Age- and Sex- Related Differences in Muscle Phosphocreatine and Oxygenation Kinetics during High Intensity Exercise in Adolescents and Adults

This is the author's post-print version of an article published in Nuclear Magnetic Resonance in Biomedicine, 2010, Vol. 23, Issue 6, pp. 569–577 Copyright © 2010 Wiley-Blackwell. The definitive version is available at www3.interscience.wiley.comThe aim of this investigation was to examine the adaptation of the muscle phosphates (e.g. phosphocreatine (PCr) and ADP) implicated in regulating oxidative phosphorylation, and oxygenation at the onset of high intensity exercise in children and adults. The hypotheses were threefold: primary PCr kinetics would be faster in children than adults; the amplitude of the PCr slow component would be attenuated in children; and the amplitude of the deoxyhaemoglobin/myoglobin (HHb) slow component would be reduced in children. Eleven children (5 girls, 6 boys, 13 ± 1 years) and 11 adults (5 women, 6 men, 24 ± 4 years) completed two to four constant work rate exercise tests within a 1.5 T MR scanner. Quadriceps muscle energetics during high intensity exercise were monitored using 31P-MRS. Muscle oxygenation was monitored using near-infrared spectroscopy. The time constant for the PCr response was not significantly different in boys (31 ± 10 s), girls (31 ± 10 s), men (44 ± 20 s) or women (29 ± 14 s, main effects: age, p = 0.37, sex, p = 0.25). The amplitude of the PCr slow component relative to end-exercise PCr was not significantly different between children (23 ± 23%) and adults (17 ± 13%, p = 0.47). End-exercise [PCr] was significantly lower, and [ADP] higher, in females (18 ± 4 mM and 53 ± 16 µM) than males (23 ± 4 mM, p = 0.02 and 37 ± 11 µM, p = 0.02), but did not differ with age ([PCr]: p = 0.96, [ADP]: p = 0.72). The mean response time for muscle tissue deoxygenation was significantly faster in children (22 ± 4 s) than adults (27 ± 7 s, p = 0.01). The results of this study show that the control of oxidative metabolism at the onset of high intensity exercise is adult-like in 13-year-old children, but that matching of oxygen delivery to extraction is more precise in adults

DNMTs are required for delayed genome instability caused by radiation

This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited - Copyright @ 2012 Landes Bioscience.The ability of ionizing radiation to initiate genomic instability has been harnessed in the clinic where the localized delivery of controlled doses of radiation is used to induce cell death in tumor cells. Though very effective as a therapy, tumor relapse can occur in vivo and its appearance has been attributed to the radio-resistance of cells with stem cell-like features. The molecular mechanisms underlying these phenomena are unclear but there is evidence suggesting an inverse correlation between radiation-induced genomic instability and global hypomethylation. To further investigate the relationship between DNA hypomethylation, radiosensitivity and genomic stability in stem-like cells we have studied mouse embryonic stem cells containing differing levels of DNA methylation due to the presence or absence of DNA methyltransferases. Unexpectedly, we found that global levels of methylation do not determine radiosensitivity. In particular, radiation-induced delayed genomic instability was observed at the Hprt gene locus only in wild-type cells. Furthermore, absence of Dnmt1 resulted in a 10-fold increase in de novo Hprt mutation rate, which was unaltered by radiation. Our data indicate that functional DNMTs are required for radiation-induced genomic instability, and that individual DNMTs play distinct roles in genome stability. We propose that DNMTS may contribute to the acquirement of radio-resistance in stem-like cells.This study is funded by NOTE, BBSRC and the Royal Society Dorothy Hodgkin Research Fellowship

Accumulating exercise and postprandial health in adolescents

ArticleCopyright © 2015 Published by Elsevier Inc.Purpose: To examine the influence of exercise intensity on postprandial health outcomes in adolescents when exercise is accumulated throughout the day. Methods: 19 adolescents (9 male, 13.7 ± 0.4 y) completed three 1-day trials in a randomised order: 1) rest (CON); or four bouts of 2) 2 x 1 min cycling at 90% peak power with 75 s recovery (high-intensity interval exercise; HIIE); or 3) cycling at 90% of the gas exchange threshold (moderate-intensity exercise; MIE), which was work-matched to HIIE. Each bout was separated by 2 hours. Participants consumed a high fat milkshake for breakfast and lunch. Postprandial triacylglycerol (TAG), glucose, systolic blood pressure (SBP) and fat oxidation were assessed throughout the day. Results: There was no effect of trial on total area under the curve (TAUC) for TAG (P=0.87). TAUC-glucose was lower in HIIE compared to CON (P=0.03, ES=0.42) and MIE (P=0.04, ES=0.41), with no difference between MIE and CON (P=0.89, ES=0.04). Postprandial SBP was lower in HIIE compared to CON (P=0.04, ES=0.50) and MIE (P=0.04, ES=0.40), but not different between MIE and CON (P=0.52, ES=0.11). Resting fat oxidation was increased in HIIE compared to CON (P=0.01, ES=0.74) and MIE (P=0.05, ES=0.51), with no difference between MIE and CON (P=0.37, ES=0.24). Conclusion: Neither exercise trial attenuated postprandial lipaemia. However, accumulating brief bouts of HIIE, but not MIE, reduced postprandial plasma glucose and SBP, and increased resting fat oxidation in adolescent boys and girls. The intensity of accumulated exercise may therefore have important implications for health outcomes in youth.Sport and Health Sciences Research Committee, College of Life and Environmental Sciences, University of Exeter

Sequential and counter-selectable cassettes for fission yeast

BACKGROUND: Fission yeast is one of the most commonly used model organisms for studying genetics. For selection of desirable genotypes, antibiotic resistance cassettes are widely integrated into the genome near genes of interest. In yeasts, this is achieved by PCR amplification of the cassette flanked by short homology sequences, which can be incorporated by homology directed repair. However, the currently available cassettes all share the same tef promoter and terminator sequences. It can therefore be challenging to perform multiple genetic modifications by PCR-based targeting, as existing resistance cassettes in strains can be favored for recombination due to shared homology between the cassettes. RESULTS: Here we have generated new selection cassettes that do not recombine with those traditionally used. We achieved this by swapping the tef promoter and terminator sequences in the established antibiotic resistance MX6 cassette series for alternative promoters and/or terminators. The newly created selection cassettes did not recombine with the tef-containing MX6 cassettes already present in the genome, allowing for sequential gene targeting using the PCR-based method. In addition, we have generated a series of plasmids to facilitate the C-terminal tagging of genes with desired epitopes. We also utilized the anti-selection gene HSV-TK, which results in cell death in strains grown on the drug 5-Fluoro-2'-deoxyuridine (FdU, Floxuridin or FUDR). By fusing an antibiotic resistance gene to HSV-TK, we were able to select on the relevant antibiotic as well as counter-select on FdU media to confirm the desired genomic modification had been made. We noted that the efficiency of the counter selection by FdU was enhanced by treatment with hydroxyurea. However, a number of DNA replication checkpoint and homologous recombination mutants, including rad3∆, cds1∆, rad54∆ and rad55∆, exhibited sensitivity to FdU even though those strains did not carry the HSV-TK gene. To remove counter-selectable markers, we introduced the Cre-loxP irreversible recombination method. Finally, utilizing the negative selectable markers, we showed efficient induction of point mutations in an endogenous gene by a two-step transformation method. CONCLUSIONS: The plasmid constructs and techniques described here are invaluable tools for sequential gene targeting and will simplify construction of fission yeast strains required for study

Longitudinal Changes in the Oxygen Uptake Kinetic Response to Heavy-Intensity Exercise in 14- to 16-Year-Old Boys

There is another ORE record for this publication: http://hdl.handle.net/10036/3830This study examined longitudinal changes in the pulmonary oxygen uptake (p(V) over dotO(2)) kinetic response to heavy-intensity exercise in 14-16 yr old boys. Fourteen healthy boys (age 14.1 +/- 0.2 yr) completed exercise testing on two occasions with a 2-yr interval. Each participant completed a minimum of three 'step' exercise transitions, from unloaded pedalling to a constant work rate corresponding to 40% of the difference between the (p(V) over dotO(2)) (2), at the gas exchange threshold and peak (p(V) over dotO(2)) , (40% A). Over the 2-yr period a significant increase in the phase II time constant (25 5 vs. 30 +/- 5 s; p = .002, omega(2) = 0.34), the relative amplitude of the (p(V) over dotO(2)) slow component (9 +/- 5 vs. 13 +/- 4%; p = .036, omega(2) = 0.14) and the(p(V) over dotO(2)) gain at end-exercise (11.6 +/- 0.6 vs. 12.4 +/- 0.7 mL.min(-1).W-1; p < .001, omega(2) = 0.42) were observed. These data indicate that the control of oxidative phosphorylation in response to heavy-intensity cycling exercise is age-dependent in teenage boys

The effects of two weeks high-intensity interval training on fasting glucose, glucose tolerance and insulin resistance in adolescent boys: a pilot study

This is the final version. Available on open access from BMC via the DOI in this recordAvailability of data and materials: The datasets generated and analysed during the current study are not publicly available due to ethical restrictions but are available from the corresponding author upon reasonable request.Background Current evidence of metabolic health benefits of high-intensity interval training (HIIT) are limited to longer training periods or conducted in overweight youth. This study assessed 1) fasting and postprandial insulin and glucose before and after 2 weeks of HIIT in healthy adolescent boys, and 2) the relationship between pre intervention health outcomes and the effects of the HIIT intervention. Methods Seven healthy boys (age:14.3 ± 0.3 y, BMI: 21.6 ± 2.6, 3 participants classified as overweight) completed 6 sessions of HIIT over 2 weeks. Insulin resistance (IR) and blood glucose and insulin responses to a Mixed Meal Tolerance Test (MMTT) were assessed before (PRE), 20 h and 70 h after (POST) the final HIIT session. Results Two weeks of HIIT had no effect on fasting plasma glucose, insulin or IR at 20 h and 70 h POST HIIT, nor insulin and glucose response to MMTT (all P > 0.05). There was a strong negative correlation between PRE training IR and change in IR after HIIT (r = − 0.96, P < 0.05). Conclusion Two weeks of HIIT did not elicit improvements to fasting or postprandial glucose or insulin health outcomes in a group of adolescent boys. However the negative correlation between PRE IR and improvements after HIIT suggest that interventions of this type may be effective in adolescents with raised baseline IR.National Institute for Health Research (NIHR)Northcott Devon Medical Foundatio

Establishing maximal oxygen uptake in young people during a ramp cycle test to exhaustion

addresses: Barker, AR, Univ Exeter, Sch Sport & Hlth Sci, Bioenerget & Human Performance Res Grp, Exeter, EX1 2LU, Devon, EnglandCopyright BMJ Publishing GroupObjectives This study tested the hypotheses that (1) secondary criteria (respiratory exchange ratio (RER), heart rate, blood [ lactate]) traditionally used to verify the determination of maximum oxygen uptake (O-2max) in children can result in the acceptance of a 'submaximal' O-2max or falsely reject a 'true' O-2max and (2) the O-2peak recorded during a ramp test in children is comparable to the O-2peak achieved during supramaximal testing.Methods Thirteen children (9-10 years) completed a ramp cycle test to exhaustion to determine their O-2peak. After 15 min of recovery, the participants performed a supramaximal cycle test to exhaustion at 105% of their ramp test peak power.Results Compared with the O-2peak during the ramp test, a significantly lower O-2 was recorded at a RER of 1.00 (1.293 litre/min (SD 0.265) vs 1.681 litre/min (SD 0.295), pO-2peak that was not significantly different from the ramp test (1.615 litre/min (SD 0.307) vs 1.690 litre/min (SD 0.284), p=0.090, respectively).Conclusions The use of secondary criteria to verify a maximal effort in young people during ramp cycling exercise may result in the acceptance of a submaximal O-2max. As supramaximal testing elicits a O-2peak similar to the ramp protocol, thus satisfying the plateau criterion, the use of such tests is recommended as the appropriate method of confirming a 'true' O-2max with children

A life threatening uterine inversion and massive post partum hemorrhage caused by placenta accrete during Caesarean section in a primigravida: a case report

Epoxide hydrolases catalyze the cofactor-independent hydrolysis of reactive and toxic epoxides. They play an essential role in the detoxification of various xenobiotics in higher organisms and in the bacterial degradation of several environmental pollutants. The first x-ray structure of one of these, from Agrobacterium radiobacter AD1, has been determined by isomorphous replacement at 2.1-Å resolution. The enzyme shows a two-domain structure with the core having the α/β hydrolase-fold topology. The catalytic residues, Asp107 and His275, are located in a predominantly hydrophobic environment between the two domains. A tunnel connects the back of the active-site cavity with the surface of the enzyme and provides access to the active site for the catalytic water molecule, which in the crystal structure, has been found at hydrogen bond distance to His275. Because of a crystallographic contact, the active site has become accessible for the Gln134 side chain, which occupies a position mimicking a bound substrate. The structure suggests Tyr152/Tyr215 as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen.

Relationship between (non)linear phase II pulmonary oxygen uptake kinetics with skeletal muscle oxygenation and age in 11 to 15 y olds

This is the author accepted manuscript. the final version is available from Wiley via the DOI in this recordThis study investigated in nineteen male youth (mean age: 13.6 ± 1.1 y, range: 11.7 – 15.7 y) the relationship between pulmonary oxygen uptake ( o2) and muscle deoxygenation kinetics during moderate‐ and very heavy‐intensity ‘step’ cycling initiated from unloaded pedaling (i.e. U→M and U→VH) and moderate‐to‐very heavy‐ intensity step cycling (i.e. M→VH). Pulmonary o2 was measured breath‐by‐breath and tissue oxygenation index (TOI) of the vastus lateralis using near‐infrared spectroscopy. There were no significant differences in the phase II time constant (τ o2p) between U→M and U→VH (23 ± 6 s vs. 25 ± 7 s; P = 0.36); however, the τ o2p was slower during M→VH (42 ± 16 s) compared to other conditions (P < 0.001). Quadriceps TOI decreased with a faster (P < 0.01) mean response time (MRT; i.e. time delay + τ) during U→VH (14 ± 2 s) compared to U→M (22 ± 4 s) and M→VH (20 ± 6 s). The difference (Δ) between the τ o2p and MRT‐TOI was greater during U→VH compared to U→M (12 ± 7 vs. 2 ± 7 s, P < 0.001) and during M→VH (23 ± 15 s) compared to other conditions (P < 0.02), suggesting an increased proportional speeding of fractional O2 extraction. The slowing of the τ o2p during M→VH relative to U→M and U→VH correlated positively with chronological age (r = 0.68 and 0.57, respectively, P < 0.01). In youth, “work‐to‐work” transitions slowed microvascular O2 delivery‐to‐O2 utilization with alterations in phase II o2 dynamics accentuated between the ages of 11 to 15 y

LARP7 family proteins have conserved function in telomerase assembly

Understanding the intricacies of telomerase regulation is crucial due to the potential health benefits of modifying its activity. Telomerase is composed of an RNA component and reverse transcriptase. However, additional factors required during biogenesis vary between species. Here we have identified fission yeast Lar7 as a member of the conserved LARP7 family, which includes the Tetrahymena telomerase-binding protein p65 and human LARP7. We show that Lar7 has conserved RNA-recognition motifs, which bind telomerase RNA to protect it from exosomal degradation. In addition, Lar7 is required to stabilise the association of telomerase RNA with the protective complex LSm2-8, and telomerase reverse transcriptase. Lar7 remains a component of the mature telomerase complex and is required for telomerase localisation to the telomere. Collectively, we demonstrate that Lar7 is a crucial player in fission yeast telomerase biogenesis, similarly to p65 in Tetrahymena, and highlight the LARP7 family as a conserved factor in telomere maintenance