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PURPOSE: Hyperpolarized 13C MRI is a powerful technique to study dynamic metabolic processes in vivo; but it has predominantly been used in mammals, mostly humans, pigs, and rodents. METHODS: In the present study, we use this technique to characterize the metabolic fate of hyperpolarized [1- 13C]pyruvate in Burmese pythons (Python bivittatus), a large species of constricting snake that exhibits a four- to tenfold rise in metabolism and large growth of the visceral organs within 24-48 h of ingestion of their large meals. RESULTS: We demonstrate a fivefold elevation of the whole-body lactate-to-pyruvate ratio in digesting snakes, pointing to a large rise in lactate production from pyruvate. Consistent with the well-known metabolic stimulation of digestion, measurements of mitochondrial respiration in hepatocytes in vitro indicate a marked postprandial upregulation of mitochondrial respiration. We observed that a low SNR of the hyperpolarized 13C produced metabolites in the python, and this lack of signal was possibly due to the low metabolism of reptiles compared with mammals, preventing quantification of alanine and bicarbonate production with the experimental setup used in this study. Spatial quantification of the [1- 13C]lactate was only possible in postprandial snakes (with high metabolism), where a statistically significant difference between the heart and liver was observed. CONCLUSION: We confirm the large postprandial rise in the wet mass of most visceral organs, except for the heart, and demonstrated that it is possible to image the [1- 13C]pyruvate uptake and intracellular conversion to [1- 13C]lactate in ectothermic animals.

BackgroundDNA methylation of cytosines at CpG dinucleotides is a widespread epigenetic mark; but genome-wide variation has been relatively unexplored due to the limited representation of variable CpGs on commercial high-throughput arrays.ObjectiveTo explore this hidden portion of the epigenome, we combined whole-genome bisulfite sequencing (WGBS) with in silico evidence of gene regulatory regions to design a custom array of high-value CpGs. We focused these studies in airway epithelial cells from children with and without allergic asthma because these cells mediate the effects of inhaled microbes, pollution, and allergens on asthma and allergic disease risk.MethodsWe identified differentially methylated regions (DMRs) from WGBS in nasal epithelial cell (NEC) DNA from 39 children with and without allergic asthma of both European and African ancestries. We selected CpGs from DMRs, previous allergy or asthma Epigenome-Wide Association Studies (EWAS), or Genome-Wide Association Study (GWAS) loci, and overlapped them with functional annotations for inclusion on a custom Asthma&Allergy array. Using both the Custom and EPIC arrays, we performed EWAS of allergic sensitization (AS) in NEC DNA from children in the URECA birth cohort and using the Custom array in the INSPIRE birth cohort. We assigned each CpG on the arrays to its nearest gene and its promotor capture Hi-C interacting gene and performed expression quantitative trait methylation (eQTM) studies for both sets of genes.ResultsCustom array CpGs were enriched for intermediate methylation (IM) levels compared to EPIC CpGs. IM CpGs were further enriched among those associated with AS and for eQTMs on both arrays.ConclusionsOur study revealed signature features of high-value CpGs and evidence for epigenetic regulation of genes at AS EWAS loci that are robust to race/ethnicity, ascertainment, age, and geography.Clinical ImplicationsThese studies identified allergic sensitization-associated differentially methylated CpGs and their target genes in airway epithelium, providing potential epigenetic mechanisms in the development of allergic diseases and suggesting novel drug targets.Capsule SummaryThis study of previously unexplored regions of the airway epithelial methylome revealed novel epigenetic mechanisms regulating genes previously implicated in the pathogenesis of asthma and allergic diseases.

HyperpolarizedIn the present study, we use this technique to characterize the metabolic fate of hyperpolarized [1-We demonstrate a fivefold elevation of the whole-body lactate-to-pyruvate ratio in digesting snakes, pointing to a large rise in lactate production from pyruvate. Consistent with the well-known metabolic stimulation of digestion, measurements of mitochondrial respiration in hepatocytes in vitro indicate a marked postprandial upregulation of mitochondrial respiration. We observed that a low SNR of the hyperpolarizedWe confirm the large postprandial rise in the wet mass of most visceral organs, except for the heart, and demonstrated that it is possible to image the [1

Aim: Diabetes Mellitus is a global public health challenge with major and potentially devastating complications, and concomitant complications include retinopathy and neuropathy due to hypoxia and microvascular dysfunction. In this study, we investigated the effect of hyperbaric oxygen therapy as a method to transiently improve tissue oxygenation on diabetic retinopathy and neuropathy in a streptozotocin induced type-1 diabetic rat model (Wistar). Methods: Streptozotocin induced type-1 diabetic rats received 10 sessions of 2-h hyperbaric oxygen exposures (pO2 = 309 kPa) over 2 weeks. Animals were exposed to light stimuli to produce light evoked potentials to estimate the effect of oxygen treatment on diabetic retinopathy. Sciatic nerves were exposed and stimulated to produce muscle evoked potential, which were recorded in the muscles of the foot and subsequently used to evaluate the effect of oxygen treatment on diabetic neuropathy. Results: We found significantly shorter light evoked potential latency and increased amplitude in hyperbaric oxygen treated animals. No change was found in nerve conduction. Conclusions: This study showed that hyperbaric oxygen therapy is a potentially effective treatment for diabetic retinopathy, improving both latency and amplitude of light evoked potentials.

Genetic variants have been associated with myriad molecular phenotypes that provide new insight into the range of mechanisms underlying genetic traits and diseases. Identifying any particular genetic variant's cascade of effects, from molecule to individual, requires assaying multiple layers of molecular complexity. We introduce the Enhancing GTEx (eGTEx) project that extends the GTEx project to combine gene expression with additional intermediate molecular measurements on the same tissues to provide a resource for studying how genetic differences cascade through molecular phenotypes to impact human health.

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of disease.

The ejection fraction of the trabeculated cardiac ventricle of reptiles has not previously been measured. Here, we used the gold standard clinical methodology - electrocardiogram-gated flow magnetic resonance imaging (MRI) - to validate stroke volume measurements and end diastolic ventricular blood volume. This produced an estimate of ejection fraction in our study species, the red footed tortoise

Introduction: While pressurisation and decompression may provoke detrimental physiologic effects in humans and animals, many underlyingphysiological phenomena remain unknown. Most studies on e.g. decompression sickness are conducted following pressurisation and decompression, in part because it is intrinsically difficult to study humans or animals inside a pressure chamber. We have developed a preclinical pressure chamber system compatible with CT, PET and MR-imaging during pressurisation up to 1.013 mPa, which allows for anatomical visualisations and measurements of certain physiological processes in vivo during pressurisation.Material and methods: Anaesthetised rats (simulated diving and control groups) underwent the following imaging protocols: First, a 3T clinical MRI-system was employed to evaluate in vivo cerebral relaxation parameters (T1, T2 and T2*). MRI was performed before, during 709 kPa pressurisation, and repeatedly after 500 kPa/min decompression. After MRI, venous bubble development was monitored using ultrasound. Second, preclinical μCT, PET/MRI, and high-field 9.4 T MR-Imaging systems evaluated changes in cerebral standard uptake value (SUV) of F-FDG, changes in cerebral blood flow (deltaCBF), T and T * relaxation, and blood brain barrier (BBB) breakage 90 min following decompression.Results: Simulated diving induced significant changes in all measured parameters during 3 T MRI. SUV of F-FDG was unchanged in both groups,although following μCT-examination revealed intracranial bubbles. deltaCBF was significantly lower in dived vs. control rats, while T1-relaxation - but not T *-relaxation - was significantly elevated in both groups. No BBB-breakage was observed.Conclusion: Our preliminary results obtained using CT-, PET- and MR-imaging of rats indicate that these non-invasive imaging techniques are sensitive to subtle cerebral changes during and following simulated diving. We believe that the described system is useful in many areas of experimental baromedical research.

Rare genetic variants are abundant in humans and are expected to contribute to individual disease risk1,2,3,4. While genetic association studies have successfully identified common genetic variants associated with susceptibility, these studies are not practical for identifying rare variants1,5. Efforts to distinguish pathogenic variants from benign rare variants have leveraged the genetic code to identify deleterious protein-coding alleles1,6,7, but no analogous code exists for non-coding variants. Therefore, ascertaining which rare variants have phenotypic effects remains a major challenge. Rare non-coding variants have been associated with extreme gene expression in studies using single tissues8,9,10,11, but their effects across tissues are unknown. Here we identify gene expression outliers, or individuals showing extreme expression levels for a particular gene, across 44 human tissues by using combined analyses of whole genomes and multi-tissue RNA-sequencing data from the Genotype-Tissue Expression (GTEx) project v6p release12. We find that 58% of underexpression and 28% of overexpression outliers have nearby conserved rare variants compared to 8% of non-outliers. Additionally, we developed RIVER (RNA-informed variant effect on regulation), a Bayesian statistical model that incorporates expression data to predict a regulatory effect for rare variants with higher accuracy than models using genomic annotations alone. Overall, we demonstrate that rare variants contribute to large gene expression changes across tissues and provide an integrative method for interpretation of rare variants in individual genomes.

We describe the development of a novel preclinical rodent-sized pressure chamber system compatible with computed tomography (CT), positron emission tomography (PET) and magnetic resonance imaging (MRI) that allows continuous uncompromised and minimally invasive data acquisition throughout hyperbaric exposures. The effect of various pressures on the acquired image intensity obtained with different CT, PET and MRI phantoms are characterised.Tissue-representative phantom models were examined with CT, PET or MRI at normobaric pressure and hyperbaric pressures up to 1.013 mPa. The relationships between the acquired image signals and pressure were evaluated by linear regression analysis for each phantom.CT and PET showed no effect of pressure per se, except for CT of air, demonstrating an increase in Hounsfield units in proportion to the pressure. For MRI, pressurisation induced no effect on the longitudinal relaxation rate (R₁), whereas the transversal relaxation rate (R₂) changed slightly. The R₂ data further revealed an association between pressure and the concentration of the paramagnetic nuclei gadolinium, the contrast agent used to mimic different tissues in the MRI phantoms.This study demonstrates a pressure chamber system compatible with CT, PET and MRI. We found that no correction in image intensity was required with pressurisation up to 1.013 mPa for any imaging modality. CT, PET or MRI can be used to obtain anatomical and physiological information from pressurised model animals in this chamber.

Many freshwater turtles are extremely tolerant to the lack of oxygen and can survive the winter submerged in anoxic mud in ice-covered lakes. The pronounced anoxia-tolerance resides with a considerable depression of cellular metabolism and the ability to use the shell to buffer the acidosis arising from anaerobic metabolism (1). Infusion of microspheres has shown that the shell receives almost half of the cardiac output in turtles made anoxic at low temperatures (2). However, the vasculature of the turtle shell remains to be described. To visualise the vasculature within the carapace and plastron of the turtle Trachemys scripta, we perfused terminally anaesthetised turtles with different contrast enhancing agents (Microfil [lead n/a]), barium sulphate [250 mg/kg], and iodine [15-250 mg/kg]), and the animals were then scanned by both single source as well as dual energy Computed Tomographic systems, to create three dimensional representations. Dual energy Computed Tomography provided good visual contrast between blood vessels and bony tissue inside the turtle shell due to differences in energy level from the two simultaneously acquired x-ray sources. However, inadequate resolution of the clinical scanners prevented visualisation of the smaller vessels with a diameter below approximately 600 μm. Thus, our technique clearly revealed the larger intracortical vessel embedded in the carapace and plastron of Trachemys, and future studies should seek to determine whether this vascularisation is altered during prolonged anoxia. 1. Jackson, DC. (2011). Life in a shell: a physiologist´s view of a turtle. Harvard University Press, USA. 2. Stecyk, JAW., Overgaard, J., Farrell, AP., Wang, T. (2004). α-Adrenergic regulation of systemic peripheral resistance and blood flow distribution in the turtle Trachemys scripta during anoxia submergence at 5˚C and 21˚C. J. Exp. Biol. 207, 269-283.

Several traditional handbooks and web-based databases exist with descriptions of animal anatomy, providing dissection photographies or hand drawn images in explanatory figures. In recent years sophisticated databases have been developed providing unique 2D and 3D visualisations of the internal and external structure of living and extinct vertebrates. Biological visualisation libraries such as www.digimorph.org hold terabytes of images of various species, with its core focus on high-resolution computed tomographic (CT) cranial imaging. Although impressive, CT imaging is mostly suited to visualise bone anatomy, and there is a considerable lack of non-interpreted soft-tissue visualisation in figures of anatomical literature and databases. This study aims to present the endless possibilities of high resolution visualisation of both soft and hard structures in animals using magnetic resonance imaging (MRI) and CT.Various species (tarantula, horseshoe crab, carp, haddock, lungfish, axolotl) were subjected to multi-slice MRI and CT protocols to produce 2D images of body slices, followed by volume rendering producing 3D digital models of animal anatomy with applications for visualising specific organs of interest. A number of animals (american alligator, ball python, swamp eel, red eared slider, monitor lizard, bearded dragon) were additionally perfused with MRI and CT contrast agents enabling a 3D visualisation of vascular arrangements.We were in hours able to produce high resolution 3D digital models of animal soft and hard tissue anatomy in quality similar or superior to time consuming dissection, and we propose MRI and CT as valuable tools in future studies of animal anatomy in research and education.

Natural birch sap contains around 1% of sugars, mainly glucose and fructose and a relatively high concentration of amino acids. The sap has been used in Russia and the Baltics for centuries as a food additive and in resent years has experienced a renaissance as an additive in high quality beers and as sweetener in ice cream (Skarø Is). It has been claimed to have a flavour enhancing effect when combined with fresh fruit thereby reducing the need for sugar additionto sorbets and other fruit containing ice creams. One of the main problems when using birch sap in industrial productions is storage. Birch sap can only be drawn during the period from spring thaw to the birch come into leaf, roughly from March to late April. Ice cream production on the other hand peak from late May through August. As birch sap besides a perfect blend of sugars and amino acids also contains micronutrients it is a perfect medium for many microorganisms like different strains of lactobacillus and can thus only be stored in frozen condition. This adds to the cost of using birch sap in industry. Two possible solutions to reduce storage costs seem viable if addition of preservatives is tobe avoided. One is to reduce the birch sap volume by concentrating the sap with reverse osmosis (RO) to around 15%.thus reducing the frozen storage volume. The other is to increase the sugar concentration to around 60-70% making thesap self-preserving as done with birch syrup. Concentration of birch sap to birch syrup is traditionally carried out asevaporation at around 106 ºC giving an undesirable dark brown syrup due to Maillard reactions [4]. This is notacceptable in an additive for ice cream production both because of the colour but also because of the change in flavour.The aim of the work presented here was to produce a 60% birch sap concentrate using low temperature direct contactmembrane distillation (DCMD) combined with microfiltration (MF) and reverse osmosis (RO)

Pythons, renowned for their abilities to fast for manybmonths and ingest very large meals, exhibit extreme physiological adaptations to their “sit-and-wait predator” lifestyle. In particular, the size and function of their visceral organs are rapidly up- and downregulated during the transitions between feast and famine. In this study, we have employed computer tomography (CT) and magnetic resonance imaging (MRI) to successively portrait the complete digestion cycle in a completely non-invasive way. Two- and threedimensional graphical reconstructions were performed, specifically addressing the visceral organs and intestines. Fasting Burmese pythons (Python molurus) were scanned before and at 2, 16, 24, 40, 48, 72 and 132 hours after ingestion of one rat. Acquired images revealed a gradual disappearance of the meal accompanied by an overall expansion of the intestine, shrinking of the gallbladder, and a 30% increase in heart volume. These immediate responses following ingestion are consistent with previous invasive studies of pythons. In conclusion, our study showed that MRI and CT are capable to repeatedly and non-invasively image the phenotypic flexibility of internal organs in vertebrates.

Introduction: Regeneration is a widespread phenomenon functioning to maintain and restore normal form and function of cells, tissues, and sometimes organs or appendages. Mammalian species typically employed in regenerative research generally process limited regenerative potential. Conversely, lower vertebrates such as the urodele amphibians (salamanders and newts), are excellent animal models for regenerative studies. The iconic Mexican axolotl (Ambystoma mexicanum) is capable of regenerating whole limbs, tail, jaw, and many inner organs, by dedifferentiation of cells to form a blastema (collection of stem cells), that proliferate and regenerate lost tissue completely without scar formation.The objective of this study was to evaluate if the positive effect of HBOT in wound healing translates to whole limb regeneration in a non-necrotic environment. The effect of two different constitutive HBOT-regimes was tested in regenerating axolotl limbs. Materials and Methods: Left forelimb was amputated at the site of mid-humerus in six anaesthetised axolotls. Two HBOT groups (HBOT1 and HBOT2) underwent daily HBOT for 120 minutes at 3 ATA in a pressure chamber. HBOT1 were pressurized in oxygen saturated water, whereas HBOT2 were pressurized in gaseous oxygen with 100% relative humidity. Control animals were exposed to normobaric conditions in water. Regeneration rate was evaluated as the increasing length of the regenerating appendage. Animals were kept at 20°C at all times. Results: Due to a group n = 2, the data is only indicative. At present, 80 constitutive days of HBOT has been performed for HBOT1. HBOT2 animals were not able to tolerate hyperbaric gaseous oxygen, and were excluded after 3 HBO treatments. No indicative effect of HBOT on whole limb regeneration has yet been identified.Discussion:This pilot study revealed no effect of 80 days constitutive HBOT in a regeneration-competent species, suggesting that HBOT has minor consequences on tissue regeneration in a non-necrotic environment.Keywords: whole limb regeneration, amphibian, Mexican axolotl (Ambystoma mexicanum), HBOT

Moderne skanningsteknikker giver mulighed for at gørlave virtuelle dissektioner helt uden blod. Teknikkerne har revolutioneret diagnostikken af patienter på hospitalerne, men giver også en helt unik muliughed for at opnå helt ny viden om menneskers og dyrs anatomi og fysiologi.

OBJECTIVES: We describe the development of a novel preclinical rodent-sized pressure chamber system compatible with computed tomography (CT), positron emission tomography (PET) and magnetic resonance imaging (MRI) that allows continuous uncompromised and minimally invasive data acquisition throughout hyperbaric exposures. The effect of various pressures on the acquired image intensity obtained with different CT, PET and MRI phantoms are characterised.MATERIAL AND METHODS: Tissue-representative phantom models were examined with CT, PET or MRI at normobaric pressure and hyperbaric pressures up to 1.013 mPa. The relationships between the acquired image signals and pressure were evaluated by linear regression analysis for each phantom.RESULTS: CT and PET showed no effect of pressure per se, except for CT of air, demonstrating an increase in Hounsfield units in proportion to the pressure. For MRI, pressurisation induced no effect on the longitudinal relaxation rate (R₁), whereas the transversal relaxation rate (R₂) changed slightly. The R₂ data further revealed an association between pressure and the concentration of the paramagnetic nuclei gadolinium, the contrast agent used to mimic different tissues in the MRI phantoms.CONCLUSION: This study demonstrates a pressure chamber system compatible with CT, PET and MRI. We found that no correction in image intensity was required with pressurisation up to 1.013 mPa for any imaging modality. CT, PET or MRI can be used to obtain anatomical and physiological information from pressurised model animals in this chamber.