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Oxygen fluctuations are common in marine waters, and hypoxia–reoxygenation (H–R) stress can negatively affect mitochondrial metabolism. The long-lived ocean quahog, Arctica islandica, is known for its hypoxia tolerance associated with metabolic rate depression, yet the mechanisms that sustain mitochondrial function during oxygen fluctuations are not well understood. We used top-down metabolic control analysis (MCA) to determine aerobic capacity and control over oxygen flux in the mitochondria of quahogs exposed to short-term hypoxia (24 h

Supercritical fluid chromatography (SFC) holds the potential to become an orthogonal method to HPLC/UHPLC in xenobiotic metabolism studies, due to its outstanding capacity to simultaneously separate highly similar (as HPLC) and physicochemically different analytes (problematic using HPLC). Paucity of guideline-conform validation, however, has been a major obstacle to clinical application of SFC, even in cases where biotransformation yields chemically dissimilar metabolites that require more than one HPLC method for comprehensive analysis. Here, a method based on supercritical fluid chromatography coupled to single quadrupole MS detection was developed to simultaneously quantify the divisive analgesic flupirtine and its acidic and basic metabolites, represented by 4-fluorohippuric acid (4-FHA) and the active metabolite D-13223 respectively, using custom-made synthetic internal standards. Experimental data on the fundamental retention mechanisms under supercritical conditions, indicating the importance of halogen and π-π-bonding for specific retention on polysaccharide-based stationary-phases, is discussed. Compared to previous HPLC methods, the novel method offers higher versatility in terms of the target metabolite range (addressing both acidic and basic metabolites within a singular method), faster analysis (7.5 min), and compliance with green chemistry principles. Validation was performed according to EMA criteria on bioanalytical method validation, demonstrating selectivity, carry-over, calibration curve parameters (LLOQ, range, and linearity), within- and between-run accuracy and precision, dilution integrity, matrix effect and stability. For proof-of-concept, the SFC method was applied to clinical samples of human urine obtained after single intravenous (100 mg), single oral (100 mg), and repeated oral administration (400 mg). Flupirtine, D-13223, and 4-FHA could be quantified, shedding light on the extent of oxidative flupirtine metabolism in humans in the context of the unresolved biotoxification that has led to the withdrawal of specific neuronal KV7 openers.

The potassium channel openers flupirtine and retigabine have proven to be valuable analgesics or antiepileptics. Their recent withdrawal due to occasional hepatotoxicity and tissue discoloration, respectively, leaves a therapeutic niche unfilled. Metabolic oxidation of both drugs gives rise to the formation of electrophilic quinones. These elusive, highly reactive metabolites may induce liver injury in the case of flupirtine and blue tissue discoloration after prolonged intake of retigabine. We examined which structural features can be altered to avoid the detrimental oxidation of the aromatic ring and shift oxidation toward the formation of more benign metabolites. Structure-activity relationship studies were performed to evaluate the KV 7.2/3 channel opening activity of 45 derivatives. Sulfide analogues were identified that are devoid of the risk of quinone formation, but possess potent KV 7.2/3 opening activity. For example, flupirtine analogue 3-(3,5-difluorophenyl)-N-(6-(isobutylthio)-2-(pyrrolidin-1-yl)pyridin-3-yl)propanamide (48) has 100-fold enhanced activity (EC50 =1.4 nm), a vastly improved toxicity/activity ratio, and the same efficacy as retigabine in vitro.

The highly structurally similar drugs flupirtine and retigabine have been regarded as safe and effective for many years but lately they turned out to exert intolerable side effects. While the twin molecules share the mode of action, both stabilize the open state of voltage-gated potassium channels, the form and severity of adverse effects is different. The analgesic flupirtine caused drug-induced liver injury in rare but fatal cases, whereas prolonged use of the antiepileptic retigabine led to blue tissue discoloration. Because the adverse effects seem unrelated to the mode of action, it is likely, that both drugs that occupied important therapeutic niches, could be replaced. Reasons for the clinically relevant toxicity will be clarified and future substitutes for these drugs presented in this review.

Drug induced liver injury (DILI) and tissue discoloration led to the recent discontinuation of the therapeutic use of the closely related drugs flupirtine and retigabine, respectively. Experience gained with these drugs strongly suggests that heterotetramer, voltage-gated potassium channels 2 and 3 (KV7.2/3) are valid targets for effective treatment of pain and epilepsy. Because the adverse effects are not related to the mechanism of action, it appears promising to investigate chemical modifications of these clinically validated, drug-like leads. In the present retro-metabolic drug design study, a series of 43 compounds were synthesized and characterized with regard to KV7.2/3 opening activity and efficacy. The most active compound 22d displays excellent potency (EC50 = 4 nM) and efficacy (154%) as a KV7.2/3 opener. Limited aqueous solubility hampered toxicity testing at concentrations higher than 63 μM, but this concentration was nontoxic to two hepatocellular cell lines (HEP-G2 and TAMH) in culture. The slightly less active but more soluble compound 25b (EC50 = 11 nM, efficacy 111%) showed an improved toxicity/activity ratio compared to flupirtine by three orders of magnitude and represents an attractive lead structure for the development of safer analgesics and antiepileptics.

Ectotherms are exposed to a range of environmental temperatures and may face extremes beyond their upper thermal limits. Such temperature extremes can stimulate aerobic metabolism toward its maximum, a decline in aerobic substrate oxidation, and a parallel increase of anaerobic metabolism, combined with ROS generation and oxidative stress. Under these stressful conditions, marine organisms recruit several defensive strategies for their maintenance and survival. However, thermal tolerance of ectothermic organisms may be increased after a brief exposure to sub-lethal temperatures, a process known as "hardening". In our study, we examined the ability of M. galloprovincialis to increase its thermal tolerance under the effect of elevated temperatures (24, 26 and 28 °C) through the "hardening" process. Our results demonstrate that this process can increase the heat tolerance and antioxidant defense of heat hardened mussels through more efficient ETS activity when exposed to temperatures beyond 24 °C, compared to non-hardened individuals. Enhanced cell protection is reflected in better adaptive strategies of heat hardened mussels, and thus decreased mortality. Although hardening seems a promising process for the maintenance of aquacultured populations under increased seasonal temperatures, further investigation of the molecular and cellular mechanisms regulating mussels’ heat resistance is required.

The thermal tolerance of marine decapod crustacea is defined through their capacities for oxygen uptake and distribution. High ambient CO2levels were previously shown to reduce hemolymph oxygen levels at enhanced cardiac performance during warming. This study investigated the impacts of warming under two CO2levels on ventilation and hemolymph circulation in edible crabsCancer pagurus. It also highlights changes in the ventilatory and cardiac pauses displayed by Decapoda under routine metabolism. Animals were exposed to step-wise, sub-critical warming (12–20°C over 5 days) under control (470 μatm) and high (1,350 μatm) waterPCO2. Flow-through respirometry was combined with magnetic resonance imaging and infra-red photoplethysmography to allow for simultaneous, non-invasive measurements of metabolic rates (M˙O2), ventilation and cardiovascular performance. Crabs spent significantly more time in a lowM˙O2state (metabolic pause), when experiencing high CO2conditions above 16°C, compared to normocapnic warming. Heart rates leveled off beyond 18°C at any CO2level. Cardiac output continued to increase with high-CO2-warming, due to elevated cardiac stroke volumes. Consequently, temperature-dependent branchial hemolymph flow remained unaffected by CO2. Instead, a suppressing effect of CO2on ventilation was found beyond 16°C. These results indicate constrained oxygen uptake at stable cardiovascular performance in a decapod crustacean.Cancer pagurus: urn:lsid:zoobank.org:act:B750F89A-84B5-448B-8D80-EBD724A1C9D4

Psychological risk assessment is a legal obligation for companies and part of occupational safety and employment protection in Germany. However, data from psychological risk assessments in nursing staff are scarce, although this population is at increased risk for secondary traumatic stress by patient experienced trauma. Therefore, our study aimed at examining the frequency of reported secondary trauma events, secondary traumatic stress, and its possible consequences for psychological well-being and work ability in nurses. N = 320 nurses (n = 280 female) were assessed at a University Hospital in Germany as part of the psychological risk assessment. Secondary traumatic events, secondary traumatic stress, and symptoms of depression and anxiety were measured using self-report questionnaires (PHQ-2, GAD-2), and work ability was assessed using a modified version of the questionnaire for workplace analysis (KFZA). Of 320 nurses, 292 (91.2%) experienced secondary trauma, and 74 nurses (25.3%) reported secondary traumatic symptoms. Nurses with secondary traumatic symptoms reported higher depression (p < 0.001) and anxiety scores (p < 0.001) compared to nurses without secondary trauma experience, and to nurses with secondary trauma experience but without secondary traumatic stress (both p < 0.001). Further, nurses with secondary traumatic stress reported significantly reduced work ability, social support and control over work, and increased emotional strain and labor time. Nurses with secondary traumatic stress may be at increased risk of developing major depression and anxiety disorders, and particularly need support in overcoming secondary traumatic experiences. Psychological risk assessment is a useful tool to identify groups at risk, and pave the way to implement strategies to improve mental well-being and prevent work ability in high risk groups.

International audience; Radioisotopes have been used in earth and environmental sciences for over 150 years and provide unique tools to study environmental processes in great detail from a cellular level through to an oceanic basin scale. These nuclear techniques have been employed to understand coastal and marine ecosystems via laboratory and field studies to understand how aquatic organisms respond to environmental stressors including temperature, pH, nutrients, metals, organic anthropogenic compounds and biological toxins. Global marine issues, such as ocean warming, deoxygenation, plastic pollution, ocean acidification, increased duration and intensity of toxic harmful algal blooms (HABs), and coastal contamination are all impacting marine environments, thereby imposing various environmental and economic risks. Being able to reliably assess the health of coastal and marine ecosystems, and how they may respond to future disturbances, can provide vital information for society in the sustainable management of their marine environments. This paper summarises the historical use of radiotracers in these systems, describes how existing techniques of radioecological tracing can be developed for specific current environmental issues and provides information on emerging issues that would benefit from current and new radiotracer methods. Current challenges with using radioecological tracers and opportunities are highlighted as well as opportunities to maximise the uptake of these methods to greatly increase the ability of environmental managers to conduct evidence-based management of coastal and marine ecosystems

BackgroundBrachyuran crabs can effectively modulate cardiac stroke volume independently of heart rate in response to abiotic drivers. Non-invasive techniques can help to improve the understanding of cardiac performance parameters of these animals. This study demonstrates the in vivo quantification of cardiac performance parameters through magnetic resonance imaging (MRI) on the edible crabCancer pagurus. Furthermore, the suitability of signal integrals of infra-red photoplethysmographs as a qualitative tool is assessed under severe hypoxia.ResultsMulti-slice self-gated cardiac cinematic (CINE) MRI revealed the structure and motion of the ventricle to quantify heart rates, end-diastolic volume, end-systolic volume, stroke volume and ejection fraction. CINE MRI showed that stroke volumes increased under hypoxia because of a reduction of end-systolic volumes at constant end-diastolic volumes. Plethysmograph recordings allowed for automated heart rate measurements but determination of a qualitative stroke volume proxy strongly depended on the position of the sensor on the animal. Both techniques revealed a doubling in stroke volumes after 6 h under severe hypoxia (waterPO2 = 15% air saturation).ConclusionsMRI has allowed for detailed descriptions of cardiac performance in intact animals under hypoxia. The temporal resolution of quantitative non-invasive CINE MRI is limited but should encourage further refining. The stroke volume proxy based on plethysmograph recordings is feasible to complement other cardiac measurements over time. The presented methods allow for non-destructive in vivo determinations of multiple cardiac performance parameters, with the possibility to study neuro-hormonal or environmental effects on decapod cardio physiology.

Flupirtine, an opener of neuronal voltage gated potassium channels (KV7.2/3), has been used as a therapeutic alternative for pain treatment in patients refractory to NSAIDs and opioids. Because flupirtine is associated with rare but fatal drug-induced liver injury that may result from the formation of toxic metabolites upon metabolic oxidation, we synthesized novel derivatives with the goal of identifying equally active and ultimately safer KV7.2/3 channel openers. Four thioether analogues were designed to lack a nitrogen atom that would be a prerequisite for the formation of toxic para-quinone diimines, and form sulfoxide and sulfone metabolites instead. KV7.2/3 channel opening activity and hepatotoxicity data of twelve novel flupirtine analogues, four thioethers and their respective sulfoxide and sulfone metabolites are reported.

With ongoing climate change, rising ocean temperature is usually accompanied by falling oxygen levels (hypoxia) and increasing CO 2 concentration (hypercapnia). Both drivers may impose constraints on physiological mechanisms that define thermal limits resulting in increased vulnerability towards warming in marine ectotherms. The present study aimed to detect differences in thermal tolerance by investigating the underlying metabolic responses in the green abalone ( Haliotis fulgens ) under conditions of hypoxia and hypercapnia. Juvenile abalones were exposed to a temperature ramp (+ 3 °C day − 1 ) under hypoxia (50% air saturation) and hypercapnia (~ 1000 μatm p CO 2 ), both individually and in combination. Impacts on energy metabolism were assessed by analyzing whole animal respiration rates and metabolic profiles of gills and hepatopancreas via 1 H NMR spectroscopy. While hypercapnia had a minor impact on the results of the temperature treatment, hypoxia strongly increased the vulnerability to warming, indicated by respiration rates falling below values expected from an exponential increase and by the onset of anaerobic metabolism suggesting a downward shift of the upper critical temperature. Warming under combined hypoxia and hypercapnia elicited a severe change in metabolism involving a strong accumulation of amino acids, osmolytes and anaerobic end products at intermediate temperatures, followed by declining concentrations at warmer temperatures. This matched the limited capacity to increase metabolic rate, loss of attachment and mortality observed under these conditions suggesting a strong narrowing of the thermal window. In all cases, the accumulation of free amino acids identified proteins as a significant energy source during warming stress.

We examined the relationship between individuals' perceived violation of rights and interests and their participation in collective action. In addition, we examined the mediation effects of perceived relative deprivation and political interest. We used data from the Chinese General Social Survey conducted in 2010, which resulted in a sample size of 11,121. Results showed that, after controlling for demographic variables, perceived violation of rights and interests significantly increased the probability of individuals participating in collective action. Further testing showed that perceived relative economic deprivation had a significant mediation effect. Although perceived violation of rights and interests also increased individuals' perceived relative social deprivation, the perception of relative social deprivation did not have an effect on collective action participation. Political interest, however, had a significant mediation effect. Practical implications for public administrators in China are discussed.

Object: Dynamic in vivo 31P-NMR spectroscopy in combination with Magnetic Resonance Imaging (MRI) was used to study muscle bioenergetics of boreal and Arctic scallops (Pecten maximus and Chlamys islandica) to test the hypothesis that future Ocean Warming and Acidification (OWA) will impair the performance of marine invertebrates. Materials & methods: Experiments were conducted following the recommendations for studies of muscle bioenergetics in vertebrates. Animals were long-term incubated under different environmental conditions: controls at 0 °C for C. islandica and 15 °C for P. maximus under ambient PCO2 of 0.039 kPa, a warm exposure with +5 °C (5 °C and 20 °C, respectively) under ambient PCO2 (OW group), and a combined exposure to warmed acidified conditions (5 °C and 20 °C, 0.112 kPa PCO2, OWA group). Scallops were placed in a 4.7 T MR animal scanner and the energetic status of the adductor muscle was determined under resting conditions using in vivo 31P-NMR spectroscopy. The surplus oxidative flux (Qmax) was quantified by recording the recovery of arginine phosphate (PLA) directly after moderate swimming exercise of the scallops. Results: Measurements led to reproducible results within each experimental group. Under projected future conditions resting PLA levels (PLArest) were reduced, indicating reduced energy reserves in warming exposed scallops per se. In comparison to vertebrate muscle tissue surplus Qmax of scallop muscle was about one order of magnitude lower. This can be explained by lower mitochondrial contents and capacities in invertebrate than vertebrate muscle tissue. Warm exposed scallops showed a slower recovery rate of PLA levels (kPLA) and a reduced surplus Qmax. Elevated PCO2 did not affected PLA recovery further. Conclusion: Dynamic in vivo 31P-NMR spectroscopy revealed constrained residual aerobic power budgets in boreal and Arctic scallops under projected ocean warming and acidification indicating that scallops are susceptible to future climate change. The observed reduction in muscular PLArest of scallops coping with a warmer and acidified ocean may be linked to an enhanced energy demand and reduced oxygen partial pressures (PO2) in their body fluids. Delayed recovery from moderate swimming at elevated temperature is a result of reduced PLArest concentrations associated with a warm-induced reduction of a residual aerobic power budget.

An approach is presented for high-field MRI studies of the cardiovascular system (CVS) of a marine crustacean, the edible crab Cancer pagurus, submerged in highly conductive seawater. Structure and function of the CVS were investigated at 9.4 T. Cardiac motion was studied using self-gated CINE MRI. Imaging protocols and radio-frequency coil arrangements were tested for anatomical imaging. Haemolymph flow was quantified using phase-contrast angiography. Signal-to-noise-ratios and flow velocities in afferent and efferent branchial veins were compared with Student’s t test (n = 5). Seawater induced signal losses were dependent on imaging protocols and RF coil setup. Internal cardiac structures could be visualized with high spatial resolution within 8 min using a gradient-echo technique. Variations in haemolymph flow in different vessels could be determined over time. Maximum flow was similar within individual vessels and corresponded to literature values from Doppler measurements. Heart contractions were more pronounced in lateral and dorso-ventral directions than in the anterior–posterior direction. Choosing adequate imaging protocols in combination with a specific RF coil arrangement allows to monitor various parts of the crustacean CVS with exceptionally high spatial resolution despite the adverse effects of seawater at 9.4 T.

Stanniocalcin 1 (STC1) and parathyroid hormone-related protein (PTHrP) are calciotropic hormones in vertebrates. Here, a recently hypothesized metabolic role for these hormones is tested on European sea bass treated with: (i) teleost PTHrP(1-34), (ii) PTHrP(1-34) and anti-STC1 serum (pro-PTHrP groups), (iii) a PTHrP antagonist PTHrP(7-34) or (iv) PTHrP(7-34) and STC1 (pro-STC1 groups). Livers were analysed using untargeted metabolic profiling based on proton nuclear magnetic resonance (1H-NMR) spectroscopy. Concentrations of branched-chain amino acid (BCAA), alanine, glutamine and glutamate increased in pro-STC1 groups suggesting their mobilization from the muscle to the liver for degradation and gluconeogenesis from alanine and glutamine. In addition, only STC1 treatment decreased the concentrations of succinate, fumarate and acetate, indicating slowing of the citric acid cycle. In the pro-PTHrP groups the concentrations of glucose, erythritol and lactate decreased, indicative of gluconeogenesis from lactate. Taurine, trimethylamine, trimethylamine N-oxide and carnitine changed in opposite directions in the pro-STC1 versus the pro-PTHrP groups, suggesting opposite effects, with STC1 stimulating lipogenesis and PTHrP activating lipolysis/β-oxidation of fatty acids. These findings suggest a role for STC1 and PTHrP related to strategic energy mechanisms that involve the production of glucose and safeguard of liver glycogen reserves for stressful situations. Portuguese Foundation for Science and Technology (FCT) SFRH/BD/103185/2014 info:eu-repo/semantics/publishedVersion

In the ocean the main climate drivers affecting marine organisms are warming, hypercapnia, and hypoxia. We investigated the acute effects of warming (W), warming plus hypercapnia (WHc, ~1800 μatm CO2), warming plus hypoxia (WHo, ~12.1 kPa O2), and a combined exposure of all three drivers (Deadly Trio, DT) on king scallops (Pecten maximus). All exposures started at 14 °C and temperature was increased by 2 °C once every 48 h until the lethal temperature was reached (28 °C). Gill samples were taken at 14 °C, 18 °C, 22 °C, and 26 °C and analyzed for their metabolic response by 1H-nuclear magnetic resonance (NMR) spectroscopy. Scallops were most tolerant to WHc and most susceptible to oxygen reduction (WHo and DT). In particular under DT, scallops' mitochondrial energy metabolism was affected. Changes became apparent at 22 °C and 26 °C involving significant accumulation of glycogenic amino acids (e.g. glycine and valine) and anaerobic end-products (e.g. acetic acid and succinate). In line with these observations the LT50 was lower under the exposure to DT (22.5 °C) than to W alone (~ 25 °C) indicating a narrowing of the thermal niche due to an imbalance between oxygen demand and supply.

The interaction between ocean warming, hypoxia and hypercapnia, suggested by climate projections, may push an organism earlier to the limits of its thermal tolerance window. In a previous study on juveniles of green abalone (Haliotis fulgens), combined exposure to hypoxia and hypercapnia during heat stress induced a lowered critical thermal maximum (CTmax), indicated by constrained oxygen consumption, muscular spams and loss of attachment. Thus, the present study investigated the cell physiology in foot muscle of H. fulgens juveniles exposed to acute warming (18 °C to 32 °C at +3 °C day-1) under hypoxia (50% air saturation) and hypercapnia (~1000 μatm PCO2), alone and in combination, to decipher the mechanisms leading to functional loss in this tissue. Under exposure to either hypoxia or hypercapnia, citrate synthase (CS) activity decreased with initial warming, in line with thermal compensation, but returned to control levels at 32 °C. The anaerobic enzymes lactate and tauropine dehydrogenase increased only under hypoxia at 32 °C. Under the combined treatment, CS overcame thermal compensation and remained stable overall, indicating active mitochondrial regulation under these conditions. Limited accumulation of anaerobic metabolites indicates unchanged mode of energy production. In all treatments, upregulation of Hsp70 mRNA was observed already at 30 °C. However, lack of evidence for Hsp70 protein accumulation provides only limited support to thermal denaturation of proteins. We conclude that under combined hypoxia and hypercapnia, metabolic depression allowed the H. fulgens musculature to retain an aerobic mode of metabolism in response to warming but may have contributed to functional loss.

Chemical exchange saturation transfer (CEST) from taurine to water (TauCEST) can be used for in vivo mapping of taurine concentrations as well as for measurements of relative changes in intracellular pH (pHi ) at temperatures below 37°C. Therefore, TauCEST offers the opportunity to investigate acid-base regulation and neurological disturbances of ectothermic animals living at low temperatures, and in particular to study the impact of ocean acidification (OA) on neurophysiological changes of fish. Here, we report the first in vivo application of TauCEST imaging. Thus, the study aimed to investigate the TauCEST effect in a broad range of temperatures (1-37°C) and pH (5.5-8.0), motivated by the high taurine concentration measured in the brains of polar fish. The in vitro data show that the TauCEST effect is especially detectable in the low temperature range and strictly monotonic for the relevant pH range (6.8-7.5). To investigate the specificity of TauCEST imaging for the brain of polar cod (Boreogadus saida) at 1.5°C simulations were carried out, indicating a taurine contribution of about 65% to the in vivo expected CEST effect, if experimental parameters are optimized. B. saida was acutely exposed to three different CO2 concentrations in the sea water (control normocapnia; comparatively moderate hypercapnia OAm = 3300 μatm; high hypercapnia OAh = 4900 μatm). TauCEST imaging of the brain showed a significant increase in the TauCEST effect under the different CO2 concentrations of about 1.5-3% in comparison with control measurements, indicative of changes in pHi or metabolite concentration. Consecutive recordings of 1 H MR spectra gave no support for a concentration induced change of the in vivo observed TauCEST effect. Thus, the in vivo application of TauCEST offers the possibility of mapping relative changes in pHi in the brain of polar cod during exposure to CO2 .

Research on molluscan specimens is increasingly being carried out using high-throughput molecular techniques. Due to their efficiency, these technologies have effectively resulted in a strong bias towards genotypic analyses. Therefore, the future large-scale correlation of such data with the phenotype will require a significant increase in the output of morphological studies. Three-dimensional (3D) scanning techniques such as magnetic resonance imaging (MRI) or computed tomography (CT) can achieve this goal as they permit rapidly obtaining digital data non-destructively or even entirely non-invasively from living, fixed, and fossil samples. With a large number of species and a relatively complex morphology, the Mollusca would profit from a more widespread application of digital 3D imaging techniques. In order to provide an overview of the capacity of various MRI and CT techniques to visualize internal and external structures of molluscs, more than twenty specimens ranging in size from a few millimeters to well over one meter were scanned in vivo as well as ex vivo. The results show that all major molluscan organ systems can be successfully visualized using both MRI and CT. The choice of a suitable imaging technique depends primarily on the specimen’s life condition, its size, the required resolution, and possible invasiveness of the approach. Apart from visual examples derived from more than two dozen scans, the present article provides guidelines and best practices for digital 3D imaging of a broad range of molluscan taxa. Furthermore, a comprehensive overview of studies that previously have employed MRI or CT techniques in malacological research is given.

SummaryBackground: In the summer of 2016 an international group of biomedical and health informatics faculty and graduate students gathered for the 16th meeting of the International Partnership in Health Informatics Education (IPHIE) masterclass at the University of Utah campus in Salt Lake City, Utah. This international biomedical and health informatics workshop was created to share knowledge and explore issues in biomedical health informatics (BHI).Objective: The goal of this paper is to summarize the discussions of biomedical and health informatics graduate students who were asked to define interoperability, and make critical observations to gather insight on how to improve biomedical education.Methods: Students were assigned to one of four groups and asked to define interoperability and explore potential solutions to current problems of interoperability in health care.Results: We summarize here the student reports on the importance and possible solutions to the “interoperability problem” in biomedical informatics. Reports are provided from each of the four groups of highly qualified graduate students from leading BHI programs in the US, Europe and Asia.Conclusion: International workshops such as IPHIE provide a unique opportunity for graduate student learning and knowledge sharing. BHI faculty are encouraged to incorporate into their curriculum opportunities to exercise and strengthen student critical thinking to prepare our students for solving health informatics problems in the future.Citation: Turner AM, Facell JC, Jaspers M, Wetter T, Pfeifer D, Gatewood LC, Adam T, Li Y-C, Lin M-C, Evans RS, Beukenhorst A, van Mens H, Tensen E, Bock C, Fendrich L, Seitz P, Suleder J, Aldekhyyel R, Bridgeman K, Hu Z, Sattler A, Guo S-Y, Mohaimenul IMd, Ningrum DNA, Tung H-R, Bian J, Plasek JM, Rommel C, Burke J, Sohi H. Solving interoperability in translational health: perspectives of students from the International Partnership in Health Informatics Education (IPHIE) 2016 master class. Appl Clin Inform 2017; 8: 651–659 https://doi.org/10.4338/ACI-2017-01-CR-0012

Magnetic Resonance Imaging (MRI) and Magnetic Resonance Spectroscopy (MRS) gain increasing attention and importance as a tool in marine ecology. So far, studies were largely limited to morphological studies, e.g. for the creation of digital libraries. Here, the utility of MRI and MRS for ecologists is tested and exemplified using formalin preserved samples of the Antarctic silverfish, Pleuragramma antarctica. As this species lacks a swim bladder, buoyancy is attained by the deposition of large amounts of lipids that are mainly stored in subcutaneous and intermuscular lipid sacs. In this study MRI and MRS are not only used to study internal morphology, but additionally to investigate functional morphology and to measure parameters of high ecological interest. The data are compared with literature data obtained by means of traditional ecological methods. The results from this study show that MR scans are not only an alternative to histological sections (as shown before), but even allow the visualization of particular features in delicate soft tissues, such as Pleuragramma's lipid sacs. 3D rendering techniques proved to be a useful tool to study organ volumes and lipid content, which usually requires laborious chemical lipid extraction and analysis. Moreover, the application of MRS even allows for an analysis of lipids and fatty acids within lipid sacs, which wouldn't be possible using destructive methods. MRI and MRS, in particular when used in combination, have the capacity to provide useful data on parameters of high ecological relevance and thus have proven to be a highly valuable addition, if not alternative, to the classical methods.

Single-cell investigations of the diatoms Coscinodsicus granii and Thalassiosira pseudonana were performed using laser desorption/ionization (LDI)-MS without the addition of chemical matrices. The unique cell wall architecture of these microalgae, more precisely the biomineralized nanostructured surface, supported the ionization of cellular as well as surface-related metabolites. In model experiments with purified diatom cell walls of eight species C. granii and T. pseudonana proved to promote the ionization of the polymer polyethylene glycol most efficiently. These species were therefore chosen for further experiments. Without any additional workup, living diatom cells can be washed, can be placed on the LDI target and can immediately be profiled using LDI-MS. Characteristic signals arising from the two species were assigned to common metabolites known from diatom metabolism. Among others, chlorophyll, phospholipids and amino acids were detected. Using these fingerprint signals, we were able to perform species-specific MS imaging down to a single-cell resolution of 20 by 20 µm. The larger C. granii cells can be directly visualized, while more than one of the smaller T. pseudonana cells is needed to generate high-quality images. The introduced technique will pave the way toward a chemotyping of phytoplankton that will enable the automated annotation of microalgal species. But also, an assignment of metabolic plasticity on a single-cell level that could answer fundamental questions about plankton diversity is now in reach. Copyright © 2013 John Wiley & Sons, Ltd.

The ongoing process of ocean acidification already affects marine life, and according to the concept of oxygen and capacity limitation of thermal tolerance, these effects may be intensified at the borders of the thermal tolerance window. We studied the effects of elevated CO2 concentrations on clapping performance and energy metabolism of the commercially important scallop Pecten maximus. Individuals were exposed for at least 30 days to 4 °C (winter) or to 10 °C (spring/summer) at either ambient (0.04 kPa, normocapnia) or predicted future PCO2 levels (0.11 kPa, hypercapnia). Cold-exposed (4 °C) groups revealed thermal stress exacerbated by PCO2 indicated by a high mortality overall and its increase from 55 % under normocapnia to 90 % under hypercapnia. We therefore excluded the 4 °C groups from further experimentation. Scallops at 10 °C showed impaired clapping performance following hypercapnic exposure. Force production was significantly reduced although the number of claps was unchanged between normocapnia- and hypercapnia-exposed scallops. The difference between maximal and resting metabolic rate (aerobic scope) of the hypercapnic scallops was significantly reduced compared with normocapnic animals, indicating a reduction in net aerobic scope. Our data confirm that ocean acidification narrows the thermal tolerance range of scallops resulting in elevated vulnerability to temperature extremes and impairs the animal’s performance capacity with potentially detrimental consequences for its fitness and survival in the ocean of tomorrow.

Magnetic resonance imaging (MRI) is a noninvasive imaging technique that today constitutes one of the main pillars of preclinical and clinical imaging. MRI’s capacity to depict soft tissue in whole specimens ex vivo as well as in vivo, achievable voxel resolutions well below (100 μm)3, and the absence of ionizing radiation have resulted in the broad application of this technique both in human diagnostics and studies involving small animal model organisms. Unfortunately, MRI systems are expensive devices and have so far only sporadically been used to resolve questions in zoology and in particular in zoomorphology. However, the results from two recent studies involving systematic scanning of representative species from a vertebrate group (fishes) as well as an invertebrate taxon (sea urchins) suggest that MRI could in fact be used more widely in zoology. Using novel image data derived from representative species of numerous higher metazoan clades in combination with a comprehensive literature survey, we review and evaluate the potential of MRI for systematic taxon scanning. According to our results, numerous animal groups are suitable for systematic MRI scanning, among them various cnidarian and arthropod taxa, brachiopods, various molluscan taxa, echinoderms, as well as all vertebrate clades. However, various phyla in their entirety cannot be considered suitable for this approach mainly due to their small size (e.g., Kinorhyncha) or their unfavorable shape (e.g., Nematomorpha), while other taxa are prone to produce artifacts associated either with their biology (e.g., Echiura) or their anatomy (e.g., Polyplacophora). In order to initiate further uses of MRI in zoology, we outline the principles underlying various applications of this technique such as the use of contrast agents, in vivo MRI, functional MRI, as well as magnetic resonance spectroscopy. Finally, we discuss how future technical developments might shape the use of MRI for the study of zoological specimens.

Acidification of ocean surface waters by anthropogenic carbon dioxide (CO(2)) emissions is a currently developing scenario that warrants a broadening of research foci in the study of acid-base physiology. Recent studies working with environmentally relevant CO(2) levels, indicate that some echinoderms and molluscs reduce metabolic rates, soft tissue growth and calcification during hypercapnic exposure. In contrast to all prior invertebrate species studied so far, growth trials with the cuttlefish Sepia officinalis found no indication of reduced growth or calcification performance during long-term exposure to 0.6 kPa CO(2). It is hypothesized that the differing sensitivities to elevated seawater pCO(2) could be explained by taxa specific differences in acid-base regulatory capacity. In this study, we examined the acid-base regulatory ability of S. officinalis in vivo, using a specially modified cannulation technique as well as (31)P NMR spectroscopy. During acute exposure to 0.6 kPa CO(2), S. officinalis rapidly increased its blood [HCO(3)(-)] to 10.4 mM through active ion-transport processes, and partially compensated the hypercapnia induced respiratory acidosis. A minor decrease in intracellular pH (pH(i)) and stable intracellular phosphagen levels indicated efficient pH(i) regulation. We conclude that S. officinalis is not only an efficient acid-base regulator, but is also able to do so without disturbing metabolic equilibria in characteristic tissues or compromising aerobic capacities. The cuttlefish did not exhibit acute intolerance to hypercapnia that has been hypothesized for more active cephalopod species (squid). Even though blood pH (pHe) remained 0.18 pH units below control values, arterial O(2) saturation was not compromised in S. officinalis because of the comparatively lower pH sensitivity of oxygen binding to its blood pigment. This raises questions concerning the potentially broad range of sensitivity to changes in acid-base status amongst invertebrates, as well as to the underlying mechanistic origins. Further studies are needed to better characterize the connection between acid-base status and animal fitness in various marine species.

High-resolution proton magnetic resonance spectroscopy was performed on tissue specimens from 33 patients with astrocytic tumors (22 astrocytomas, 11 glioblastomas) and 13 patients with meningiomas. For all patients, samples of primary tumors and their first recurrences were examined. Increased anaplasia, with respect to malignant transformation, resulting in a higher malignancy grade, was present in 11 recurrences of 22 astrocytoma patients. Spectroscopic features of tumor types, as determined on samples of the primary occurrences, were in good agreement with previous studies. Compared with the respective primary astrocytomas, characteristic features of glioblastomas were significantly increased concentrations of alanine (Ala) (p = 0.005), increased metabolite ratios of glycine (Gly)/total creatine (tCr) (p = 0.0001) and glutamate (Glu)/glutamine (Gln) (p = 0.004). Meningiomas showed increased Ala (p = 0.02) and metabolite ratios [Gly, total choline (tCho), Ala] over tCr (p = 0.001) relative to astrocytomas, and N-acetylaspartate and myo-inositol were absent. Metabolic changes of an evolving tumor were observed in recurrent astrocytomas: owing to their consecutive assessments, more indicators of malignant degeneration were detected in astrocytoma recurrences (e.g. Gly, p = 0.029; tCho, p = 0.034; Glu, p = 0.015; tCho/tCr, p = 0.001) in contrast to the comparison of primary astrocytomas with primary glioblastomas. The present investigation demonstrated a correlation of the tCho-signal with tumor progression. Significantly elevated concentrations of Ala (p = 0.037) and Glu (p = 0.003) and metabolite ratio tCho/tCr (p = 0.005) were even found in recurrent low-grade astrocytomas with unchanged histopathological grading (n = 11). This may be related to an early stage of malignant transformation, not yet detectable morphologically, and emphasizes the high sensitivity of 1H NMR spectroscopy in elucidating characteristics of brain tumor metabolism.

In vivo (31)P-nuclear magnetic resonance (NMR) spectroscopy was used to measure the levels of ATP, phospho-l-arginine (PLA), and inorganic phosphate in the adductor muscle of the Antarctic scallop Adamussium colbecki and two temperate species, Aequipecten opercularis and Pecten maximus. Graded exercise regimes from light (one to two contractions) to exhausting (failing to respond to further stimulation) were imposed on animals of each species at its habitat temperature (0 degrees vs. 12 degrees C, respectively). NMR spectroscopy allowed noninvasive measurement of metabolite levels and intracellular pH at high time resolution (30-120-s intervals) during exercise and throughout the recovery period. Significant differences were shown between the magnitude and form of the metabolic response with increasing levels of exercise in each species. After exhaustion, short-term (first 15 min) muscle alkalosis was followed by acidosis of up to 0.2 pH units during the recovery process. Aequipecten opercularis had similar resting muscle PLA levels compared with either P. maximus or A. colbecki but used a fivefold greater proportion of this store per contraction and was able to perform only half as many claps (maximum of 24) as the other species before exhaustion. All species regenerated their PLA store at a similar rate despite different environmental temperatures. These findings argue for some cold compensation of muscular performance and recovery capacities in the Antarctic scallop, albeit at levels of performance similar to scallops with low activity lifestyles from temperate latitudes.

Microstructure and thermal evolution of sea-ice brine inclusions were investigated with magnetic resonance imaging (MRI) techniques. Ice samples were kept at temperatures between −2°C and −25°C during 1H imaging in a 4.7-T magnet at 200 MHz. Measurements were completed in a 20-cm diameter cylindrical probe and actively shielded gradient coils (max. 50 mT m−1, pixel dimensions >0.2 mm, slice thicknesses >1 mm), and for higher resolution in a mini-imaging unit with a 9-cm diameter probe with gradient coils of 200 mT m−1 (pixel dimensions

T2*-weighted imaging at 4.7 T was used to identify the cortical areas activated by electrical stimulation of the forepaw and hindpaw of alpha-chloralose anesthetized rats. Variation of the coronal slice position relative to the bregma, showed that the forepaw representation in the somatosensory cortex is more frontal and lateral than that of the hindpaw. Overlap between both activation areas was observed only in a small region in the slice at the level of the bregma. Documented localizations of both representations are in good agreement with earlier observations using invasive techniques. The determination of the separate areas of both paws indicates the feasibility of more complex activation studies in anesthetized animals, such as combined stimulations for the investigation of potentiation or depression effects on individual stimuli.

A fast version of the arterial spin tagging technique for the detection of cerebral perfusion is presented. Based on adiabatic spin inversion in combination with snapshot FLASH imaging our technique allows the recording of perfusion changes with a temporal resolution of about 3 s. Differences of cerebral perfusion dependent on the choice of anesthesia were observed in rat brain. Furthermore, with this arterial spin tagging method we demonstrated perfusion increases in the somatosensory cortex of anaesthetized rats during forepaw stimulation. Comparison of the activated areas in the T 2 * -weighted BOLD images and the perfusion-weighted images showed good spatial correspondence, but the sensitivity to the functional activation was more than ten times higher in the perfusion technique.

Mechanisms responsive to hypercapnia (elevated CO(2) concentrations) and shaping branchial energy turnover were investigated in isolated perfused gills of two Antarctic Notothenioids (Gobionotothen gibberifrons, Notothenia coriiceps). Branchial oxygen consumption was measured under normo- versus hypercapnic conditions (10,000 ppm CO(2)) at high extracellular pH values. The fractional costs of ion regulation, protein and RNA synthesis in the energy budgets were determined using specific inhibitors. Overall gill energy turnover was maintained under pH compensated hypercapnia in both Antarctic species as well as in a temperate zoarcid (Zoarces viviparus). However, fractional energy consumption by the examined processes rose drastically in G. gibberifrons (100-180%), and to a lesser extent in N. coriiceps gills (7-56%). In conclusion, high CO(2) concentrations under conditions of compensated acidosis induce cost increments in epithelial processes, however, at maintained overall rates of branchial energy turnover.

We examined the energy status, nitrogen metabolism and hepatic glutamate dehydrogenase activity in the African lungfish Protopterus annectens during aestivation in normoxia (air) or hypoxia (2% O(2) in N(2)), with tissues sampled on day 3 (aerial exposure with preparation for aestivation), day 6 (entering into aestivation) or day 12 (undergoing aestivation). There was no accumulation of ammonia in tissues of fish exposed to normoxia or hypoxia throughout the 12-day period. Ammonia toxicity was avoided by increased urea synthesis and/or decreased endogenous N production (as ammonia), but the dependency on these two mechanisms differed between the normoxic and the hypoxic fish. The rate of urea synthesis increased 2.4-fold, with only a 12% decrease in the rate of N production in the normoxic fish. By contrast, the rate of N production in the hypoxic fish decreased by 58%, with no increase in the rate of urea synthesis. Using in vivo (31)P NMR spectroscopy, it was demonstrated that hypoxia led to significantly lower ATP concentration on day 12 and significantly lower creatine phosphate concentration on days 1, 6, 9 and 12 in the anterior region of the fish as compared with normoxia. Additionally, the hypoxic fish had lower creatine phosphate concentration in the middle region than the normoxic fish on day 9. Hence, lowering the dependency on increased urea synthesis to detoxify ammonia, which is energy intensive by reducing N production, would conserve cellular energy during aestivation in hypoxia. Indeed, there were significant increases in glutamate concentrations in tissues of fish aestivating in hypoxia, which indicates decreases in its degradation and/or transamination. Furthermore, there were significant increases in the hepatic glutamate dehydrogenase (GDH) amination activity, the amination/deamination ratio and the dependency of the amination activity on ADP activation in fish on days 6 and 12 in hypoxia, but similar changes occurred only in the normoxic fish on day 12. Therefore, our results indicate for the first time that P. annectens exhibited different adaptive responses during aestivation in normoxia and in hypoxia. They also indicate that reduction in nitrogen metabolism, and probably metabolic rate, did not occur simply in association with aestivation (in normoxia) but responded more effectively to a combined effect of aestivation and hypoxia.

Earlier work found cuttlefish (Sepia officinalis) ventilatory muscle tissue to progressively switch to an anaerobic mode of energy production at critical temperatures (T (c)) of 7.0 and 26.8 degrees C. These findings suggested that oxygen availability limits thermal tolerance. The present study was designed to elucidate whether it is the ventilatory apparatus that sets critical temperature thresholds during acute thermal stress. Routine metabolic rate (rmr) rose exponentially between 11 and 23 degrees C, while below (8 degrees C) and above (26 degrees C) this temperature range, rmr was significantly depressed. Ventilation frequency (f (V)) and mean mantle cavity pressure (MMP) followed an exponential relationship within the entire investigated temperature range (8-26 degrees C). Oxygen extraction from the ventilatory current (EO(2)) decreased in a sigmoidal fashion with temperature, falling from90% at 8 degrees C to 32% at 26 degrees C. Consequently, ventilatory minute volume (MV(V)) increased by a factor of 20 from 7 to 150% body weight min(-1) in the same temperature interval. Increases in MMP and MV(V) resulted in ventilatory muscle power output (P (out)) increasing by a factor of80 from 0.03 to 2.4 mW kg(-1) animal. Nonetheless, costs for ventilatory mechanics remain below 1.5% rmr in the natural thermal window of the population (English Channel, 9-17 degrees C), owing to very low MMPs of0.05 kPa driving the ventilatory stream, and may maximally rise to 8.6% rmr at 26 degrees C. Model calculations suggest that the ventilatory system can maintain high arterial PO(2) values of14 kPa over the entire temperature interval. We therefore conclude that the cuttlefish ventilation system is probably not limiting oxygen transfer during acute thermal stress. Depression of rmr, well before critical temperatures are being reached, is likely caused by circulatory capacity limitations and not by fatigue of ventilatory muscle fibres.

Venous blood flow was measured for the first time in a cephalopod. Blood velocity was determined in the anterior vena cava (AVC) of cuttlefish S. officinalis with a Doppler, while simultaneously, ventilatory pressure oscillations were recorded in the mantle cavity. In addition, magnetic resonance imaging (MRI) was employed to investigate pulsatile flow in other major vessels. Blood pulses in the AVC are obligatorily coupled to ventilatory pressure pulses, both in frequency and phase. AVC peak blood velocity (v(AVC)) in animals of 232 (+/- 30 SD) g wet mass at 15 degrees C was found to be 14.2 (+/- 7.1) cm s(-1), AVC stroke volume (SV(AVC)) was 0.2 (+/- 0.1) ml stroke(-1), AVC minute volume (MV(AVC)) amounted to 5.5 (+/- 2.8) ml min(-1). Intense exercise bouts of 1-2 min resulted in 2.2-fold increases in MV(AVC), enabled by 1.6-fold increments in both, AVC pulse frequency (f (AVC)) and v(AVC). As increases in blood flow occurred delayed in time by 1.7 min with regard to exercise periods, we concluded that it is not direct mantle cavity pressure conveyance that drives venous return in this cephalopod blood vessel. However, during jetting at high pressure amplitude (1 kPa), AVC blood flow and mantle cavity pressure pulse shapes completely overlap, suggesting that under these conditions, blood transport must be driven passively by mantle cavity pressure. MRI measurements at 15 degrees C also revealed that under resting conditions, f (AVC )and ventilation frequency (f (V)) match at 31.6 (+/- 2.1) strokes min(-1). In addition, rates of pulsations in the cephalic artery and in afferent branchial vessels did not significantly differ from f (AVC) and f (V). It is suggested that these adaptations are beneficial for high rates of oxygen extraction observed in S. officinalis and the energy conserving mode of life of the cuttlefish ecotype in general.

The hypothesis of an oxygen-limited thermal tolerance due to restrictions in cardiovascular performance at extreme temperatures was tested in Atlantic cod, Gadus morhua (North Sea). Heart rate, changes in arterial and venous blood flow, and venous oxygen tensions were determined during an acute temperature change to define pejus (“getting worse”) temperatures that border the thermal optimum range. An exponential increase in heart rate occurred between 2 and 16°C (Q10= 2.38 ± 0.35). Thermal sensitivity was reduced beyond 16°C when cardiac arrhythmia became visible. Flow-weighted magnetic resonance imaging (MRI) measurements of temperature-dependent blood flow revealed no exponential but a hyperbolic increase of blood flow with a moderate linear increase at temperatures >4°C. Therefore, temperature-dependent heart rate increments are not mirrored by similar increments in blood flow. Venous Po2(PvO2), which reflects the quality of oxygen supply to the heart of cod (no coronary circulation present), followed an inverse U-shaped curve with highest PvO2levels at 5.0 ± 0.2°C. Thermal limitation of circulatory performance in cod set in below 2°C and beyond 7°C, respectively, characterized by decreased PvO2. Further warming led to a sharp drop in PvO2beyond 16.1 ± 1.2°C in accordance with the onset of cardiac arrhythmia and, likely, the critical temperature. In conclusion, progressive cooling or warming brings cod from a temperature range of optimum cardiac performance into a pejus range, when aerobic scope falls before critical temperatures are reached. These patterns might cause a shift in the geographical distribution of cod with global warming.

Adaptational physiology studies how animals cope with their environment, even if this environment is subject to permanent fluctuations such as tidal or seasonal variations. Aquatic organisms are generally more prone to be exposed to osmotic, hypoxic and temperature challenges than terrestrial animals. Some of these challenges are more restraining in an aquatic environment. To date, very few studies have used in vivo magnetic resonance imaging (MRI) to uncover the physiological mechanisms that respond to or compensate for these challenges. This paper provides an overview of what has been accomplished thus far by using MRI to study the environmental physiology of fish. It introduces the reader to the use of small teleost fish such as carp (12 cm, 60 g) and eelpout (25 cm, 50 g) as models for such research and to provide new perceptions into the applicability of MRI tools based on new insights into the nature of MRI contrast. Representative MRI studies have made contributions to the identification of the lack of cell volume repair in stenohaline fish during osmotic stress. They have studied the underlying physiological mechanisms of brain anoxia tolerance in fish and have qualified the role of the cardio-circulatory system in setting thermal tolerance windows of fish.

In vivo Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS) experiments were carried out in non-anaesthetized marine fish with a maximum body length of 40 cm using a 4.7 T horizontal magnet. In flow-through animal chambers long-term investigations could be carried out for several days in a sessile benthic zoarcid species, the eelpout Zoarces viviparus, as well as in a pelagic gadid species, the cod Gadus morhua. Sea water adapted probes and optimised excitation and refocusing pulse shapes reduced the negative effects of the highly conductive medium. In both species, in vivo 31P-NMR spectra were collected within 5 minutes with a reasonable signal to noise ratio (PCr/Pi ratio > 40 in cod) and typical line widths in the range of 20 to max. 40 Hz. Magnetic resonance images were recorded in the sessile species without movement artefacts. In these fish it was even possible to measure the blood flow of different vessels with a flow compensated gradient echo sequence and to carry out localized in vivo 1H-NMR spectroscopy in different organs.

In vivo magnetic resonance imaging (MRI) and angiography were applied to the marine spider crab Maja squinado for a study of temperature effects and thermal tolerance. Ventilation and haemolymph circulation were investigated during progressive cooling from 12 degrees C to 2 degrees C. The anatomical resolution of MR images from Maja squinado obtained with a standard spin echo sequence were suitable to resolve the structures of various internal organs. The heart of the animal could be depicted without movement artifacts. The use of a flow compensated gradient echo sequence allowed simultaneous observations of ventilation, reflected by water flow through the gill chambers as well as of haemolymph flow. Simultaneous investigation of various arteries was possible by use of flow weighted MRI. In addition to those accessible by standard invasive flow sensitive doppler sensors, flow changes in gill, leg arteries and the venous return could be observed. Both ventilation and haemolymph flow decreased during progressive cooling and changes in haemolymph flow varied between arteries. Haemolymph flow through the Arteria sternalis, some gill and leg arteries was maintained at low temperatures indicating a reduced thermal sensitivity of flow in selected vessels. In support of previous invasive studies of haemolymph flow as well as heart and ventilation rates, the results demonstrate that the operation of gills and the maintenance of locomotor activity are critical for cold tolerance. A shift in haemolymph flow between arteries likely occurs to ensure the functioning of locomotion and ventilation in the cold.

Temperature-dependent adjustments of intracellular pH are thought to play a major role in the maintenance of protein function. Comparative studies were carried out in two species from the same fish family (Zoarcidae), the stenothermal Antarctic eelpout (Pachycara brachycephalum) and the eurythermal eelpout (Zoarces viviparus), to find out whether pH regulation is modified by temperature in the closely related species and to what extent the respective pattern differs between eurytherms and stenotherms. Previous invasive studies had compared individual animals sampled at various temperatures and suggested that a decrease in intracellular pH (pHi) values occurs at rising temperatures, as predicted by the alpha-stat hypothesis of acid-base regulation. The present study used non-invasive in vivo 31P-NMR spectroscopy in non-anaesthetized, unrestrained fish for long-term online recordings in individual specimens. Control spectra obtained at T= 0°C for P. brachycephalum and at 12°C for Z. viviparus indicated low stress conditions, as well as a high stability of energy and acid-base status over time periods longer than 1 week. Temperature changes had no influence on the concentration of high-energy phosphates like phos- phocreatine or ATP. Temperature-induced pH changes were monitored continuously in a range between 0 and 6°C for polar, and 12 and 18°C for temperate eelpout. A pHi change of around -0.015 pH units/°C was observed within both species, in accordance with the alpha-stat hypothesis; however, extrapolation to the same temperature revealed different set points of pH regulation in the two species. These findings confirm that an alpha-stat pattern of pH regulation can be found in stenothermal Antarctic animals, at set points deviating from an alpha-stat pattern, however, in a between-species comparison.

Somatosensory evoked potentials (SEP) and T2*-weighted nuclear magnetic resonance (NMR) images were recorded simultaneously during somatosensory stimulation of rat to investigate the relationship between electrical activation of the brain tissue and the signal intensity change in functional NMR imaging. Electrical forepaw stimulation was performed in Wistar rats anesthetized with alpha-chloralose. SEPs were recorded with calomel electrodes at stimulation frequencies of 1.5, 3, 4.5, and 6 Hz. At the same time, T2*-weighted imaging was performed, and the signal intensity increase during stimulation was correlated with the mean amplitude of the SEP. Both the stimulation-evoked signal intensity increase in T2*-weighted images and the amplitude of SEPs were dependent on the stimulation frequency, with the largest signals at a stimulation frequency of 1.5 Hz and decreasing activations with increasing frequencies. The feasibility of simultaneous, artifact-free recordings of T2*-weighted NMR images and of evoked potentials is proved. Furthermore, the study demonstrates-in the intact brain-the validity of functional magnetic resonance imaging for estimating the intensity of electrocortical activation.

Functional activation of somatosensory cortex was studied in alpha-chloralose anesthetized rats by functional magnetic resonance imaging (fMRI), using both perfusion-weighted and T2*-weighted (blood oxygenation level dependent, BOLD) imaging. The sensitivity of functional activation was altered by ventilating animals for 3 minutes with 6% CO2. Before hypercapnic conditioning, electrical stimulation of the left forepaw at a frequency of 3 Hz led to an increase of signal intensity (relative to the unstimulated baseline condition) in the right somatosensory cortex by 6+/-2% (means+/-SD) in T2*-weighted images and by 45%+/-48% in perfusion-weighted images. After hypercapnic conditioning the signal intensity increase in perfusion-weighted images doubled to 91%+/-62% (P=0.034), whereas that of T2*-weighted images only marginally increased to 7+/-4% (not significant). This different behavior in both imaging modalities is interpreted as evidence for an increased flow response in combination with a higher oxygen extraction. Thus, the fMRI data reflect hypercapnia-induced resetting of the functional-metabolic coupling of the tissue during activation.

The dependence of functional MRI contrast on the repetition rate (1.5-9 Hz) of a sensory stimulus was investigated with a T2*-weighted gradient echo method during forepaw stimulation of alpha-chloralose anesthetized rats (n = 5). An activation area was observed in the left or right somatosensory cortex dependent on the stimulation of the right or left forepaw, respectively. The activation intensity decreased for stimulation frequencies above 3 Hz, and was negligible at 9 Hz. The interpretation is that at low stimulation rates the neurons can respond to each stimulus, but at higher rates there is insufficient recovery time so that the response is progressively occluded.

Extracellular acidosis has been demonstrated to play a key role in the process of metabolic depression under long-term environmental stress, exemplified in the marine invertebrate Sipunculus nudus. These findings led to the hypothesis that acid–base regulation is associated with a visible cost depending on the rate and mode of H+-equivalent ion exchange. To test this hypothesis, the effects of different ion-transport inhibitors on the rate of pH recovery during hypercapnia, on energy turnover and on steady-state acid–base variables were studied in isolated body wall musculature of the marine worm Sipunculus nudus under control conditions (pHe 7.90) and during steady-state extracellular acidosis (pHe 7.50 or 7.20) by in vivo31P-NMR and oxygen consumption analyses. During acute hypercapnia (2 % CO2), recovery of pHi was delayed at pHe 7.5 compared with pHe 7.9. Inhibition of the Na+/H+-exchanger by 5-(N,N-dimethyl)-amiloride (DMA) at pHe 7.5 delayed recovery even further. This effect was much smaller at pHe 7.9. Inhibition of anion exchange by the addition of the transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) prevented pH recovery at pHe 7.5 and delayed recovery at pHe 7.9, in accordance with an effect on Na+-dependent Cl−/HCO3− exchange. The effects of ouabain, DIDS and DMA on metabolic rate were reduced at low pHe, thereby supporting the conclusion that acidosis caused the ATP demand of Na+/K+-ATPase to fall. This reduction occurred via an inhibiting effect on both Na+/H+- and Na+-dependent Cl−/HCO3− (i.e. Na+/H+/Cl−/HCO3−) exchange in accordance with a reduction in the ATP demand for acid–base regulation during metabolic depression. Considering the ATP stoichiometries of the two exchangers, metabolic depression may be supported by the predominant use of Na+/H+/Cl−/HCO3− exchange under conditions of extracellular acidosis.