Time perception, pacing and exercise intensity: maximal exercise distorts the perception of time

Introduction Currently there are no data examining the impact of exercise on the perception of time, which is surprising as optimal competitive performance is dependent on accurate pacing using knowledge of time elapsed. Methods With institutional ethics approval, 12 recreationally active adult participants (f = 7, m = 5) undertook both 30 s Wingate cycles and 20 min (1200 s) rowing ergometer bouts as short and long duration self-paced exercise trials, in each of three conditions on separate occasions: 1) light exertion: RPE 11, 2) heavy exertion: RPE 15, 3) maximal exertion: RPE 20. Participants were unaware of exercise duration and were required to verbally indicate when they perceived (subjective time) 1) 25%, 2) 50%, 3) 75% and 4) 100% of each bout's measured (chronological) time had elapsed. Results In response to the Wingate task, there was no difference between durations of subjective time at the 25%, nor at the 50% interval. However, at the 75% and 100% intervals, the estimate for the RPE 20 condition was shortest (P < 0.01). In response to rowing, there were no differences at the 25% interval, but there was some evidence that the RPE 20 condition was perceived shorter at 50%. At 75% and 100%, the RPE 20 condition was perceived to be shorter than both RPE 15 (P = 0.04) and RPE 11 (P = 0.008) conditions. Conclusion This study is the first to empirically demonstrate that exercise intensity distorts time perception, particularly during maximal exercise. Consequently external feedback of chronological time may be an important factor for athletes undertaking maximal effort tasks or competitions

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37°C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 μl tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy

Selection and Validation of Reference Genes for miRNA Expression Studies during Porcine Pregnancy

MicroRNAs comprise a family of small non-coding RNAs that modulate several developmental and physiological processes including pregnancy. Their ubiquitous presence is confirmed in mammals, worms, flies and plants. Although rapid advances have been made in microRNA research, information on stable reference genes for validation of microRNA expression is still lacking. Real time PCR is a widely used tool to quantify gene transcripts. An appropriate reference gene must be chosen to minimize experimental error in this system. A small difference in miRNA levels between experimental samples can be biologically meaningful as these entities can affect multiple targets in a pathway. This study examined the suitability of six commercially available reference genes (RNU1A, RNU5A, RNU6B, SNORD25, SCARNA17, and SNORA73A) in maternal-fetal tissues from healthy and spontaneously arresting/dying conceptuses from sows were separately analyzed at gestation day 20. Comparisons were also made with non-pregnant endometrial tissues from sows. Spontaneous fetal loss is a prime concern to the commercial pork industry. Our laboratory has previously identified deficits in vasculature development at maternal-fetal interface as one of the major participating causes of fetal loss. Using this well-established model, we have extended our studies to identify suitable microRNA reference genes. A methodical approach to assessing suitability was adopted using standard curve and melting curve analysis, PCR product sequencing, real time PCR expression in a panel of gestational tissues, and geNorm and NormFinder analysis. Our quantitative real time PCR analysis confirmed expression of all 6 reference genes in maternal and fetal tissues. All genes were uniformly expressed in tissues from healthy and spontaneously arresting conceptus attachment sites. Comparisons between tissue types (maternal/fetal/non-pregnant) revealed significant differences for RNU5A, RNU6B, SCARNA17, and SNORA73A expression. Based on our methodical assessment of all 6 reference genes, results suggest that RNU1A is the most stable reference gene for porcine pregnancy studies

Nerve Agent Hydrolysis Activity Designed into a Human Drug Metabolism Enzyme

Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning

Morphologic and functional correlates of synaptic pathology in the cathepsin D knockout mouse model of congenital neuronal ceroid lipofuscinosis

Mutations in the cathepsin D (CTSD) gene cause an aggressive neurodegenerative disease (congenital neuronal ceroid lipofuscinosis) that leads to early death. Recent evidence suggests that presynaptic abnormalities play a major role in the pathogenesis of CTSD deficiencies. To identify the early events that lead to synaptic alterations, we investigated synaptic ultrastructure and function in pre-symptomatic CTSD knock-out (Ctsd(−/−)) mice. Electron microscopy revealed that there were significantly greater numbers of readily releasable synaptic vesicles present in Ctsd(−/−) mice than in wild-type control mice as early as postnatal day 16. The size of this synaptic vesicle pool continued to increase with disease progression in the hippocampus and thalamus of the Ctsd(−/−) mice. Electrophysiology revealed a markedly decreased frequency of miniature excitatory postsynaptic currents (EPSCs) with no effect on pair-pulse modulation of the evoked EPSPs in the hippocampus of Ctsd(−/−) mice. The reduced miniature EPSC frequency was observed before the appearance of epilepsy or any morphological sign of synaptic degeneration. Taken together, the data indicate that CTSD is required for normal synaptic function, and that a failure in synaptic trafficking or recycling may be an early and important pathological mechanism in Ctsd(−/−) mice; these presynaptic abnormalities may initiate synaptic degeneration in advance of subsequent neuronal loss

Potential novel habitat created by holdfasts from cultivated Laminaria digitata : assessing the macroinvertebrate assemblages

A.M.W. is currently funded by the Dr. Tony Ryan Research Trust, NUI Galway.Interest in the cultivation of native kelp species is increasing within Europe. Observations of seaweed farms suggest that they may act as a habitat for associated species, potentially altering the richness of the local area. Previous studies have generally focused on species associated with wild kelps, showing the holdfast to be relatively species-rich. Little research has, however, been conducted on the species associated with cultivated kelps. The habitat created by cultivated kelp holdfasts may act as a novel habitat and not simply an expansion of existing kelp habitat, due to differences in holdfast age, holdfast morphology and holdfast position in the water column. Laminaria digitata from the west of Ireland were examined to test if these differences result in the fauna of cultivated (suspended) holdfasts being distinct from wild (benthic) stands. To place the results in a broader context, patterns were compared to holdfast-richness relationships observed in comparable studies from the NE Atlantic. Total abundance of holdfast epifauna was similar across benthic and suspended holdfasts from the west of Ireland, although species richness was higher in suspended samples. Richness and abundance in suspended kelp holdfasts were consistent with the range of values recorded in other wild kelp studies. There were significant differences in assemblage composition between holdfast types (ANOSIM; R = 0.383, p <0.05). The distributions of faunal feeding types did not, however, vary between suspended and benthic kelps. Suspended holdfasts in the west of Ireland represented a novel habitat with higher species richness and a different species assemblage when compared to adjacent benthic kelps.Publisher PDFPeer reviewe