymmetric time lag limits over evolutionary time is thought to reflect the higher fitness costs associated with transitioning to the gregarious morph in error. However, it is important to consider the relationship between asymmetric limits and the mechanisms that regulate gene expression changes. If stimuli frequency, gene expression patterns and the speed of phenotypic transition are typically linked, type and frequency of salient stimuli, as well as gene regulatory dynamics, could be used to predict plasticity limits and potential costs to plasticity evolution. Conversely, selection could shape the nature of underlying gene regulatory plasticity mechanisms.In some cases where individuals can shift phenotypes bidirectionally, or multiple times within their lifespan, transitions are asymmetric, occurring more rapidly or more easily in one direction than another (a form of ‘lag time limit’ to plasticity evolution). Asymmetry can result in a temporary mismatch between environment and phenotype, a limit to plasticity evolutionAstrotilapia burtoni is a cichlid fish in which males can transition between dominant and subordinate states that differ in behaviour, body colouration and testis size. Behaviour changes very rapidly regardless of the direction of transition19. Studies examining a small set of transcription factors showed that the same transient increase in immediate early gene expression is involved in both ascent to and descent from dominance. However, brain regional patterns of gene expression differentiate the direction of social transition82. Thus, similar genes are used in transition and expressed with the same temporal rules, but the location of expression is direction specific. Altering the regional location of variation in brain genomic state could differentially impact other phenotypes regulated by those same brain regions, perhaps imposing a cost to plasticity that extends beyond the focal behavioural phenotype.Even when phenotypic transitions are symmetric, important differences in underlying mechanisms could reveal hidden direction-specific constraints. 9 is a plasticity limit that occurs when environmental inputs occur following the window of a developmental critical period. In the case of morphology, if these inputs occur late in the development of a trait, they may not have the capacity to fully alter the developing structure. Similarly, for behaviour, late inputs may not be robustly assimilated because major periods of brain development have lapsed. The idea of critical periods may also apply in important ways in the context of gene expression. Developmental time periods are critical periods for chromatin modifications in some cases11, and inputs during these periods may set the capacity for additional plasticity8. Such epigenetic processes, however, are also hormonally responsive throughout life, and particularly plastic in the nervous system11. More work is needed to determine, for example, whether periods of hormonal shifts  may serve as additional critical periods for epigenetic reprogramming, with temporal consequences. These possibilities suggest the epiphenotype problem may apply beyond morphological contexts, and could be considered a limit in the context of behavioural plasticity.The ‘epiphenotype problem’Behaviour presents unique challenges to phenotypic plasticity theory owing to its exceptional capacity for change. However, behavioural plasticity is far from unlimited, due at least in part to substantial variation in the timescales of environmental effects on behavioural expression. Here we explored evidence for the idea that genomic mechanisms that entrain environmental experience at different timescales are non-independent. If so, the temporal properties of the mechanisms that regulate gene expression could be used to predict the relative stability of environmental effects on behaviour. This is especially true if gene expression dynamics track behavioural dynamics and reflect the integration of one or several environmental inputs. Finally, from an evolutionary perspective, mechanistic knowledge may uncover the characteristics of and ultimately predict circumstances under which individuals integrate or prioritize of environmental inputs over time. However, there remain a number of outstanding questions and experimental challenges that are limiting our understanding of temporal rules of genomic regulatory mechanisms, how information from environmental cues is integrated over time, and how a focus on individual behavioural traits can be expanded to consider multiple correlated phenotypes.46. The intuitive hypothesis that relatively high-stability behavioural phenotypes are underpinned by chromatin modifications, while more transient shifts in behaviour are associated with more labile expression mechanisms may not be supported, at least in this most simplistic form.While there is reasonably strong evidence for overlapping, non-independent patterns of gene expression across timescales of behavioural variation, evidence that specific regulatory mechanisms encode time and thus can be used to predict gene expression temporal dynamics is lacking. More experimental evidence is needed to link certain types of regulatory mechanisms  to particular time frames for stability in gene expression and behaviour61, which raises the possibility that methylation may not necessarily confer stability. Assessing first the temporal dynamics of gene expression and behaviour, and then assessing regulatory mechanisms for target genes may provide more meaningful insights about regulatory mechanisms that confer gene expression stability. A second approach could be to explicitly test the reversibility of presumed long-lasting or stable behavioural phenotypes and gene regulatory mechanisms83. For instance, only in certain cases have the reversibility of social experiences during developmental critical periods been investigated, particularly for social effects on the genome.Fortunately, with the increased capacity to generate transcriptomic data, it is possible to evaluate the temporal component of gene expression and behavioural dynamics simultaneously in order to gain a better understanding of how plasticity at these two levels is linked. This information could then be used for more targeted assessment of regulatory mechanisms associated with highly labile vs. more stable patterns of behaviour and gene expression. For example, in the honey bee, genes involved in energy metabolism are highly methylated, but also highly responsive to environmental inputs throughout life . However, faced with a monetary trade-off between generating transcriptomic data at multiple time points and other –omics data that address underlying regulatory mechanisms, e.g., bisulfite sequencing data to examine patterns of DNA methylation, temporal expression data, even for a subset of genes, may be more valuable to the questions posed herein.We argue that in addition to generating –omics data to assess epigenetic marks and their behavioural correlates, there is value in focusing explicitly on gene expression temporal dynamics86), while in some cases, networks are relatively robust to perturbation. Exploring the relative robustness of gene expression networks may be a powerful tool however, if it reveals that certain subnetworks are more resilient to change than others. Such a finding could help narrow the focus to the most labile genes87. More simplified approaches that target candidate genes and pathways associated with certain behavioural traits may also prove insightful10.In addition to monetary considerations, there are important analytical challenges to assessing temporal dynamics of gene expression and behaviour. For one, gene expression patterns are highly dependent on sampling time point, and it can be difficult across organisms and behavioural contexts to select a relevant sampling strategy. Moreover, because behavioural phenotypes are often polygenic and reflect epistatic interactions among loci, evaluating expression dynamics may require gene network-level data and analyses. For instance, network or epistatic relationships among genes can be malleable . It is inherently easier to find evidence of overlapping gene expression profiles across timescales than to say definitively that expression profiles are unique. This means that the ability to identify cases where completely unique sets of genes give rise to the same behavioural phenotype, the alternative hypothesis to non-independent mechanisms, is limited. Similarly, even in cases of gene expression overlap, there are always some genes that are context-specific, and it is not always possible to identify which genes are most critical to patterns of behavioural variation. Finally, certain genes may play discrete roles in modulating trait means and trait variability88, and the relationships between gene expression values and phenotypic change may also shift over the lifetime due to critical changes in regulatory interactions among genes87. All of these are important considerations when assessing patterns of genomic similarity across timescale.There are some notable analytical considerations with respect to identifying mechanistic commonalities across timescales , immune function2, and energy metabolism90 can show relatively rapid shifts over time, but are also subject to some of the same timescale properties as behaviour. Some of these traits, like immune function, are even activational in nature, similar to behaviour. Investigating the gene expression principles discussed herein in the context of non-behaviour phenotypes has the added value of addressing whether or not the nature of expression dynamics and regulatory mechanisms are specific to the brain, or if these are patterns that can be extrapolated to other tissues.Temporally linking RNA abundance and behaviour may point to cases in which higher levels of biological organization  play an important role in modulating behavioural phenotypes with respect to time, especially if changes in brain structure or function manifest as a predictable mismatch between gene expression and behavioural dynamics Box . Importa1. However, few models in this area account for situations in which environmental experience not only affects the organism’s informational status, but also directly impacts lasting somatic traits of the organism, e.g., body size or strength1. Incorporating measures of genomic state, particularly if gene expression dynamics show predictable temporal patterns, could be a relatively straightforward way to track changes in somatic or physiological state, and thus integrate mechanistic data into these models.A growing body of literature applies modelling frameworks to understand how organisms integrate environmental information from multiple time pointsModels could also consider mechanisms for information integration, and how the nature of these mechanisms could feedback to influence behavioural trajectories over time. For instance, development can serve as a critical period of environmental influence that irrevocably shapes an organism’s physiology and behaviour throughout life, even with additional real-time environmental information. Though this type of critical period is presumably a target of selection, it may also be a generalized form of plasticity constraint. Adding mechanistic data about temporal plasticity constraints may improve information integration theory, and more generally, theory about plasticity evolution.91. Genetic correlations can be sensitive to normal environmental variation93, as well as more exceptional contexts like inbreeding or stress96. This environmental sensitivity means that spatial or temporal fluctuation in genetic correlations could substantially influence the evolution of behavioural traits. If traits depend on shared molecular mechanisms, independent optimization of the two traits might not be possible, at least over short to intermediate evolutionary time periods.Here we have largely focused on independent behavioural traits, but gene expression mechanisms associated with timescale also have implications for correlated traits. Evolution in response to selection on a suite of correlated traits depends on genetic variation and covariation among the traitsWork in behavioural genomics has laid the foundation to analyse sophisticated relationships between molecular dynamics and behavioural state at different timescales. Future work in this field could greatly benefit from even relatively simple experiments that more clearly define how the environment influences the expression of non-independent and behaviourally relevant sets of genes. Gene expression, like behaviour, shows a high degree of environmental sensitivity. Tracking changes at both levels across time may provide new insights into behavioural regulation at intermediate levels of biological organization, and provide predictive value about the relative stability of environmental influences on behaviour.97. One vexing reality is that some of these social effects are easily remediated while others persist throughout life30. Understanding the timescales of genomic effects may point to interventions that interrupt detrimental social effects on mental and physical health97.In the social and health sciences, there is a growing focus on the physiological and behavioural impacts of the social environment, which can influence susceptibility to illnesses including stroke, heart disease and depressionStress reactivity is a phenotype that is associated with physical and behavioural health problems throughout life—depression, heart disease, diabetes, stroke and suicide. It is also informed by multiple sources of inherited and environmentally derived information98. Rat pups that are experimentally handled for a short time period (3–15 min) early in life receive high levels of maternal licking and grooming upon return to the nest, and as a result show a diminished level of stress reactivity later in life. Licking and grooming results in increased expression of stress hormone receptors, mediated by epigenetic mechanisms96, and resilience to stressful situations, e.g., exposure to novel environments. Longer-term (180 min), but still relatively temporary experimental handling and separation from the mother, in contrast, has opposite effects on stress reactivity, resulting in a chronic upregulation of the stress response systems and a fear of novelty99. Thus, similar types of stressors, with minor differences in severity relative to the lifespan, have context-dependent effects on stress reactivity. Moreover, maternal care behaviours, as well as pre-natal maternal stress exposure, can override heritable genetic variation in stress reactivity96. Stress experienced at all of these time points is mediated by the same hormonal mechanism, which allows for the non-independent integration of information from multiple sources.22. The temporal dynamics that describe behaviour and genomic state following a signalling event are complicated by the fact that these two levels of organization interact and are often bi-directional , assessed at the transcriptomic38, metabolomic54 and enzymatic38 levels. Thus, this system also provides a unique opportunity to study how genomic state corresponds to functional changes at higher levels of biological organization that also influence behavioural plasticity.Work in the honey bee indicates that variation in aggression in multiple contexts is associated with shifts in brain energy metabolism (reviewed in ref.


Through their dam-building activities and subsequent water storage, beaver have the potential to restore riparian ecosystems and offset some of the predicted effects of climate change by modulating streamflow. Thus, it is not surprising that reintroducing beaver to watersheds from which they have been extirpated is an often-used restoration and climate-adaptation strategy. Identifying sites for reintroduction, however, requires detailed information about habitat factors—information that is not often available at broad spatial scales. Here we explore the potential for beaver relocation throughout the Snohomish River Basin in Washington, USA with a model that identifies some of the basic building blocks of beaver habitat suitability and does so by relying solely on remotely sensed data. More specifically, we developed a generalized intrinsic potential model that draws on remotely sensed measures of stream gradient, stream width, and valley width to identify where beaver could become established if suitable vegetation were to be present. Thus, the model serves as a preliminary screening tool that can be applied over relatively large extents. We applied the model to 5,019 stream km and assessed the ability of the model to correctly predict beaver habitat by surveying for beavers in 352 stream reaches. To further assess the potential for relocation, we assessed land ownership, use, and land cover in the landscape surrounding stream reaches with varying levels of intrinsic potential. Model results showed that 33% of streams had moderate or high intrinsic potential for beaver habitat. We found that no site that was classified as having low intrinsic potential had any sign of beavers and that beaver were absent from nearly three quarters of potentially suitable sites, indicating that there are factors preventing the local population from occupying these areas. Of the riparian areas around streams with high intrinsic potential for beaver, 38% are on public lands and 17% are on large tracts of privately-owned timber land. Thus, although there are a large number of areas that could be suitable for relocation and restoration using beavers, current land use patterns may substantially limit feasibility in these areas. Castor canadensis) have long been recognized as ecosystem engineers, creating diverse and resilient wetland and riverine systems [Oncorhynchus kisutch), have been partially attributed to the loss of beaver ponds [North American beaver  by using geomorphic variables that are less prone to change through time. Intrinsic potential models have been previously used to inform fish habitat restoration work , and somHere, we develop and apply a beaver intrinsic potential model that can predict where high quality beaver habitat currently exists and where colonization will occur as population levels increase or if management changes are made . We developed our BIP model within the Snohomish River Basin, Washington State (USA), and validated it within the Skykomish River sub-basin. Our study had four primary objectives. The first objective was to develop a BIP model parameterized with readily available, remotely sensed public data, thus facilitating the transferability of the approach to other regions. The second objective was to evaluate the effectiveness of our BIP model within a large basin that has high levels of hydrogeomorphic complexity and highly variable beaver population densities. The third objective was to assess the potential for continued population expansion by assessing the degree to which beaver occupied areas with high intrinsic potential. Finally, we aimed to explore the potential constraints or barriers to colonization and occupancy of areas with high intrinsic potential by assessing land use in riparian areas with high intrinsic potential.2), located on the west slope of the Cascade Range in Washington State, was selected for BIP model development, and the Skykomish River sub-basin  was used for model validation  using ArcGIS. We obtained hydrography data layers consisting of a combination of field-verified and digital elevation model (DEM)-derived stream segments from King and Snohomish Counties, Washington. We derived stream slope, bankfull width, discharge, and stream segment breaks using the methodology outlined by Davies et al. . The valTo assist in identifying the range of intrinsic potential habitat within our study basin, we selected 501 stream segments showing signs of current or recently abandoned beaver ponding using Google Earth and U.S. National Agriculture Imagery Program (NAIP) orthographic imagery. These segments were sampled from the Snohomish watershed but excluded the Skykomish sub-basin where model validation occurred. We described stream slope, stream width, and valley width within each segment to identify the range of conditions present at sites that beavers colonized .Based on the range of conditions present at potentially suitable sites, we assigned a ranked value from 0–4 to each of these variables, commensurate with their level of intrinsic potential according to the criteria in Following completion of model development, we compared the spatial distribution of modeled intrinsic potential to land use in the Snohomish Basin to identify how habitat was distributed across the landscape and where it might be at odds with existing human use. Land use was identified within 30-m buffers of all modeled streams using Snohomish County zoning GIS data.We field-verified the BIP model in the Skykomish River sub-basin, testing how well the modeled intrinsic potential predicted field-assessed intrinsic potential at surveyed sites. We limited survey locations to those that were within stream segments that had a relatively homogenous gradient and stream width within the mapped stream segment. Using a quasi-random approach, we selected 100 stream segments from each of the four BIP classes (0–3) for a total of 400 survey sites, which were reduced to 352 due to access limitations in some cases. We conducted a blind assessment of conditions at each field location during base flow conditions . In instances where there was uncertainty, we revisited the site during higher flow conditions. The same metrics used to construct model scores within mapped stream segment, stream slope, width, and valley width, were evaluated in the field. A field score for each segment was found using the same methods used in the model . TogethePrior to validating model scores with field conditions, we established an intrinsic potential threshold, or a cutoff, separating high IP model scores from those thought to be less suitable using methods in . We estaWe performed a validation test for the BIP model’s ability to predict the site’s intrinsic habitat potential. Validation was conducted by comparing modeled suitability with observed BIP at 352 stream segments using a contingency table. Overall model accuracy, sensitivity, and specificity were assessed.In addition to comparing our model predictions to BIP assessed in the field, we determined the degree to which BIP based on remotely sensed data predicted beaver occupancy. We surveyed the same 352 sites used in our validation effort for signs of both current and historical occupation. Site occupancy was determined by the presence of recent beaver sign, such as freshly chewed sticks, cut logs, fresh scent mounds, or presence of actively maintained dam or lodge structures (following Snodgrass and Meffe ). In areHigh, Moderate, Low, or No BIP) to 48,397 stream segments comprising 5,019 stream km within the Snohomish River Basin  have thOur study demonstrates that there is a large amount of potential habitat within the Snohomish watershed that remains unoccupied by beavers, much of which is in fragmented landscapes and in ownership patterns to which it is not easy to apply beaver restoration . AlthougCastor fibre) demonstrates that in areas where beaver populations are depressed, but unexploited, they will increase rapidly towards carrying capacity [In the Skykomish sub-basin, site assessments showed no sign or evidence of past beaver presence in many stream segments categorized as geomorphologically suitable beaver habitat by the BIP model and site surveys. Field surveys indicate that approximately 75 percent of geomorphologically suitable sites in the basin are vacant, raising the question: why are there no colonies in these areas? A recent study of the European beaver . Duringsensu ,50. WhilS1 Table(TXT)Click here for additional data file.


Hemodynamic instability during renal replacement therapy (HIRRT) in the form of intradialytic hypotension (IDH) is a frequent complication of hemodialysis in end-stage kidney disease (ESKD), and most studies have focused on this chronic population. However, HIRRT is also an important concern for critically ill ICU patients with acute kidney injury (AKI), complicating an estimated 30% of dialysis treatments in this population. HIRRT can exacerbate organ hypoperfusion in the setting of critical illness and may negatively impact renal recovery in the AKI population. This is a protocol for a systematic review to synthesize the evidence surrounding dialysis-related interventions used to minimize HIRRT in critically ill patients with RRT-requiring AKI. This protocol has been registered with the International Prospective Register of Systematic Reviews (PROSPERO) database.We will search MEDLINE, EMBASE, and CENTRAL databases in collaboration with a health information specialist using a comprehensive search strategy. We will also supplement our search with a scan of the “gray literature” to identify relevant ongoing trials or conference abstracts. Observational studies and clinical trials will be included in our analysis. Our outcomes will include the incidence of HIRRT, dialysis-related complications, in-hospital mortality, and renal recovery. Prior to our search, we performed an initial search of these databases and PROSPERO, which yielded no prior or ongoing systematic reviews on this topic. Two reviewers will independently screen the list of identified abstracts using pre-defined inclusion and exclusion criteria. Two reviewers will then independently extract data from selected studies and undertake an assessment of their quality using validated tools.HIRRT is a common complication of renal replacement therapy not only in ESKD but also in the critically ill AKI population. It can result in early discontinuation of dialysis, further organ injury from hypoperfusion, and possibly negatively impact renal recovery. This systematic review will synthesize the existing evidence on the interventions employed to predict or prevent episodes HIRRT in critically ill patients with RRT-requiring AKI. This systematic review will allow for an understanding the current evidence for interventions to limit HIRRT in AKI and, in doing so, may also highlight areas in need of further research.CRD42016037754PROSPERO The online version of this article (doi:10.1186/s13643-017-0512-9) contains supplementary material, which is available to authorized users. Episodes of hemodynamic instability during renal replacement therapy (HIRRT) in the form of intradialytic hypotension (IDH) frequently complicates maintenance hemodialysis treatments in end-stage kidney disease (ESKD) patients . Minor cMost studies of interventions to prevent or limit HIRRT have focused specifically on IDH in maintenance hemodialysis patients with ESKD; however, IDH is only one form of HIRRT which is also an important concern amongst critically ill patients who require hemodialysis or other forms of RRT  or continuous renal replacement therapy (CRRT)) in the context of acute kidney injury (AKI). AKI that necessitates RRT is a frequent and serious complication of critical illness with an incidence of 2 to 3% amongst all patients admitted to the intensive care unit (ICU) . One stuThe goal of this study is to summarize all observational and trial evidence related to interventions to prevent or limit HIRRT in AKI patients. This encompasses AKI that is treated with the most commonly utilized RRT modalities in the setting of critical illness .The primary objective of this systematic review is to synthesize the evidence surrounding interventions to limit HIRRT in critically ill patients with AKI. In particular, we wish to assess the impact of various dialysis-related interventions that have been studied to minimize its occurrence.What is the effectiveness of interventions or strategies employed to limit HIRRT in AKI?This protocol is reported in accordance with the PRISMA-P 2015 checklist   statement . This prAll studies that assess a dialysis-related intervention to limit or prevent HIRRT in AKI populations, including interventional studies  and observational  studies, will be eligible. In addition, to be eligible for inclusion, studies must meet the following criteria.Studies with critically ill adult (18 years of age or older) patients with AKI treated with RRT  will be included.Studies with a dialysis-related intervention (or a modifiable factor) related to the application of RRT  will be included. .Only studies that also included critically ill adult patients with AKI treated with RRT that did not receive the exposure or intervention, as a comparitor group, will be included. .Primary outcome: HIRRT. Any definition used is acceptable provided that it is defined by the study’s methods. HIRRT encompasses not only changes in blood pressure (as in IDH) but also the initiation or increased dosing of vasopressors during RRT and/or any other criteria used to define hemodynamic instability during RRT (such as the need for fluid boluses). Studies that report HIRRT as an outcome but without any definition will be excluded.Secondary outcomes: death (any time points reported); ICU and hospital lengths of stay; renal recovery ; increased vasopressor use; need for interventions to treat HIRRT ; cardiovascular events; system clotting; bleeding; dialysis adequacy; treatment-related symptoms .An initial search of MEDLINE and PubMed was performed to assess for prior systematic reviews on this subject. Prospero was searched for any currently registered systematic review on this topic. No prior or ongoing systematic review was identified.In collaboration with other members of the research team, a health information specialist (LS) has constructed and will implement a comprehensive search strategy (refer to MEDLINE (1950–Feb 2016)EMBASE (1980–Feb 2016)CENTRAL As our search strategy, we will search the following databases:https://www.isrctn.com/). As well, this will include a review of abstracts from past 10 years from relevant scientific meetings:American Society of Nephrology Kidney WeekSociety for Critical Care Medicine (included in EMBASE search)International Symposium on Intensive Care and Emergency Medicine (included in EMBASE search)We will supplement our search with a manual scan of bibliographies of all included studies. In addition, a search of the “gray literature” will be performed. This includes a search using “Google Scholar” and a scan of clinical trial registries for ongoing clinical studies  for screening by two authors .Original data from an original study .Articles must specifically address HIRRT in patients with AKI.Any form of RRT for AKI is eligible except peritoneal dialysis.Only studies with titles published in English or French will be considered eligible for inclusion.Data from multiple reports assessing the same population or dataset will only be included once. If multiple reports are believed to represent the same population/dataset but report different outcomes or numbers of participants, then clarification will be sought from the corresponding author(s) regarding the totality of reporting for that population/dataset.An initial screen of all identified abstracts will be conducted by two investigators (EC and AD) to determine whether the articles are eligible for further review. The articles considered for further review must have the following requirements:The same investigators will then conduct the full-text search to determine whether retrieved articles fulfill inclusion or exclusion criteria. Disagreements will be resolved by consensus where possible and by a third reviewer (SH) if not.Study characteristics: first author, year of publication, geographic location, setting , design , duration of follow-upSample characteristics: numbers of participants in each arm, age, sex, inclusion and exclusion criteria, baseline imbalances across study arms, other possible confoundersDetails regarding interventions and co-interventions: dose/type of intervention, blinding, dropouts, and study withdrawalsOutcomes assessed: primary outcome of HIRRT vs other, definition of HIRRT and other outcomes assessed, description of measurement tools/devices.Effect of interventions/exposures: data describing categorical or continuous efficacy variables for the included outcomes, adverse eventsPrior to duplicate extraction by two independent reviewers , a data extraction form will be created and piloted. The data extracted will include:Data will be entered into Review Manager (RevMan 5.3) by one reviewer (AD) with verification by the second reviewer (EC).Two independent investigators (EC and AD) will assess the included studies for potential bias and the quality of reporting.Observational cohort studies will be assessed using the Cochrane Risk of Bias Assessment Tool for Non-Randomized Studies of Interventions (ACROBAT-NRSI) . QualityAny disagreements will be resolved by consensus or, if that is not possible, a third reviewer (SH).RevMan 5.3 will be used for all analyses. For continuous data, we will report the mean differences between groups as well as the 95% confidence intervals.I2 statistic with thresholds for interpretation defined in accordance with the Cochrane Handbook for Systematic Reviews of Interventions [Data will only be pooled for studies of similar design and limited heterogeneity; however, we expect that significant heterogeneity in interventions, comparator populations, and outcomes will prevent meta-analysis. We plan to examine potential sources of clinical and methodological heterogeneity according to study design, patient population, and outcomes measured as well as intervention and comparator characteristics. Should it be possible to do so, statistical heterogeneity will be assessed using the chi-squared test and defined using the ventions .Outcomes of interestCause of AKIIndication for RRTTreatment setting Methodological qualityPublication dataExtent of adjustment for confoundingShould meta-analysis not be possible or appropriate, the data will be primarily interpreted into a narrative synthesis. This synthesis will be organized according to the intervention/exposure(s) used to attempt to reduce or prevent HIRRT. For each intervention/exposure, they may be subgrouped according to the type of RRT used . Other groupings and subgroupings that may be considered include the following:Frequency of HIRRTMortalityICU and hospital length of stayRenal recoveryWhen possible, data will be interpreted into a quantitative synthesis. Data from included studies will be pooled whenever possible to explore the effect of any identified intervention on the primary or secondary outcomes:Assessment of reporting biases will be performed using a funnel plot (the standard error of the intervention effect estimate relative to the study size) if there are more than 10 studies included for analysis .An assessment of the quality of the evidence for the primary outcome from “very low” to “high” will be made in accordance with the criteria suggested by the Grading of Recommendations Assessment, Development and Evaluation (GRADE) Workgroup .HIRRT is a common complication of renal replacement therapy not only in ESKD but also in the critically ill AKI population. The consequences of HIRRT can range from minor to serious events including bowel ischemia, myocardial infarction, and access thrombosis . HIRRT cThis review proposes to use a systematic approach to identify, gather, and summarize the currently existing evidence on interventions to prevent HIRRT in critically ill patients with AKI on RRT from interventional and observational studies as well any ongoing trials. We aim to assess the effectiveness of strategies used to limit or prevent HIRRT in AKI by assessing specific, clinically important outcomes. In addition to exploring strategies to limit HIRRT in AKI, we will also review the available evidence for major outcomes associated with HIRRT, which includes mortality, ICU/hospital length of stay, and renal recovery. This summary of evidence can help guide clinical decisions regarding RRT parameters in AKI.We expect that most of the evidence will come from observational studies rather than RCTs. As there is a higher risk of bias in observational studies, we have chosen to implement in our protocol three accepted assessment tools, the Cochrane ACROBAT-NRSI , the STR


Cognitive Research: Principles includes articles aiming to formalize and test the effectiveness of embodied learning in STEM. The collection provides guideposts, staking out the terrain that should be surveyed before larger-scale efforts are undertaken. This introduction provides a broader context concerning mechanisms that can support embodied learning and make it especially well suited to the STEM disciplines.Embodied learning approaches emphasize the use of action to support pedagogical goals. A specific version of embodied learning posits an action-to-abstraction transition supported by gesture, sketching, and analogical mapping. These tools seem to have special promise for bolstering learning in science, technology, engineering, and mathematics (STEM) disciplines, but existing efforts need further theoretical and empirical development. The topical collection in  Gibson  speculatTheorists have distinguished between embodied cognition as incorporating the role of the body in providing sensory inputs to the cognitive system—a definition not incompatible with the computational account of cognition—and embodied cognition in which the body provides a constitutive component of cognition  building analogies between sensorimotor and abstract domains; 2) using gesture as a linking and abstraction tool; 3) improving cognitive skills and abilities; 4) off-loading cognitive processes and representations into the body or environment; and 5) constructing and interpreting visual representations.Analogies aid learning by mapping an unfamiliar domain onto a familiar one to encourage inferences or reach new conclusions Gentner, . AnalogiThe symbol grounding problem, as described by Glenberg  and BarsNathan and Walkington 4 proposeDove  and ChatApplying these ideas to STEM learning, Hayes and Kraemer 5 link thOne way analogies can connect abstract concepts with concrete sensory representations is through gesture. Gestures are nonverbal representational movements, usually of the hands, and usually accompanying speech. Gesture plays a role in maintaining or recalling visual imagery, simulating action, and representing the speaker’s nonverbal thoughts . By offloading, the learner may be able to use the extra cognitive resources to focus on problem solving, making inferences, or explaining to others. Embodied actions have long been thought to offload cognitive process onto the world or the body. Margaret Wilson’s  synthesiHow might action help offload information? Kirsh and Maglio  have disObservational research in the geosciences has demonstrated that offloading cognition plays a role in problem solving among geology students , and engineering, understudied disciplines in interdisciplinary cognitive science.


The burden of pleural diseases has substantially increased in the past decade because of a rise in the incidence of pleural space infections and pleural malignancies in a patient population that is older and more immunocompromised and has more comorbidities. This complexity increasingly requires minimally invasive diagnostic options and tailored management. Implications for patients are such that the limitations of current diagnostic methods need to be addressed by multidisciplinary teams of investigators from the fields of imaging, biology, and engineering. Ignored for a long time as an epiphenomenon at the crossroad of many unrelated medical problems, pleural diseases are finally getting the attention they deserve and have spurred a vibrant and exciting field of research. Interestingly, surgical removal of the pleura, or chemical fusion of the visceral and parietal membranes for therapeutic purposes (pleurodesis), seems to have little physiologic effect on lung function or work of breathing in long-term human studies and this raises the question of why such a complex structure was selected for in the first place5.The term pleura refers to the membrane separating the lungs from the chest wall, which is constituted by two leaflets attached to the outer surface of the lung  and the inside of the chest wall , joining at the hilum of the lungs, hence forming a genuine sac surrounding both lungs. The small amount of pleural fluid separating visceral from parietal pleural membranes serves as a lubricant, optimizing respiratory mechanics by facilitating synchronous chest wall and lung movements during respiratory efforts6. Approximately 150,000 of these effusions are caused by malignancy—lung and breast cancer are by far the most common offenders—generally due to parietal pleural involvement in advanced disease7. Other common causes of pleural effusions include congestive heart failure, pleural space infections, pulmonary embolism, and manifestations of connective tissue diseases such as lupus or rheumatoid arthritis. It should be intuitively obvious, given the profound implications of such diagnoses for patients, that timely and accurate diagnosis of unexplained pleural effusions is of considerable importance. Pleural fluid aspiration followed by application of the time-honored Light’s criteria remains a remarkably useful step in the diagnostic algorithm of pleural effusions, although an expanding panel of diagnostic tools in the past decade has slowly transformed our approach to pleural diseases, the increasing complexity of which has motivated the development of multidisciplinary pleural subspecialty programs at many institutions in recent years12. In this brief report, we will attempt to summarize what we believe are the most significant and promising recent advances in the diagnosis of pleural diseases and highlight current research efforts in the field.Notwithstanding this lack of clear physiologic function, pleural disorders represent one of the most common reasons for consultation of lung specialists, and an estimated 1.5 million new pleural effusions identified every year in the US alone are caused by more than 60 distinct disease processes16. In fact, the evidence for improved outcomes and reduced complication rates with ultrasound for thoracic procedures is so overwhelming that its use is now universally considered the standard of care18. Bedside ultrasound examination of the pleura can easily confirm the presence of pleural fluid when conventional chest x-ray findings are equivocal. In addition, it allows an estimation of its volume and complexity and occasionally identifies pleural nodules or masses suggestive of pleural malignancy19. While most physicians use ultrasound merely to mark the site for needle insertion before setting up for the procedure, real-time ultrasound using a sterile sleeve allows sampling of previously unreachable small loculated effusions and occasionally allows parietal pleural biopsies. Furthermore, recent evidence suggests that pre-procedural identification of the intercostal artery, the course of which can be unpredictable, is easily achieved with a high-frequency probe and could help avoid rare but potentially fatal bleeding complications22. Ultrasound examination immediately after the procedure can indirectly suggest the occurrence of pneumothorax by the disappearance of the typical “sliding” sign, representing the sliding of visceral and parietal pleura over each other. Rare but potentially serious bleeding complications from injury of the intercostal artery or one of its collateral vessels may also be identified in the form of rapid re-accumulation of echogenic pleural fluid24.Thoracic ultrasound, widely available in the form of increasingly sophisticated and affordable portable and handheld units, has unquestionably changed the paradigm in bedside medical evaluation. Basic ultrasound education now complements traditional physical examination training at many medical schools, residency, and fellowship programs. Point-of-care ultrasound for diagnosis and management of thoracic diseases in particular is recommended and endorsed by most relevant scientific societies, and there are established training and certification standards26. An intriguing recent report suggests that ultrasound may allow the identification of unexpandable lung before thoracentesis. In some cases, such as when chronic inflammatory effusions have resulted in the creation of a thick peel around the lung, fluid drainage is not accompanied by lung re-expansion. The vacuum generated can expose patients to complications including pain, pneumothorax, and re-expansion pulmonary edema. Transmission of the heartbeat results in different lung movement and deformation changes that can be identified by using specific ultrasound modes such as the M-mode and speckle tracking27. In this small study, ultrasound in fact performed better than pleural manometry, long regarded as the gold standard for the diagnosis of unexpandable lung and the utility of which is being assessed in an ongoing randomized clinical trial .Interestingly, there has been relatively little research on pleural ultrasound as a predictive tool. Two small studies suggest that a highly organized pleural space with loculations and septations during pleural space infection predicts poorer outcomes, but these studies are small and unadjusted for possible confounders28. However, in the absence of such specific findings, computed tomography appears neither sensitive nor specific enough to obviate the need for invasive diagnostic interventions30. Positron emission tomography is often used to assess the probability of malignant pleural effusion but with modest sensitivity and specificity, which in a recent meta-analysis were estimated to be 81% and 74%, respectively31. Recently, magnetic resonance imaging has been proposed as a potentially superior imaging modality for pleural effusions in general and malignant pleural mesothelioma in particular but remains largely underutilized compared with computed tomography34. Radiomic approaches to thoracic diseases represent an early and exciting field in imaging science, and very preliminary work in quantitative imaging for the diagnosis of malignant pleural effusion—and staging and diagnosis of mesothelioma in particular—is generating considerable interest36.There have been comparatively less recent data on other imaging modalities. Whereas loculations (sequestrations of fluid in non-dependent areas) are well identified on computed tomography, the degree of organization within the effusion is better appreciated on ultrasound. In addition, the presence of any of the four following radiologic signs is highly predictive of malignancy: parietal pleural thickening of more than 1 cm, thickened mediastinal pleura, circumferential thickening, and the presence of pleural nodules11. Briefly, at the risk of oversimplifying complex processes, transudative effusions typically result from local alterations of the Starling rules that govern capillary microcirculation in the parietal pleura with increased hydrostatic  or decreased oncotic  pressures, whereas exudative effusions suggest a disease process involving the parietal pleura itself, whether from malignancy, infection, or inflammatory diseases. However, these processes are complex and overlaps are frequent in terms of both pathophysiology and pleural fluid analysis. For example, an estimated 25% of effusions due to congestive heart failure may classify as exudates (“pseudo-exudates”), particularly if the patient is on diuretics37, and conversely a small percentage of documented malignant pleural effusions may classify as transudative effusions. While a high index of suspicion should lead to pleural biopsies in the latter scenario, recent data suggest that the pleural fluid–to–serum albumin gradient could help clarify the etiology of heart failure–related pseudo-exudative pleural effusions38. Pleural and serum brain natriuretic peptide and amino-terminal pro-brain natriuretic peptide levels are also useful to establish heart failure as the cause of the effusion39. Pleural space infections, potentially life-threatening conditions, are usually characterized by typical pleural fluid biochemistry and hence are more straightforward from a diagnostic standpoint, and an interesting recent observation is that the yield of pleural fluid cultures can be increased by 20% by simply inoculating blood culture bottles with pleural fluid at the bedside rather than sending the fluid directly to the laboratory40.The “diagnostic separation of transudates and exudates” using a combination of pleural fluid protein and lactate dehydrogenase levels was published in 1972  and to this day remains the single most important step in determining the etiology of a pleural effusion41, although some reports suggest much lower estimates42. Others focusing on modern immunohistochemistry techniques seem much more optimistic, particularly for mesothelioma, a primary pleural malignancy which traditionally requires pleural biopsies for definitive diagnosis44. In addition, pleural fluid has been shown to be a biospecimen suitable for the majority of molecular analyses required for targeted therapy45. High-throughput techniques and modern molecular techniques promise to identify biomarkers expressed by cancer cells or their environment that could ultimately transform our diagnostic approach. Novel proposed techniques include single-cell mechanophenotyping which evaluates the deformability of pleural cells46, circulating tumor cells and cell-free tumor DNA48, and metabolic-based assays to identify non-leukocyte metabolically active tumor cells49. These recent developments, though exciting, remain preliminary and are still closer to the bench than they are to the clinic. The search for optimal biomarkers has been hampered by a lack of standardized methodology and failure to externally validate promising results, as in the case of fibulin-3, a biomarker once anticipated to transform our approach to malignant mesothelioma53. Large ongoing research projects are attempting to identify reliable alternative candidates.The diagnostic utility of pleural fluid analysis for malignant pleural effusions has been the object of several recent studies. The sensitivity of cytology for malignancy is estimated around 60%, and there is a 15% increase with a second procedure54. A large multicenter observational prospective study to validate this score is ongoing. Similarly, the LENT  score is a clinical prediction model that provides estimates of survival for patients with malignant pleural effusions, which could prove very useful in subject selection in clinical trials and ultimately individualized medical or surgical management of pleural effusions55.An unprecedented number of well-designed randomized controlled studies published in the last decade by a growing international pleural research network have clarified and sometimes transformed patient management for malignant pleural effusions and pleural space infections in particular. However, one major obstacle faced by researchers and clinicians has been the lack of clinical prediction models allowing appropriate patient selection and stratification. The RAPID  score was derived and validated from two large datasets and proposes to risk-stratify patients with pleural space infections, and ultimately its purpose is to individualize management, which currently is subject to local preferences and expertise rather than patient characteristics56. An example from a Bayesian approach to diagnosis might be helpful to illustrate the limitations of this approach. Given a sensitivity for cytology of 60% and a specificity of 100%, a patient with a 50% pre-test probability of pleural malignancy would have, after a negative cytology, a remaining 27% post-test probability of disease. After a second thoracentesis, this number decreases to only approximately 18% (a generous estimate as the same test is used twice). Given the profound implications of a diagnosis of malignant pleural effusion, it should be intuitively obvious that moving forward with additional, ideally minimally invasive, diagnostic tests would be desirable.Approximately 25% of exudative pleural effusions remain without identifiable causes after pleural fluid analysis and cytology. Parietal pleural biopsies are the recommended next step in the diagnostic assessment but often are not pursued as they sometimes require thoracic surgery which, though less invasive since the advent of video-assisted thoracoscopic surgery, remains a major operation requiring general anesthesia, double-lumen endotracheal intubation, large-bore chest tube placement, and a hospital stay. Hence, clinicians often default to observation after an unfruitful pleural fluid analysis61. While pleuroscopy is slowly gaining traction in the US, centers offering the procedure have exponentially increased over the past decade in Europe and other parts of the world and presumably this is due to more favorable regulatory environments and sometimes more selective access to thoracic surgery. The development of dedicated interventional pulmonology training programs in the US, with accreditation standards that include pleuroscopy training endorsed by all relevant medical societies, may facilitate a more widespread adoption of this safe and effective diagnostic modality15. Even less invasive interventions, such as “mini-thoracoscopy”, using increasingly smaller instruments, have been proposed62.Reclaiming a procedure initially introduced by Hans Christian Jacobeus, a Swedish Professor of Internal Medicine at the Karolinska Institute in Stockholm from 1916 to 1937, interventional pulmonologists and pleural specialists have popularized minimally invasive, so-called “local anesthetic” pleuroscopy, which allows pleural exploration and biopsies in awake patients and often is performed on an outpatient basis63.When focal pleural lesions can be identified by computed tomography or ultrasound, percutaneous image-guided biopsies performed by interventional radiologists or pulmonary specialists are a useful and minimally invasive approach to diagnosis which in recent studies has had a yield similar to that of thoracoscopy but without the option to offer definitive treatment in the same setting The rising burden of pleural disease in an increasingly complex patient population demands a more tailored approach to diagnosis and management than ever before. Thoracic ultrasound, the application of new bioassays in addition to foundational biochemical analysis of pleural fluid, the development of models for prognosis and prediction of treatment response, and the resurgence of medical thoracoscopy-pleuroscopy comprise recent advances in pleural disease, and there is a great need for further basic, translational, and clinical research in this field.