Expression of prolyl hydroxylase domains, the upstream regulators of HIF, in the brain of the anoxia-tolerant crucian carp during anoxia-reoxygenation.

The hypoxia-inducible factor (HIF) is considered key in the transcriptional response to low oxygen. Yet, the role of HIF in the absence of oxygen (anoxia) and in preparation for reoxygenation remains unclear. Recent studies suggest that mounting a HIF response may be counterproductive for anoxia survival. We here studied one of the champions of anoxia survival, the crucian carp (Carassius carassius), and hypothesized that expression of prolyl hydroxylase domains (PHDs; the upstream regulators of HIF) are upregulated to circumvent an energy-costly activation of HIF in anoxia and to prepare for reoxygenation. We measured whole brain mRNA and protein levels of the three isoforms PHD1, PHD2, and PHD3, coded for by multiple paralogs of the genes egln2, egln1, and egln3, using quantitative PCR and Western blotting in the brain of crucian carps exposed to 5 days normoxia or anoxia, and 5 days anoxia followed by 3 or 24 h of reoxygenation. The mRNA levels of most egln paralogs were increased in anoxia and upon reoxygenation, with egln3 showing the largest increase in mRNA level (up to 17-fold) and highest relative mRNA abundance (up to 75% of expressed egln). The protein level of all PHDs was maintained in anoxia and increased upon reoxygenation. We then explored PHD distribution in different brain regions and found PHD immunoreactivity to be associated with axonal branches and showing region-specific changes during anoxia-reoxygenation. Our results support an overall upregulation of egln under prolonged anoxia and PHDs upon reoxygenation in crucian carp, likely aimed at suppressing HIF responses, although regional differences are apparent in such a complex organ as the brain.NEW & NOTEWORTHY We report a profound upregulation of most egln paralog mRNA levels in anoxia and upon reoxygenation, with egln3ii showing the largest, a 17-fold increase, and highest relative mRNA abundance. The relative abundance of prolyl hydroxylase domain (PHD) proteins was maintained during anoxia and increased at reoxygenation. PHD immunoreactivity was localized to axonal branches with region-specific changes during anoxia-reoxygenation. These dynamic and regional changes in crucian carp, champion of anoxia tolerance, are most likely adaptive and call for further mechanistic studies.

Rapid physiological and transcriptomic changes associated with oxygen delivery in larval anemonefish suggest a role in adaptation to life on hypoxic coral reefs.

Connectivity of coral reef fish populations relies on successful dispersal of a pelagic larval phase. Pelagic larvae must exhibit high swimming abilities to overcome ocean and reef currents, but once settling onto the reef, larvae transition to endure habitats that become hypoxic at night. Therefore, coral reef fish larvae must rapidly and dramatically shift their physiology over a short period of time. Taking an integrative, physiological approach, using swimming respirometry, and examining hypoxia tolerance and transcriptomics, we show that larvae of cinnamon anemonefish (Amphiprion melanopus) rapidly transition between "physiological extremes" at the end of their larval phase. Daily measurements of swimming larval anemonefish over their entire early development show that they initially have very high mass-specific oxygen uptake rates. However, oxygen uptake rates decrease midway through the larval phase. This occurs in conjunction with a switch in haemoglobin gene expression and increased expression of myoglobin, cytoglobin, and neuroglobin, which may all contribute to the observed increase in hypoxia tolerance. Our findings indicate that critical ontogenetic changes in the gene expression of oxygen-binding proteins may underpin the physiological mechanisms needed for successful larval recruitment to reefs.

Two decades of research on anoxia tolerance - mitochondria, -omics and physiological diversity.

Just over two decades ago, Bob Boutilier published a much-cited Review in this journal on the mechanisms of cell survival in hypoxia and hypothermia. Here, we celebrate this important Review by describing how our knowledge of the mechanisms behind anoxia tolerance have progressed since 2001, including new key roles of mitochondria, something Boutilier had started exploring. Evidence now suggests that, in anoxia-tolerant brains, mitochondria initiate responses aimed at suppressing electrical activity and energy use. These responses are largely dependent on gamma-aminobutyric acid (GABA) release. Animals that survive anoxia must also tolerate reoxygenation - a major challenge that could cause a massive production of damaging reactive oxygen species (ROS). Here, the handling of succinate, which builds up during anoxia, is critical. Interestingly, there are clear species differences in succinate handling among anoxia-tolerant vertebrates (Trachemys and Chrysemys turtles and crucian carp, Carassius carassius). Trachemys turtles suppress succinate build-up during anoxia, presumably to limit ROS production during reoxygenation. By contrast, in crucian carp, reduction of fumarate to succinate during anoxia appears to be essential for keeping their mitochondria charged and viable. Consequently, during anoxia, crucian carp accumulate much more succinate than Trachemys turtles. Moreover, during anoxia, succinate is apparently transported from crucian carp brain and heart to the liver, which handles succinate upon reoxygenation. This is one example of the striking physiological diversity among vertebrates that survive long-term anoxia. More examples are given, and we argue that -omics approaches are, and will be, helpful in providing new insight and moving the field forward.

Surviving without oxygen involves major tissue specific changes in the proteome of crucian carp (Carassius carassius).

The crucian carp (Carassius carassius) can survive complete oxygen depletion (anoxia) for several months at low temperatures, making it an excellent model for studying molecular adaptations to anoxia. Still, little is known about how its global proteome responds to anoxia and reoxygenation. By applying mass spectrometry-based proteome analyses on brain, heart and liver tissue from crucian carp exposed to normoxia, five days anoxia, and reoxygenation, we found major changes in particularly cardiac and hepatic protein levels in response to anoxia and reoxygenation. These included tissue-specific differences in mitochondrial proteins involved in aerobic respiration and mitochondrial membrane integrity. Enzymes in the electron transport system (ETS) decreased in heart and increased massively in liver during anoxia and reoxygenation but did not change in the brain. Importantly, the data support a special role for the liver in succinate handling upon reoxygenation, as suggested by a drastic increase of components of the ETS and uncoupling protein 2, which could allow for succinate metabolism without excessive formation of reactive oxygen species (ROS). Also during reoxygenation, the levels of proteins involved in the cristae junction organization of the mitochondria changed in the heart, possibly functioning to suppress ROS formation. Furthermore, proteins involved in immune (complement) system activation changed in the anoxic heart compared to normoxic controls. The results emphasize that responses to anoxia are highly tissue-specific and related to organ function.

Loss of pulp vitality correlated with the duration of the interim restoration and the experience of the dentist: A retrospective study.

STATEMENT OF PROBLEM: The second most common biological complication in fixed prosthodontics is loss of pulp vitality, which may lead to restoration loss. While reasons for loss of pulp vitality are unclear, 2 potential contributing factors, duration of the interim restoration and operator experience, have not been fully investigated. PURPOSE: The purpose of this retrospective study was to investigate whether the duration of the interim restoration or the experience of the dentist was correlated with loss of pulp vitality. MATERIAL AND METHODS: Fixed prosthetic restorations placed between 2005 and 2012 were retrospectively analyzed. Abutment teeth supporting single-unti or multiunit restorations were evaluated regarding loss of pulp vitality. The Mann-Whitney U test and simple logistic regression were used, with α=.05 for the subsequent multiple logistic regression. The experience of dental professionals was defined by the number of treatments performed and coupled with failure rate by using an analysis of variance. RESULTS: One hundred seventy-four dentists made 15 879 restorations, of which 1136 failed during the observation period, a failure rate of 7.2%. Two hundred fifty restorations were randomly selected from the failed restorations, and a corresponding 250 restorations were randomly selected from nonfailed restorations for the control group. Increased duration with interim replacement was linked to a higher risk of loss of pulp vitality (P<.001). Failure rate in the dentist group varied from 0% to 100%. No significant differences in failure rate were found among dentists who did few restorations and those who performed larger numbers of restorations. CONCLUSIONS: The results of the present study suggest that operator experience does not affect failure rate. However, extended time with an interim restoration was a contributing factor to the loss of pulp vitality.

Enigma of the cholesterol paradox in acute myocardial infarction: lessons from an 8-year follow-up of all-cause mortality in an age-matched and sex-matched case-control study with controls from the patients' recruitment area.

OBJECTIVE: To assess the impact of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) on long-term all-cause mortality (ACM) in patients with acute myocardial infarction (AMI) and controls. DESIGN: Matched case-control study with 8-year follow-up. SETTING: Vastmanland County Hospital, Vasteras, Sweden. PARTICIPANTS: Consecutive patients with AMI admitted to the coronary care unit from March 2005 to May 2010 and age-matched and sex-matched controls from the general population. OUTCOME MEASURES: ACM. RESULTS: Person-year at risk among patients with AMI and controls was 11 667 (cases: 5780 and controls: 5887). During follow-up, 199 patients and 84 controls died, implying 3.4 deaths among patients and 1.4 among controls per 100 person-years at risk. Unadjusted Cox analyses showed significantly increasing mortality by decreasing TC and LDL-C levels in both patients (HR=0.70, 95% CI 0.62 to 0.79, p<0.001, and HR=0.64, 95% CI 0.56 to 0.74, p<0.001) and controls (HR=0.73, 95% CI 0.60 to 0.89, p=0.002, and HR=0.74, 95% CI 0.59 to 0.93, p=0.010). After adjusting for clinical variables, the results for the patients remained significant. Cox analyses of the relations between mortality and TC and LDL-C below and above their respective medians revealed the following pattern. PATIENTS: below medians were TC and LDL-C levels significantly inversely related to mortality; above medians there were no relations with mortality. CONTROLS: below medians were TC and LDL-C levels significantly inversely related to mortality; above medians were LDL-C levels significantly positively related to mortality. Mean LDL-C level in patients with blood sampled >12 hours after symptom onset was 0.41 mmol/L lower than that in patients with blood sampled ≤12 hours (p=0.030). This LDL-C decrease was reasonably caused by ongoing AMI and reflects the difference in LDL-C levels between patients and controls. CONCLUSIONS: In patients with AMI, lower TC and LDL-C levels independently predict higher ACM. In their controls, LDL-C levels above the median independently predict higher ACM. This study adds to the body of evidence supporting the existence of a cholesterol paradox.

The Metabolomic Response of Crucian Carp (Carassius carassius) to Anoxia and Reoxygenation Differs between Tissues and Hints at Uncharacterized Survival Strategies.

The anoxia-tolerant crucian carp (Carassius carassius) has been studied in detail for numerous years, with particular focus on unravelling the underlying physiological mechanisms of anoxia tolerance. However, relatively little work has been focused on what occurs beyond anoxia, and often the focus is a single organ or tissue type. In this study, we quantified more than 100 metabolites by capillary electrophoresis-mass spectrometry (CE-MS) in brain, heart, liver, and blood plasma from four experimental groups, being normoxic (control) fish, anoxia-exposed fish, and two groups that had been exposed to anoxia followed by reoxygenation for either 3 h or 24 h. The heart, which maintains cardiac output during anoxia, unexpectedly, was slower to recover compared to the brain and liver, mainly due to a slower return to control concentrations of the energy-carrying compounds ATP, GTP, and phosphocreatine. Crucian carp accumulated amino acids in most tissues, and also surprisingly high levels of succinate in all tissues investigated during anoxia. Purine catabolism was enhanced, leading to accumulation of uric acid during anoxia and increasing urea formation that continued into 24 h of reoxygenation. These tissue-specific differences in accumulation and distribution of the metabolites may indicate an intricate system of transport between tissues, opening for new avenues of investigation of possible mechanisms aimed at reducing the generation of reactive oxygen species (ROS) and resultant tissue damage during reoxygenation.

H2S-producing enzymes in anoxia-tolerant vertebrates: Effects of cold acclimation, anoxia exposure and reoxygenation on gene and protein expression.

To lend insight into the potential role of the gasotransmitter hydrogen sulfide (H2S) in facilitating anoxia survival of anoxia-tolerant vertebrates, we quantified the gene expression of the primary H2S-synthesizing enzymes, 3-mercaptopyruvate sulfurtransferase (3MST), cystathionine γ-lyase (CSE) and cystathionine ß-synthase (CBS), in ventricle and brain of normoxic, anoxic and reoxygenated 21 °C- and 5 °C-acclimated freshwater turtles (Trachemys scripta) and 10 °C-acclimated crucian carp (Carassius carassius). Semi-quantitative Western blotting analysis was also conducted to assess 3MST and CBS protein abundance in ventricle and brain of 5 °C turtles and 10 °C crucian carp subjected to normoxia, anoxia and reoxygenation. We hypothesized that if H2S was advantageous for anoxia survival, expression levels would remain unchanged or be upregulated with anoxia and/or reoxygenation. Indeed, for both species, gene and protein expression were largely maintained with anoxia exposure (24 h, 21 °C; 5 d, 10 °C; 14 d, 5 °C). With reoxygenation, 3MST expression was increased in turtle and crucian carp brain at the protein and gene level, respectively. Additionally, the effect of cold acclimation on gene expression was assessed in several tissues of the turtle. Expression levels were maintained in most tissues, but decreased in others. The maintenance of gene and protein expression of the H2S-producing enzymes with anoxia exposure and the up-regulation of 3MST with reoxygenation suggests that H2S may facilitate anoxic survival of the two champions of vertebrate anoxia survival. The differential effects of cold acclimation on H2S enzyme expression may influence blood flow to different tissues during winter anoxia.

Neural effects of elevated CO2 in fish may be amplified by a vicious cycle.

Maladaptive behavioural disturbances have been reported in some fishes and aquatic invertebrates exposed to projected future CO2 levels. These disturbances have been linked to altered ion gradients and neurotransmitter function in the brain. Still, it seems surprising that the relatively small ionic changes induced by near-future CO2 levels can have such profound neural effects. Based on recent transcriptomics data, we propose that a vicious cycle can be triggered that amplifies the initial disturbance, explaining how small pH regulatory adjustments in response to ocean acidification can lead to major behavioural alterations in fish and other water-breathing animals. The proposed cycle is initiated by a reversal of the function of some inhibitory GABAA receptors in the direction of neural excitation and then amplified by adjustments in gene expression aimed at suppressing the excitation but in reality increasing it. In addition, the increased metabolic production of CO2 by overexcited neurons will feed into the cycle by elevating intracellular bicarbonate levels that will lead to increased excitatory ion fluxes through GABAA receptors. We also discuss the possibility that an initiation of a vicious cycle could be one of the several factors underlying the differences in neural sensitivity to elevated CO2 displayed by fishes.

Depression-like state behavioural outputs may confer beneficial outcomes in risky environments.

Recent theories in evolutionary medicine have suggested that behavioural outputs associated with depression-like states (DLS) could be an adaptation to unpredictable and precarious situations. In animal models, DLS are often linked to diverse and unpredictable stressors or adverse experiences. Theoretically, there are a range of potential fitness benefits associated with behavioural inhibition (typical to DLS), as opposed to more active/aggressive responses to adverse or uncontrollable events. This stance of evolutionary medicine has to our knowledge not been tested empirically. Here we address a possible key benefit of behavioural inhibition in a comparative model for social stress (territorial rainbow trout). By treating fish with the fast-acting antidepressant ketamine, we reversed the behavioural inhibition (i.e. stimulated an increase in activity level) in subordinate fish. During confrontation with a previously unfamiliar larger, aggressive and dominant individual, this increase in activity led to higher amounts of received aggression compared to sham-treated subordinates. This suggests that the behavioural inhibition characterizing animal models of DLS is indeed an effective coping strategy that reduces the risk of injuries in vulnerable social situations.

The expression of genes involved in excitatory and inhibitory neurotransmission in turtle (Trachemys scripta) brain during anoxic submergence at 21 °C and 5 °C reveals the importance of cold as a preparatory cue for anoxia survival.

We investigated if transcriptional responses are consistent with the arrest of synaptic activity in the anoxic turtle (Trachemys scripta) brain. Thirty-nine genes of key receptors, transporters, enzymes and regulatory proteins of inhibitory and excitatory neurotransmission were partially cloned and their expression in telencephalon of 21 °C- and 5 °C-acclimated normoxic, anoxic (24 h at 21 °C; 1 and 14 days at 5 °C) and reoxygenated (24 h at 21 °C; 13 days at 5 °C) turtles quantified by real-time RT-PCR. Gene expression was largely sustained with anoxia at 21 °C and 5 °C. However, the changes in gene expression that did occur were congruous with the decline in glutamatergic activity and the increase in GABAergic activity observed at cellular and whole organism levels. Moreover, at 21 °C, the alterations in gene expression with anoxia induced a distinct gene expression pattern compared to normoxia and reoxygenation. Strikingly, acclimation from 21 °C to 5 °C in normoxia effectuated substantial transcriptional responses. Most prominently, 56% of the excitatory neurotransmission genes were down-regulated, including most of the ones encoding the subunits composing excitatory N-methyl-d-aspartate (NMDA) and 3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptors. By contrast, only 26% of the inhibitory neurotransmission genes were down-regulated. Consequently, the gene expression pattern of 5 °C normoxic turtles was statistically distinct compared to that of 21 °C normoxic turtles. Overall, this study highlights that key transcriptional responses are consonant with the synaptic arrest that occurs in the anoxic turtle brain. In addition, the findings reveal that transcriptional remodelling induced by decreased temperature may serve to precondition the turtle brain for winter anoxia.

Effects of lactate ions on the cardiorespiratory system in rainbow trout (Oncorhynchus mykiss).

Lactate ions are involved in several physiological processes, including a direct stimulation of the carotid body, causing increased ventilation in mammals. A similar mechanism eliciting ventilatory stimulation in other vertebrate classes has been demonstrated, but it remains to be thoroughly investigated. Here, we investigated the effects of lactate ions on the cardiorespiratory system in swimming rainbow trout by manipulating the blood lactate concentration. Lactate elicited a vigorous, dose-dependent elevation of ventilation and bradycardia at physiologically relevant concentrations at constant pH. After this initial confirmation, we examined the chiral specificity of the response and found that only l-lactate induced these effects. By removal of the afferent inputs from the first gill arch, the response was greatly attenuated, and a comparison of the responses to injections up- and downstream of the gills collectively demonstrated that the lactate response was initiated by branchial cells. Injection of specific receptor antagonists revealed that a blockade of serotonergic receptors, which are involved in the hypoxic ventilatory response, significantly reduced the lactate response. Finally, we identified two putative lactate receptors based on sequence homology and found that both were expressed at substantially higher levels in the gills. We propose that lactate ions modulate ventilation by stimulating branchial oxygen-sensing cells, thus eliciting a cardiorespiratory response through receptors likely to have originated early in vertebrate evolution.

Basic Anthropometric Measures in Acute Myocardial Infarction Patients and Individually Sex- and Age-Matched Controls from the General Population.

We compared weight, height, waist and hip circumferences (hip), body mass index (BMI), and waist-to-hip ratio in acute myocardial infarction (MI) patients and individually sex- and age-matched control subjects from the general population in the catchment area of the patients and predicted the risk of MI status by these basic anthropometric measures. The study cohort comprised 748 patients ≤80 years of age with acute MI from a major Swedish cardiac center and their individually sex- and age-matched controls. The analyses were stratified for sex and age (≤65/≥66 years). Risk of MI was assessed by conditional logistic regression. A narrow hip in men ≥66 years was the single strongest risk factor of MI among the anthropometric measures. The combination of hip and weight was particularly efficient in discriminating men ≥66 years with MI from their controls (area under the receiver operating characteristic (AUROC) curve = 0.82). In men ≤65 years, the best combination was hip, BMI, and height (AUROC = 0.79). In women ≥66 years, the best discriminatory model contained only waist-to-hip ratio (AUROC = 0.67), whereas in women ≤65 years, the best combination was hip and BMI (AUROC = 0.68). A narrow hip reasonably reflects small gluteal muscles. This finding might suggest an association between MI and sarcopenia, possibly related to deficiencies in physical activity and nutrition.

Short-term cortisol exposure alters cardiac hypertrophic and non-hypertrophic signalling in a time-dependent manner in rainbow trout.

Cardiac disease is a growing concern in farmed animals, and stress has been implicated as a factor for myocardial dysfunction and mortality in commercial fish rearing. We recently showed that the stress hormone cortisol induces pathological cardiac remodelling in rainbow trout. Wild and farmed salmonids are exposed to fluctuations and sometimes prolonged episodes of increased cortisol levels. Thus, studying the timeframe of cortisol-induced cardiac remodelling is necessary to understand its role in the pathogenesis of cardiovascular disease in salmonids. We here establish that 3 weeks of cortisol exposure is sufficient to increase relative ventricular mass (RVM) by 20% in rainbow trout. Moreover, increased RVMs are associated with altered expression of hypertrophic and non-hypertrophic remodelling markers. Further, we characterised the time course of cortisol-induced cardiac remodelling by feeding rainbow trout cortisol-containing feed for 2, 7 and 21 days. We show that the effect of cortisol on expression of hypertrophic and non-hypertrophic remodelling markers is time-dependent and in some cases acute. Our data indicate that short-term stressors and life cycle transitions associated with elevated cortisol levels can potentially influence hypertrophic and non-hypertrophic remodelling of the trout heart.

Small Non-coding RNA Expression and Vertebrate Anoxia Tolerance.

Background: Extreme anoxia tolerance requires a metabolic depression whose modulation could involve small non-coding RNAs (small ncRNAs), which are specific, rapid, and reversible regulators of gene expression. A previous study of small ncRNA expression in embryos of the annual killifish Austrofundulus limnaeus, the most anoxia-tolerant vertebrate known, revealed a specific expression pattern of small ncRNAs that could play important roles in anoxia tolerance. Here, we conduct a comparative study on the presence and expression of small ncRNAs in the most anoxia-tolerant representatives of several major vertebrate lineages, to investigate the evolution of and mechanisms supporting extreme anoxia tolerance. The epaulette shark (Hemiscyllium ocellatum), crucian carp (Carassius carassius), western painted turtle (Chrysemys picta bellii), and leopard frog (Rana pipiens) were exposed to anoxia and recovery, and small ncRNAs were sequenced from the brain (one of the most anoxia-sensitive tissues) prior to, during, and following exposure to anoxia. Results: Small ncRNA profiles were broadly conserved among species under normoxic conditions, and these expression patterns were largely conserved during exposure to anoxia. In contrast, differentially expressed genes are mostly unique to each species, suggesting that each species may have evolved distinct small ncRNA expression patterns in response to anoxia. Mitochondria-derived small ncRNAs (mitosRNAs) which have a robust response to anoxia in A. limnaeus embryos, were identified in the other anoxia tolerant vertebrates here but did not display a similarly robust response to anoxia. Conclusion: These findings support an overall stabilization of the small ncRNA transcriptome during exposure to anoxic insults, but also suggest that multiple small ncRNA expression pathways may support anoxia tolerance, as no conserved small ncRNA response was identified among the anoxia-tolerant vertebrates studied. This may reflect divergent strategies to achieve the same endpoint: anoxia tolerance. However, it may also indicate that there are multiple cellular pathways that can trigger the same cellular and physiological survival processes, including hypometabolism.

Commutability Assessment of Candidate Reference Materials for Pancreatic α-Amylase.

BACKGROUND: Measurement standardization of the catalytic concentration of α-amylase in serum is based on 3 pillars: the primary reference measurement procedure (PRMP), reference laboratories, and suitable certified reference materials (CRMs). Commutability is a prerequisite when using a CRM for calibration and trueness control of routine methods or for value transfer from the PRMP to end-user calibrators of routine methods through a calibration hierarchy. METHODS: We performed a commutability study with 30 serum pools and 5 candidate reference materials (RMs) for pancreatic α-amylase using an automated version of the PRMP and 5 different routine methods. Four candidate RMs had an artificial matrix, each with a different composition, and 1 candidate RM was based on human serum. Data were analyzed according to a linear regression analysis with prediction interval as described in the Clinical and Laboratory Standards Institute guideline EP30-A and a difference in bias analysis as described in the recommendations of the IFCC Working Group on Commutability. RESULTS: The commutability profile of the 4 candidate RMs with an artificial matrix was variable. Only 1 candidate RM, with human serum albumin in the matrix, showed a good profile like that of the candidate RM based on serum. The comparison of both commutability assessment approaches indicated some differences because of inconclusive results for the difference in bias approach, suggesting a large uncertainty on the commutability assessment. CONCLUSIONS: A CRM for pancreatic amylase in an artificial matrix can be commutable for routine methods using the same substrate as the PRMP, but the matrix composition is crucial.

Experimental strategies to assess the biological ramifications of multiple drivers of global ocean change-A review.

Marine life is controlled by multiple physical and chemical drivers and by diverse ecological processes. Many of these oceanic properties are being altered by climate change and other anthropogenic pressures. Hence, identifying the influences of multifaceted ocean change, from local to global scales, is a complex task. To guide policy-making and make projections of the future of the marine biosphere, it is essential to understand biological responses at physiological, evolutionary and ecological levels. Here, we contrast and compare different approaches to multiple driver experiments that aim to elucidate biological responses to a complex matrix of ocean global change. We present the benefits and the challenges of each approach with a focus on marine research, and guidelines to navigate through these different categories to help identify strategies that might best address research questions in fundamental physiology, experimental evolutionary biology and community ecology. Our review reveals that the field of multiple driver research is being pulled in complementary directions: the need for reductionist approaches to obtain process-oriented, mechanistic understanding and a requirement to quantify responses to projected future scenarios of ocean change. We conclude the review with recommendations on how best to align different experimental approaches to contribute fundamental information needed for science-based policy formulation.

IFCC Working Group Recommendations for Assessing Commutability Part 1: General Experimental Design.

Commutability is a property of a reference material (RM) that relates to the closeness of agreement between results for an RM and results for clinical samples (CSs) when measured by ≥2 measurement procedures (MPs). Commutability of RMs used in a calibration traceability scheme is an essential property for them to be fit for purpose. Similarly, commutability of trueness controls or external quality assessment samples is essential when those materials are used to assess trueness of results for CSs. This report is part 1 of a 3-part series describing how to assess commutability of RMs. Part 1 defines commutability and addresses critical components of the experimental design for commutability assessment, including selection of individual CSs, use of pooled CSs, qualification of MPs for inclusion, establishing criteria for the determination that an RM is commutable, generalization of commutability conclusions to future measurements made with the MPs included in the assessment, and information regarding commutability to be included in the certificate for an RM. Parts 2 and 3 in the series present 2 different statistical approaches to commutability assessment that use fixed criteria related to the medical decisions that will be made using the laboratory test results.

IFCC Working Group Recommendations for Assessing Commutability Part 2: Using the Difference in Bias between a Reference Material and Clinical Samples.

A process is described to assess the commutability of a reference material (RM) intended for use as a calibrator, trueness control, or external quality assessment sample based on the difference in bias between an RM and clinical samples (CSs) measured using 2 different measurement procedures (MPs). This difference in bias is compared with a criterion based on a medically relevant difference between an RM and CS results to make a conclusion regarding commutability. When more than 2 MPs are included, the commutability is assessed pairwise for all combinations of 2 MPs. This approach allows the same criterion to be used for all combinations of MPs included in the assessment. The assessment is based on an error model that allows estimation of various random and systematic sources of error, including those from sample-specific effects of interfering substances. An advantage of this approach is that the difference in bias between an RM and the average bias of CSs at the concentration (i.e., amount of substance present or quantity value) of the RM is determined and its uncertainty estimated. An RM is considered fit for purpose for those MPs for which commutability is demonstrated.