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Health monitoring during offshore saturation diving is complicated due to restricted access to the divers, the desire to keep invasive procedures to a minimum, and limited opportunity for laboratory work onboard dive support vessels (DSV). In this pilot study, we examined whether measuring salivary biomarkrers in samples collected by the divers themselves might be a feasible approach to environmental stress assessment. Nine saturation divers were trained in the passive drool method for saliva collection and proceeded to collect samples at nine time points before, during, and after an offshore commercial saturation diving campaign. Samples collected within the hyperbaric living chambers were decompressed and stored frozen at −20°C onboard the DSV until they were shipped to land for analysis. Passive drool samples were collected without loss and assayed for a selection of salivary biomarkers: secretory immunoglobulin A (SIgA), C-reactive protein (CRP), tumor necrosis factor (TNF)-α, interleukins IL-6, IL-8, IL-1β, as well as cortisol and alpha-amylase. During the bottom phase of the hyperbaric saturation, SIgA, CRP, TNF-α, IL-8 and IL-1β increased significantly, whereas IL-6, cortisol and alpha-amylase were unchanged. All markers returned to pre-dive levels after the divers were decompressed back to surface pressure. We conclude that salivary biomarker analysis may be a feasible approach to stress assessment in offshore saturation diving. The results of our pilot test are consonant with an activation of the sympathetic nervous system related to systemic inflammation during hyperbaric and hyperoxic saturation.

Saturation divers work and live under high physiological and social demands for weeks on end. Even though physiological research has contributed insights to the work conditions of saturation divers, research on the qualities of the divers' psychosocial work environment is lacking. This study aimed to explore which job demands and resources are viewed as characteristic among saturation divers working within an isolated and confined environment. Based on data from 6 in-depth semi-structured interviews, template analysis was applied to map unique characteristics. By using the theoretical framework of the job demands-resources model, we found that the work environment in saturation diving was characterized by shifting demands and big contrasts, requiring adaptability in each individual diver. One major demand described by the informants was an unpredictable future, somewhat due to the changes in the oil and gas industry. Another important demand was the conflict between family and work/leisure when committing to work for extended periods in isolated environments. The monotony that characterizes the work environment is a challenge that must be managed. High wages, periods of leisure, and a prestigious job provide external motivation, while personal resources such as mental endurance and flexibility, a willingness to learn, and keeping up small personal routines, may benefit the divers' mental health. This is also affected by the quality of team climate—with features such as being sociable and considerate, having a dark sense of humor and having trust in one another.

Decompression sickness (DCS) develops due to inert gas bubble formation in bodily tissues and in the circulation, leading to a wide range of potentially serious clinical manifestations. Its pathophysiology remains incompletely understood. In this study, we aim to explore changes in the human leukocyte transcriptome in divers with DCS compared to closely matched unaffected controls after uneventful diving. Cases (n = 7) were divers developing the typical cutis marmorata rash after diving with a confirmed clinical diagnosis of DCS. Controls (n = 6) were healthy divers who surfaced from a 25 msw dive without decompression violation or evidence of DCS. Blood was sampled at two separate time points—within 8 h of dive completion and 40–44 h later. Transcriptome analysis by RNA-Sequencing followed by bioinformatic analysis was carried out to identify differentially expressed genes and relate their function to biological pathways. In DCS cases, we identified enrichment of transcripts involved in acute inflammation, activation of innate immunity and free radical scavenging pathways, with specific upregulation of transcripts related to neutrophil function and degranulation. DCS-induced transcriptomic events were reversed at the second time point following exposure to hyperbaric oxygen. The observed changes are consistent with findings from animal models of DCS and highlight a continuum between the responses elicited by uneventful diving and diving complicated by DCS. This study sheds light on the inflammatory pathophysiology of DCS and the associated immune response. Such data may potentially be valuable in the search for novel treatments targeting this disease., peer-reviewed

Commercial saturation divers are exposed to unique environmental conditions and are required to conduct work activity underwater. Consequently, divers’ physiological status is shown to be perturbed and therefore, appropriate strategies and guidance are required to manage the stress and adaptive response. This study aimed to evaluate the daily energy expenditure (DEE) of commercial saturation divers during a 21-day diving operation in the North Sea. Ten saturation divers were recruited during a diving operation with a living depth of 72 metres seawater (msw) and a maximum working dive depth of 81 msw. Doubly labelled water (DLW) was used to calculate DEE during a 10-day measurement period. Energy intake was also recorded during this period by maintaining a dietary log. The mean DEE calculated was 3030.9 ± 513.0 kcal/day, which was significantly greater than the mean energy intake (1875.3 ± 487.4 kcal; p = 0.005). There was also a strong positive correction correlation between DEE and total time spent performing underwater work (r = 0.7, p = 0.026). The results suggested saturation divers were in a negative energy balance during the measurement period with an intraindividual variability in the energy cost present that may be influenced by time spent underwater.

Endothelial dysfunction is recognized as a major contributor to atherosclerosis and has been suggested to be evident far before plaque formation. Endothelial dysfunction in small resistance arteries has been suggested to initiate long before changes in conduit arteries. In this study, we address early changes in endothelial function of atherosclerosis prone rats. Male ApoE knockout (KO) rats (11- to 13-weeks-old) were subjected to either a Western or standard diet. The diet intervention continued for a period of 20-24 weeks. Endothelial function of pulmonary and mesenteric arteries was examined in vitro using an isometric myograph. We found that Western diet decreased the contribution of cyclooxygenase (COX) to control the vascular tone of both pulmonary and mesenteric arteries. These changes were associated with early stage atherosclerosis and elevated level of plasma total cholesterol, LDL and triglyceride in ApoE KO rats. Chondroid-transformed smooth muscle cells, calcifications, macrophages accumulation and foam cells were also observed in the aortic arch from ApoE KO rats fed Western diet. The ApoE KO rats are a new model to study endothelial dysfunction during the earlier stages of atherosclerosis and could help us improve preclinical drug development. Open Access Tis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Te images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © Te Author(s) 2020

Commercial saturation divers must acclimatize to hyperbaric hyperoxia in their work environment, and subsequently readjust to breathing normal air when their period in saturation is over. In this study, we measured hemoglobin (Hb) during and following 4 weeks of heliox saturation diving in order to monitor anemia development and the time for Hb to recover post-saturation. Male commercial saturation divers reported their capillary blood Hb daily, before, and during 28 days of heliox saturation to a working depth of circa 200 m (n = 11), and for 12 days at surface post-saturation (n = 9–7), using HemoCue 201+ Hb devices. Hb remained in normal range during the bottom phase, but fell during the decompression; reaching levels of mild anemia (≤13.6 g/dl) the day after the divers’ return to the surface. Hb was significantly lower than the pre-saturation baseline (14.7 ± 1.1 g/dl) on the fifth day post-saturation (12.8 ± 1.8 g/dl, p = 0.028), before reverting to normal after 6–7 days. At the end of the 12-day post-saturation period, Hb was not statistically different from the pre-saturation baseline. The observed Hb changes, although significant, were modest. While we cannot rule out effect of other factors, the presence of mild anemia may partially explain the transient fatigue that commercial saturation divers experience post-saturation.

Cardiovascular risk is elevated in divers, but detailed information of cardiac function during diving is missing. The aim of this study was to apply an intact rat model with continuous monitoring of cardiac left ventricular (LV) function in a simulated diving experiment. Thirteen rats were inserted with a LV pressure–volume catheter and a pressure transducer in the femoral artery to measure hemodynamic variables, and randomly assigned to diving (n = 9) and control (n = 4) groups. The diving group was compressed to 600 kPa in air, maintained at pressure for 45 min (bottom phase), and decompressed to surface at 50 kPa/min. Data was collected before, during, and up to 60 min after exposure in the diving group, and at similar times in non-diving controls. During the bottom phase, stroke volume (SV) (−29%) and cardiac output (−30%) decreased, whereas LV end-systolic volume (+13%), mean arterial pressure (MAP) (+29%), and total peripheral resistance (TPR) (+72%) increased. There were no changes in LV contractility, stroke work, or diastolic function. All hemodynamic variables returned to baseline values within 60 min after diving. In conclusion, our simulated dive experiment to 600 kPa increased MAP and TPR to levels which caused a substantial reduction in SV and LV volume output. The increase in cardiac afterload demonstrated to take place during a dive is well tolerated by the healthy heart in our model, whereas in a failing heart this abrupt change in afterload may lead to acute cardiac decompensation. Copyright © 2020 Gaustad, Kondratiev, Eftedal and Tveita. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)

Purpose: Decompression sickness (DCS) is a poorly understood and complex systemic disease caused by inadequate desaturation following a reduction of ambient pressure. A previous proteomic study of ours showed that DCS occurrence but not diving was associated with changes in the plasma proteome in rats, including a dramatic decrease of abundance of the tetrameric form of Transthyretin (TTR). The present study aims to assess the impact on the human blood proteome of a dive inducing significant decompression stress but without inducing DCS symptoms. Experimental design: Twelve healthy male divers were subjected to a single dive at a depth of 18m of sea water (msw) with a 47-min bottom time followed by a direct ascent to the surface at a rate of 9msw/min. Venous blood was collected before the dive as well as 30mn and 2h following the dive. The plasma proteomes from four individuals were then analyzed by using a two-dimensional electrophoresis-based proteomic strategy. Results: No protein spot showed a significantly changed abundance (fdr< 0.1) between the tested times. Conclusion: These results strengthen the hypothesis according to which significant changes of the plasma proteome measurable with two-dimensional electrophoresis may only occur along with DCS symptoms. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. This is the authors' accepted and refereed manuscript to the article. Locked until 12 May 2018 due to copyright restrictions

Freedivers hold their breath while diving, causing blood oxygen levels to decrease (hypoxia) while carbon dioxide increases (hypercapnia). Whereas blood gas changes are presumably involved in the progression of respiratory diseases, less is known about their effect on healthy individuals. Here we have used gene expression profiling to analyze elite athletes' immune and inflammatory responses to freediving. Blood was collected before and 1 and 3 h after a series of maximal dynamic and static freediving apneas in a pool, and peripheral blood gene expression was mapped on genome-wide microarrays. Fractions of phenotypically distinct immune cells were computed by deconvolution of the gene expression data using Cibersort software. Changes in gene activity and associated biological pathways were determined using R and GeneGo software. The results indicated a temporary increase of neutrophil granulocytes, and a decrease of cytotoxic lymphocytes ; i.e., CD8+ T cells and resting NK cells. Biological pathway associations indicated possible protective reactions: genes involved in anti- inflammatory responses to proresolving lipid mediators were upregulated, whereas central factors involved in granule-mediated lymphocyte cytotoxicity were downregulated. While it remains unresolved whether freediving alters the immune system's defensive function, these results provide new insight into leukocyte responses and the protection of homeostasis in healthy athletes. freedivers dive on a single breath, and their performance hinges on their ability to suppress breathing voluntarily while floating face down (static apnea) or swimming horizontally (dynamic apnea) or vertically (constant weight apnea, free immersion apnea, no-limits apnea). To improve their performance, they use adaptive techniques that increase their lung capacity, reduce metabolic rates, and improve their tolerance to apnea, i.e., to hypoxia and hypercapnia (22). Freediving performance is often further enhanced by hyperventilation to reduce carbon dioxide levels prior to the dive and by glossopharyngeal insufflation (“lung packing”) for extra volumes of air or breathing pure oxygen to add to the body's oxygen reserves (19). During dives, an initial easy- going phase is followed by a physiological breaking point after which the urge to breath causes a struggle phase with displays of involuntary movements of the respiratory muscles that are thought to increase cardiac output (12, 31). This effect restores oxygen supply to the vital organs, such as the brain and the heart. Still, hypoxia can be severe at the end of a dive ; oxygen levels that are considered pathological in untrained individuals have been measured in freediving athletes' first expired breaths and arterial blood after diving (21, 30, 52). Adaption to hypercapnia also permits freedivers to prolong their breath holding, and the level of carbon dioxide first expired after breaking off a dive is considerably elevated (30). In light of an emerging understanding of the role of the immune system and inflammatory signaling in maintaining tissue and organ homeostasis (24), it is of interest to understand the responses of the white blood cells, leukocytes, to physiologically stressful changes in blood gas during voluntary apnea. The possibility of genome-wide measurements of gene expression on microarrays has expedited research into the molecular basis of biological states and responses. For studies of the immune system, peripheral blood is an obvious choice for gene expression analysis (5). Blood is a highly heterogeneous tissue. Of its formed elements, the erythrocytes, platelets, and leukocytes, only leukocytes have chromosome- containing nuclei ; genome-wide gene expression in peripheral blood therefore ideally represent the biological state of its leukocytes. However, the interpretation of gene expression data from blood is complicated by the heterogeneity of the leukocyte compartment, which consists of a number of phenotypically different cell types. The main leukocytes, the neutrophils, eosinophils, basophils, lymphocytes, and monocytes, are further divided into subsets of cells with different function in the immune system. Each leukocyte subset derives its phenotype from the particular set of genes it expresses, and the cell types are present in blood in variable amounts (51). In practical terms, this means that a measured change in the abundance of any transcript in blood does not immediately tell us whether the activity of its gene has changed, or whether there has been a change in the relative abundance of cells in which this gene is expressed (39). Traditional microarray analysis does not take sample composition into account, but recent papers have presented methods where transcriptome contributions from phenotypically distinct cell types are separated by signal deconvolution on the basis of cell type- specific gene expression (1, 25, 38). Deconvolution of microarray signals extracts cell type-specific information from system-wide data and has been found to corroborate results from flow cytometric phenotyping. Also, since deconvolution is done after the genome-wide data are collected, it eliminates the need for fractioning of samples and facilitates unbiased detection of cell types for which the patterns of gene expression are known. In this study we examine the effects of freediving on cells of the immune system. Genome-wide cDNA microarrays were used to analyze the peripheral blood transcriptome of elite freedivers who performed a series of dynamic and static apnea dives in a pool with their respiratory tract immersed. The proportions of major immune cell types in the participant's blood before and after dives were calculated by cell type-specific deconvolution of the microarray data. Changes in biological pathways were predicted on basis of differentially expressed genes.

Objective: The aim of this study was to determine if, after controlling for weight, age is associated with decompression sickness (DCS) in rats. Methods: Following compression-decompression, male rats aged 11 weeks were observed for DCS. After two weeks recovery surviving rats were re-dived using the same compression- decompression profile. Results: In this experiment there was a clear difference between DCS outcome at ages 11 or 13 weeks in matched rats (p=0.002). Discussion: Even with weight included in the model age was significantly associated with DCS (p=0.01), yet after removal of weight the association was much stronger (p=0.002). Conclusion: We believe that age is likely to be found associated with the probability of DCS in a larger dataset with a wider range of parameters, after accounting for the effect of weight. This is an accepted manuscript of an article published by Taylor & Francis in Archives of Physiology and Biochemistry on 12 Mar 2016, available at http://www.tandfonline.com/doi/full/10.3109/13813455.2016.1140787

Diving causes a transient reduction of vascular function, but the mechanisms behind this are largely unknown. The aim of this study was therefore to analyze genetic reactions that may be involved in acute changes of vascular function in divers. Rats were exposed to 709 kPa of hyperbaric air (149 kPa Po2) for 50 min followed by postdive monitoring of vascular bubble formation and full genome microarray analysis of the aorta from diving rats ( n = 8) and unexposed controls ( n = 9). Upregulation of 23 genes was observed 1 h after simulated diving. The differential gene expression was characteristic of cellular responses to oxidative stress, with functions of upregulated genes including activation and fine-tuning of stress-responsive transcription, cytokine/cytokine receptor signaling, molecular chaperoning, and coagulation. By qRT-PCR, we verified increased transcription of neuron-derived orphan receptor-1 ( Nr4a3), plasminogen activator inhibitor 1 ( Serpine1), cytokine TWEAK receptor FN14 ( Tnfrsf12a), transcription factor class E basic helix-loop-helix protein 40 ( Bhlhe40), and adrenomedullin ( Adm). Hypoxia-inducible transcription factor HIF1 subunit HIF1-α was stabilized in the aorta 1 h after diving, and after 4 h there was a fivefold increase in total protein levels of the procoagulant plasminogen activator inhibitor 1 (PAI1) in blood plasma from diving rats. The study did not have sufficient power for individual assessment of effects of hyperoxia and decompression-induced bubbles on postdive gene expression. However, differential gene expression in rats without venous bubbles was similar to that of all the diving rats, indicating that elevated Po2 instigated the observed genetic reactions.

Divers are exposed to circulatory stress that directly affects the endothelial lining of blood vessels, and even asymptomatic dives are associated with inflammatory responses, microparticle release and endothelial dysfunction. As humans age, there is a relative increase in the risk of cardiovascular disease, attributed in part to declining endothelial function. Whether extensive diving in the older diver increases the risk of disease as a result of accumulated circulatory stress or provides protection through processes of acclimatization remains an open question. We provide a brief review of current knowledge about the separate effects of diving and aging on the vascular endothelium in humans and rodents, and discuss the available data on their combined effects. The aim is to elucidate possible outcomes of the interplay between exogenous and endogenous stress factors for endothelial function and to question potential implications for cardiovascular health in the aging diver.

Divers are exposed to circulatory stress that directly affects the endothelial lining of blood vessels, and even asymptomatic dives are associated with inflammatory responses, microparticle release and endothelial dysfunction. As humans age there is a relative increase in the risk of cardiovascular disease, attributed in part to declining endothelial function. Whether extensive diving with rising age increases the risk of disease as a result of accumulated circulatory stress, or provides protection through processes of acclimatisation remains an open question. We provide a brief review of current knowledge about the separate effects of diving and aging on the vascular endothelium in humans and rodents, and discuss the available data on the combined effects of diving and aging. The aim is to elucidate possible outcomes of the interplay between exogenous and endogenous stress factors for endothelial function and to question potential implications for cardiovascular health in the aging diver. Copyright © 2015 The Author(s). Published by South Pacific Underwater Medicine Society (Spums)

Decompression sickness (DCS) is the collective term for an array of signs and symptoms triggered by ambient pressure reduction. It is of particular concern to divers as they decompress on ascend from depth to sea surface, but despite a long history of studies the determinants of DCS risk are incompletely understood and there are no validated biomarkers. In this issue of Proteomics Clinical Applications, Lautridou et al. [8] report on their search for DCS biomarkers in rats exposed to simulated diving. By comparing the plasma proteomes from animals showing neurological symptoms to those emerging from dives unaffected, they identified several high-abundance proteins not previously associated with DCS. The most significant finding was a near depletion of thyroxine- and vitamin A transporter transthyretin in symptomatic rats. In addition to their potential role as diagnostic biomarkers, the proteins identified in Lautridou's study may offer new pieces in the yet incomplete puzzle of DCS etiology.

PURPOSE: Decompression sickness (DCS) caused by vascular bubble formation is a major risk when diving. Prior studies have shown that physical exercise has a significant impact in both reducing and increasing bubble formation. There is limited knowledge about the mechanisms, but there are indications that exercise-induced muscle injury prior to diving may cause increased bubble formation. The purpose of this study was to investigate the role of exercise-induced muscle injury as a possible mechanism of bubble formation during diving. METHODS: Muscle injury was induced by exposing female Sprague-Dawley rats (n = 30) to a single bout of eccentric exercise, 100 min intermittent, downhill (-16°) treadmill running. Forty-eight hours later, the animals were exposed to a 50-min simulated saturation dive (709 kPa) in a pressure chamber, when the degree of muscle injury and inflammation would be the most pronounced. Bubble formation after the dive was observed by ultrasonic imaging for 4 h. RESULTS: No difference in bubble loads was found between the groups at any time despite evident muscle injury. Maximum bubble loads (bubbles cm-2 heart cycle-1) were not different, exercise: 1.6 ± 3.5 SD vs control: 2.2 ± 4.1 SD, P = 0.90, n = 15 in each group. CONCLUSIONS: Eccentric exercise performed 48 h prior to diving causes skeletal muscle injury but does not increase the amount of vascular bubbles in rats. The prevailing recommendation is that physical activity prior to diving is a risk factor of DCS. However, present and previous studies implicate that pre-dive physical activity does not increase the DCS risk. © Springer-Verlag Berlin Heidelberg 2014. This is the authors' accepted and refereed manuscript to the article

This study is part of a strategy aimed at using fluorescent polymerase chain reaction (PCR) on informative genetic microsatellite markers as a diagnostic tool in preimplantation genetic diagnosis (PGD) of severe monogenic disease. Two couples, both of whom had previously had children who were compound heterozygote for severe cystic fibrosis mutations, were offered PGD using fluorescent PCR of the highly polymorphic cystic fibrosis transmembrane conductance regulator (CFTR) intragenic microsatellite marker IVS17bTA. Cleavage-stage embryo biopsy followed by PCR resulted in transfer of one unaffected carrier embryo for each couple. This approach eliminates the need for single cell multiplex PCR strategies to detect CF compound heterozygotes. It also provides a control of chromosome 7 ploidy in the blastomeres and a selection against allele dropout by positive detection of each CFTR copy of all genotypes in preimplantation embryos from genetically informative families.

During scuba diving, the circulatory system is stressed by an elevated partial pressure of oxygen while the diver is submerged and by decompression-induced gas bubbles on ascent to the surface. This diving-induced stress may trigger decompression illness, but the majority of dives are asymptomatic. In this study we have mapped divers' blood transcriptomes with the aim of identifying genes, biological pathways, and cell types perturbed by the physiological stress in asymptomatic scuba diving. Ten experienced divers abstained from diving for >2 wk before performing a 3-day series of daily dives to 18 m depth for 47 min while breathing compressed air. Blood for microarray analysis was collected before and immediately after the first and last dives, and 10 matched nondivers provided controls for predive stationary transcriptomes. MetaCore GeneGo analysis of the predive samples identified stationary upregulation of genes associated with apoptosis, inflammation, and innate immune responses in the divers, most significantly involving genes in the TNFR1 pathway of caspase-dependent apoptosis, HSP60/HSP70 signaling via TLR4, and NF-κB-mediated transcription. Diving caused pronounced shifts in transcription patterns characteristic of specific leukocytes, with downregulation of genes expressed by CD8+ T lymphocytes and NK cells and upregulation of genes expressed by neutrophils, monocytes, and macrophages. Antioxidant genes were upregulated. Similar transient responses were observed after the first and last dive. The results indicate that sublethal oxidative stress elicits the myeloid innate immune system in scuba diving and that extensive diving may cause persistent change in pathways controlling apoptosis, inflammation, and innate immune responses. © 2013 American Physiological Society. Open access.

Incorporation of dUMP instead of dTMP is frequently used to control carryover contamination during PCR amplifications. We have tested four thermostable DNA polymerases for their ability to utilize dUTP as a substrate in PCR. Amplification of products in the presence of dUTP instead of dTTP was good with Thermus aquaticus DNA polymerase but highly inefficient with three other thermostable DNA polymerases. The latter was due to: (a) lower incorporation of dUMP relative to dTMP, (b) increased proofreading toward dUMP in DNA, (c) relative termination at dUMP residues as verified by sequencing reactions in the presence of dUTP, (d) thermostable dUTPase activity in the commercial enzyme preparation. The last point only applies to Pyrococcus furiosus DNA polymerase. This study demonstrates that various thermostable DNA polymerases utilize dTTP and dUTP with highly different efficiencies and thus the choice of DNA polymerase may be critical for amplification of DNA.

We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,000-fold and studied its biochemical properties, including sequence specificity. The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody directed towards human UDG. SDS-PAGE and western analysis indicate an apparent M(r) = 27,500. Bovine UDG has a 1.7-fold preference for single stranded over double stranded DNA as a substrate. Sequence specificity for uracil removal from dsDNA was examined for bovine and Escherichia coli UDG, using DNA containing less than one dUMP residue per 100 nucleotides and synthetic oligonucleotides containing one dUMP residue. Comparative studies involving about 40 uracil sites indicated similar specificities for both UDGs. We found more than a 10-fold difference in rates of uracil removal between different sequences. 5'-G/CUT-3' and 5'-G/CUG/C-3' were consensus sequences for poor repair whereas 5'-A/TUAA/T-3' was a consensus for good repair. Sequence specificity was verified in double stranded oligonucleotides, but not in single stranded ones, suggesting that the structure of the double stranded DNA helix has influence on sequence specificity. Rate of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches. The results indicate that sequence specific repair may be a determinant to be considered in mutagenesis.

Increased interest in the therapeutic use of human stem cells has emerged following significant progress in ongoing research. The cloning of a sheep, the isolation of human embryonic stem cells, and the discovery that adult stem cells may be reprogrammed taken together give substance to hopes that novel principles of treatment may be developed for a variety of serious conditions. Embryonic stem cells are derived from pre-embryos at the blastocyst stage and may give rise to all bodily tissues and cells. Animal models have demonstrated that embryonic stem cells when transplanted into adult hosts may differentiate and develop into cells and tissues applicable for treatment of a variety of conditions, including Parkinson's disease, multiple sclerosis, spinal injuries, cardiac stroke and cancer. Transplanted embryonic stem cells are exposed to immune reactions similar to those acting on organ transplants, hence immunosuppression of the recipient is generally required. It is, however, possible to obtain embryonic stem cells that are genetically identical to the patient's own cells by means of therapeutic cloning techniques. The nucleus from a somatic cell is transferred into an egg after removal of the egg's own genetic material. Under specific condition the egg will use genetic information from the somatic cell in organising the formation of a blastocyst which in turn generates embryonic stem cells. These cells have a genetic composition identical to that of the patient and are suitable for stem cell therapy.

Hyperbaric oxygen preconditioning (HBO-PC) can protect the heart from injury during subsequent ischemia. The presence of high loads of venous gas emboli (VGE) induced by a rapid ambient pressure reduction on ascent from diving may cause ischemia and acute heart failure. The aim of this study was to investigate the effect of diving-induced VGE formation on cardiac stress marker levels and the cardioprotective effect of HBO-PC. To induce high loads of VGE, 63 female Sprague-Dawley rats were subjected to a rapid ambient pressure reduction from a simulated saturation dive (50 min at 709 kPa) in a pressure chamber. VGE loads were measured for 60 min in anesthetized animals by the use of ultrasonography. The animals were divided into five groups. Three groups were exposed to either diving or to HBO-PC (100% oxygen, 38 min at 303 kPa) with a 45 or 180 min interval between HBO-PC and diving. Two additional groups were used as baseline controls for the measurements; one group was exposed to equal handling except for HBO-PC and diving, and the other group was completely unexposed. Diving caused high loads of VGE, as well as elevated levels of the cardiac stress markers, cardiac troponin T (cTnT), natriuretic peptide precursor B (Nppb), and αB-crystallin, in blood and cardiac tissue. There were strong positive correlations between VGE loads and stress marker levels after diving, and HBO-PC appeared to have a cardioprotective effect, as indicated by the lower levels of stress marker expression after diving-induced VGE formation. KEYWORDS: Cardioprotection, decompression illness, diving, gas embolism, hyperbaric oxygen preconditioning (c) 2013 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

The human uracil-DNA glycosylase gene (UNG) spans approximately 13.5 kb including the promoter. UNG comprises 6 exons and 5 introns and was assigned to chromosome 12q23-q24.1 by radiation hybrid mapping. UNG exhibits typical features of housekeeping genes, including a 5' CpG island of 1.2 kb and a very GC-rich TATA-less promoter containing a number of elements involved in constitutive expression and cell cycle regulation. A smaller CpG island is located just downstream of the gene. Within the 15-kb sequence we identified 16 Alu retroposons, 2 of which contain putative competent RNA polymerase III promoters, 3 copies of medium reiteration frequency repeats, and 1 copy of a mammalian-wide interspersed repetitive element, as well as a 300-bp TA-dinucleotide repeat. In vitro methylation of the UNG promoter strongly reduced promoter activity, but methylation may not be involved in regulation of UNG in vivo since a narrow region of the 5' CpG island comprising the putative transcription factor binding region appears to be invariably methylation-free.

Two clones containing nonfunctional pseudogenes for the human uracil-DNA glycosylase gene have been isolated. The sequences of the two clones that are homologous to the UNG cDNA span 670 and 580 bp, respectively. In the longest of these, a full length Sx type Alu sequence interrupts the homologous sequence. Chromosomal mapping locates the clones to chromosomes 16 and 14. Comparison of the pseudogene sequences to the cDNA sequence indicates that the pseudogenes diverged from the functional gene approximately 31 and 22 million years ago, which is before the point in evolution when great apes and hominides separated.

Oxidative agents produce several different types of base modifications in DNA, and only a few of these have been properly characterized with respect to mechanisms of formation and biological implications. We have established a procedure using neutral thermal hydrolysis and reverse phase high-performance liquid chromatography to determine the content of the oxidation product 5-formyluracil (5-foU) in DNA. With this method, it is shown that 5-foU residues are formed with high frequency from thymine by quinone-sensitized UV-A photooxidation. Since 5-foU is also induced by ionizing radiation, it appears to be formed under conditions where thymidine radical cations are generated and react with molecular oxygen. It was previously shown that 5-foU is formed directly from [methyl-3H]thymine residues in radioactively labeled DNA by two consecutive transmutations of 3H to 3He. The theoretical basis for the kinetics of such conversion is presented in this paper, and the calculated yields are confirmed experimentally by measuring the content of 5-foU in [methyl-3H]thymine-labeled DNA aged for different time periods. Such DNA contains virtually only 5-(hydroxymethyl)uracil and 5-foU, apart from normal bases, and is therefore very useful for the investigation of repair enzyme activities involved in the repair of 5-foU-containing DNA. Using this substrate, a DNA glycosylase activity was identified in human cell extracts for the removal of 5-foU.(ABSTRACT TRUNCATED AT 250 WORDS)

The rate of removal of uracil from different positions in double-stranded DNA by uracil-DNA glycosylase from Escherichia coli varied more than 15-fold. Consensus sequences for good and poor removal were 5′-(A/T)UA(A/T)-3′ and 5′-(G/C)U(T/G/C)-3′, respectively. In general, the sequence context surrounding U was more important for the rate of removal than whether U was present in U·A pairs or U·G mispairs. Rates of removal of U from sites of amber mutations in the lacI gene, where mutation frequencies and deamination rates were known, indicated that the observed variation in removal is biologically significant.

We have expressed a human recombinant uracil-DNA glycosylase (UNG delta 84) closely resembling the mature form of the human enzyme (UNG, from the UNG gene) in Escherichia coli and purified the protein to apparent homogeneity. This form, which lacks the first seven nonconserved amino acids at the amino terminus, has properties similar to a 50% homogeneous UDG purified from human placenta except for a lower salt optimum and a slightly lower specific activity. The recombinant enzyme removed U from ssDNA approximately 3-fold more rapidly than from dsDNA. In the presence of 10 mM NaCl, Km values were 0.45 and 1.6 microM with ssDNA and dsDNA, respectively, but Km values increased significantly with higher NaCl concentrations. The pH optimum for UNG delta 84 was 7.7-8.0; the activation energy, 50.6 kJ/mol; and the pI between 10.4 and 10.8. The enzyme displays a striking sequence specificity in removal of U from UA base pairs in M13 dsDNA. The sequence specificity for removal of U from UG mismatches (simulating the situation after deamination of C) was essentially similar to removal from UA matches when examined in oligonucleotides. However, removal of U from UG mismatches was in general slightly faster, and in some cases significantly faster, than removal from UA base pairs. Immunofluorescence studies using polyclonal antibodies against UNG delta 84 demonstrated that the major fraction of UNG was located in the nucleus. Furthermore, > 98% of the total uracil-DNA glycosylase activity from HeLa cell extracts was inhibited by the antibodies, indicating that the UNG protein represents the major uracil-DNA glycosylase in the cells.

Objective Opioids are recommended by the World Health Organization for moderate to severe cancer pain. Oxycodone is one of the most commonly used opioids and is metabolized in the liver by CYP3A4 and CYP2D6 enzymes. The aim of this cross-sectional study was to assess the relationship between oxycodone pharmacokinetics, pharmacodynamics and the CYP2D6 genotypes “poor metaboliser” (PM), “extensive metaboliser” (EM) and “ultra-rapid metaboliser” (URM) in a cohort of patients with cancer pain. Methods The patients were genotyped for the most common CYP2D6 variants and serum concentrations of oxycodone and metabolites were determined. Pain was assessed using the Brief Pain Inventory (BPI). The EORTC QLQ-C30 was used to assess the symptoms of tiredness and nausea. Cognitive function was assessed by the Mini Mental State (MMS) examination. Associations were examined by analyses of variance (ANOVA) and covariance (ANCOVA), or ordinal logistic regressions with and without covariates. Results The sample consisted of 27 PM, 413 EM (including heterozygotes) and 10 URM. PM had lower oxymorphone and noroxymorphone serum concentrations and oxymorphone to oxycodone ratios than EM and URM. No differences between PM, EM and URM in pain intensity, nausea, tiredness or cognitive function was found. Conclusion CYP2D6 genotypes caused expected differences in pharmacokinetics, but they had no pharmacodynamic consequence. CYP2D6 genotypes did not influence pain control, the adverse symptoms nausea and sedation or the risk for cognitive failure in this study of patients treated with oxycodone for cancer pain.