SUMMARY Evidence for active DNA demethylation in vertebrates is accumulating, but the mechanisms and enzymes remain unclear. Using zebrafish embryos we provide evidence for 5-methylcytosine (5-meC) removal in vivo via the coupling of a 5-meC deaminase (AID, which converts 5-meC to thymine) and a G:T mismatch-specific thymine glycosylase (Mbd4). The injection of methylated DNA into embryos induced a potent DNA demethylation activity, which was attenuated by depletion of AID or the non enzymatic factor Gadd45. Remarkably, overexpression of the deaminase/glycosylase pair AID/Mbd4 in vivo caused demethylation of the bulk genome and injected methylated DNA fragments, likely involving a G:T intermediate. Furthermore, AID or Mbd4 knockdown caused the remethylation of a set of common genes. Finally, Gadd45 promoted demethylation and enhanced functional interactions between deaminase/glycosylase pairs. Our results provide evidence for a coupled mechanism of 5-meC demethylation, whereby AID deaminates 5-meC, followed by thymine base excision by Mbd4, promoted by Gadd45.

Exercise studies have suggested that the presence of carbohydrate in the human mouth activates regions of the brain that can enhance exercise performance but direct evidence of such a mechanism is limited. The first aim of the present study was to observe how rinsing the mouth with solutions containing glucose and maltodextrin, disguised with artificial sweetener, would affect exercise performance. The second aim was to use functional magnetic resonance imaging (f MRI) to identify the brain regions activated by these substances. In Study 1A, eight endurance-trained cyclists (V O 2 max 60.8 ± 4.1 ml kg −1 min −1 ) completed a cycle time trial (total work = 914 ± 29 kJ) significantly faster when rinsing their mouths with a 6.4% glucose solution compared with a placebo containing saccharin (60.4 ± 3.7 and 61.6 ± 3.8 min, respectively, P = 0.007). The corresponding f MRI study (Study 1B) revealed that oral exposure to glucose activated reward-related brain regions, including the anterior cingulate cortex and striatum, which were unresponsive to saccharin. In Study 2A, eight endurance-trained cyclists (V O 2 max 57.8 ± 3.2 ml kg −1 min −1 ) tested the effect of rinsing with a 6.4% maltodextrin solution on exercise performance, showing it to significantly reduce the time to complete the cycle time trial (total work = 837 ± 68 kJ) compared to an artificially sweetened placebo (62.6 ± 4.7 and 64.6 ± 4.9 min, respectively, P = 0.012). The second neuroimaging study (Study 2B) compared the cortical response to oral maltodextrin and glucose, revealing a similar pattern of brain activation in response to the two carbohydrate solutions, including areas of the insula/frontal operculum, orbitofrontal cortex and striatum. The results suggest that the improvement in exercise performance that is observed when carbohydrate is present in the mouth may be due to the activation of brain regions believed to be involved in reward and motor control. The findings also suggest that there may be a class of so far unidentified oral receptors that respond to carbohydrate independently of those for sweetness.

1. After severe muscular contraction in man recovery of force is largely complete in a few minutes, but is not wholly so for many hours. The long-lasting element of fatigue is found to occur primarily for low frequencies of stimulation (e.g. 20/sec), and is much less pronounced, or absent, at high frequencies (80/sec). The twitch force is an unreliable measure of the state of fatigue. 2. The long-lasting element of fatigue is not due to depletion of high-energy phosphate nor is it due to failure of electrical activity as recorded from surface electrodes. It is probably the result of an impairment of the process of excitation-contraction coupling. Its practical importance for man could be significant as an explanation of the subjective feelings of weakness following exercise.

Tomato Cf genes confer resistance to C. fulvum, reside in complex loci carrying multiple genes, and encode predicted membrane-bound proteins with extracytoplasmic leucine-rich repeats. At least two Cf-9 homologs confer novel C. fulvum resistance specificities. Comparison of 11 genes revealed 7 hypervariable amino acid positions in a motif of the leucine-rich repeats predicted to form a beta-strand/beta-turn in which the hypervariable residues are solvent exposed and potentially contribute to recognition specificity. Higher nonsynonymous than synonymous substitution rates in this region imply selection for sequence diversification. We propose that the level of polymorphism between intergenic regions determines the frequency of sequence exchange between the tandemly repeated genes. This permits sufficient exchange to generate sequence diversity but prevents sequence homogenization.

The tomato Cf-9 gene confers resistance to infection by races of the fungus Cladosporium fulvum that carry the avirulence gene Avr9. The Cf-9 gene was isolated by transposon tagging with the maize transposable element Dissociation. The DNA sequence of Cf-9 encodes a putative membrane-anchored extracytoplasmic glycoprotein. The predicted protein shows homology to the receptor domain of several receptor-like protein kinases in Arabidopsis, to antifungal polygalacturonase-inhibiting proteins in plants, and to other members of the leucine-rich repeat family of proteins. This structure is consistent with that of a receptor that could bind Avr9 peptide and activate plant defense.

Passive stretching is commonly used to increase limb range of movement prior to athletic performance but it is unclear which component of the muscle-tendon unit (MTU) is affected by this procedure. Movement of the myotendinous junction (MTJ) of the gastrocnemius medialis muscle was measured by ultrasonography in eight male participants (20.5 ± 0.9 years) during a standard stretch in which the ankle was passively dorsiflexed at 1 deg s −1 from 0 deg (the foot at right angles to the tibia) to the participants' volitional end range of motion (ROM). Passive torque, muscle fascicle length and pennation angle were also measured. Standard stretch measurements were made before (pre-) and after (post-) five passive conditioning stretches. During each conditioning stretch the MTU was taken to the end ROM and held for 1 min. Pre-conditioning the extension of the MTU during stretch was taken up almost equally by muscle and tendon. Following conditioning, ROM increased by 4.6 ± 1.5 deg (17%) and the passive stiffness of the MTU was reduced (between 20 and 25 deg) by 47% from 16.0 ± 3.6 to 10.2 ± 2.0 Nm deg −1 . Distal MTJ displacement (between 0 and 25 deg) increased from 0.92 ± 0.06 to 1.16 ± 0.05 cm, accounting for all the additional MTU elongation and indicating that there was no change in tendon properties. Muscle extension pre-conditioning was explicable by change in length and pennation angle of the fascicles but post-conditioning this was not the case suggesting that at least part of the change in muscle with conditioning stretches was due to altered properties of connective tissue.

In plants, resistance to pathogens is frequently determined by dominant resistance genes, whose products are proposed to recognize pathogen-encoded avirulence gene (Avr) products. The tomato resistance locus Cf-2 was isolated by positional cloning and found to contain two almost identical genes, each conferring resistance to isolates of tomato leaf mould (C. fulvum) expressing the corresponding Avr2 gene. The two Cf-2 genes encode protein products that differ from each other by only three amino acids and contain 38 leucine-rich repeat (LRR) motifs. Of the LRRs, 20 show extremely conserved alternating repeats. The C-terminus of Cf-2 carries regions of pronounced homology to the protein encoded by the unlinked Cf-9 gene. We suggest that this conserved region interacts with other proteins involved in activating plant defense mechanisms.

27 The present study aimed to investigate the influence of timing of pre-exercise carbohydrate 28 feeding (Part A), and carbohydrate concentration (Part B), on short-duration high-intensity 29 exercise capacity. In Part A, seventeen males, and in Part B ten males, performed a peak 30 power output (PPO) test, two familiarisation trials at 90% of PPO, and 4 (for Part A) or 3 (for 31 Part B) experimental trials involving exercise capacity tests at 90% PPO. In Part A, the 4 trials 32 were conducted following ingestion of a 6.4% carbohydrate/electrolyte sports drink ingested 30 33 (C30) or 120 (C120) minutes before exercise, or a flavour-matched placebo administered either 34 30 (P30) or 120 (P120) minutes before exercise. In Part B, the 3 trials were performed 30 35 minutes after ingestion of 0%, 2% or 12% carbohydrate solutions. All trials were performed in a 36 double blind cross-over design following and overnight fast. Dietary intake and activity in the two 37 days before trials was recorded and replicated on each visit. Glucose, lactate, heart rate and 38 mood/arousal were recorded at intervals during the trials. In Part A, C30 produced the greatest 39 exercise capacity (mean±SD; 9.0±1.9 min, P<0.01) compared with all other trials (7.7±1.5 min 40 P30, 8.0±1.7 min P120, 7.9±1.9 min C120). In Part B, exercise capacity (min) following 41 ingestion of the 2% solution (9.2±2.1) compared with 0% (8.2±0.7) and 12% (8.0±1.3) solutions 42 approached significance (p=0.09). This study provides new evidence to suggest that timing of 43 carbohydrate intake is important in short duration high-intensity exercise tasks, but a 44 concentration effect requires further exploration. 45 46 47 48 The majority of studies examining the effects of carbohydrate feeding on exercise performance 49 and exercise capacity have focused on carbohydrate ingestion during prolonged exercise, or on 50 pre-exercise carbohydrate feeding in the few hours or minutes before prolonged endurance 51 activities (for reviews see Cermak & Van Loon, 2013; Temesi et al., 2011; Karelis et al., 2010; 52 Jeukendrup & Killer, 2010). There has been limited focus on carbohydrate feeding prior to short 53 duration (<10 min), high-intensity (>85% max), exercise tasks, presumably because it is 54 acknowledged that muscle glycogen depletion will not be limiting during exercise of this nature. 55 As a result, guidelines for pre-event fuelling focus on providing information about carbohydrate 56 intake before endurance exercise tasks lasting longer than 60 minutes (Burke et al., 2011). 57 Current guidelines specify that there is no requirement for ingestion of carbohydrate before 58 events lasting less than 45 minutes. Furthermore, it is recognized that ingestion of carbohydrate 59 in the immediate pre-exercise period (30-60 minutes before exercise) can reduce liver glucose 60 output, stimulate glucose uptake and oxidation and induce a rebound hypoglycaemia in 61 susceptible individuals (Williams and Lamb, 2008; Jeukendrup & Killer, 2010). Interestingly, 62 these known me...