Anchoring of Surface Proteins to the Cell Wall of Staphylococcus aureus: sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH2-Gly3 substrates

Staphylococcus aureus sortase anchors surface proteins to the cell wall envelope by cleaving polypeptides at the LPXTG motif. Surface proteins are linked to the peptidoglycan by an amide bond between the C-terminal carboxyl and the amino group of the pentaglycine cross-bridge. We find that purified recombinant sortase hydrolyzed peptides bearing an LPXTG motif at the peptide bond between threonine and glycine. In the presence of NH2-Gly3, sortase catalyzed exclusively a transpeptidation reaction, linking the carboxyl group of threonine to the amino group of NH2-Gly3. In the presence of amino group donors the rate of sortase mediated cleavage at the LPXTG motif was increased. Hydrolysis and transpeptidation required the sulfhydryl of cysteine 184, suggesting that sortase catalyzed the transpeptidation reaction of surface protein anchoring via the formation of a thioester acyl-enzyme intermediate

Mediating worlds: the role of nurses as ritual specialists in caring for the dead and dying

Rituals are central to the everyday life of the nurse, yet the fundamental roles that rituals play in caring for the dead and dying has often been neglected. This paper explores modern palliative and post-mortem care – its practices, practitioners and arenas – against the background of long-held, global concerns regarding the dead and dying. Comparison with the archaeological and ethnographic records demonstrates©the ubiquitous and enduring practices surrounding death, and the centrality of ritual specialists to this complex social and biological process. This deep-time perspective highlights the importance of nurses, and their associated nursing rituals, in the transition of patients between life and death, and the difficult journeys that nurse, patient and family undertake in this mediation between worlds. Such a perspective not only empowers nurses in their daily practices, and places nursing rituals firmly at the centre of modern palliative care work, but demonstrates the value of archaeology and ethnography in contextualising the challenges of today

Development of a rapid, reliable and quantitative method: "SPOTi" for testing antifungal efficacy

A reference method for the antimicrobial susceptibility testing of common fungal pathogens such as dermatophytes, is currently lacking. In this study, we report the successful adaptation of solid agar-based spot culture growth inhibition assay (SPOTi) for dermatophytes, currently being used as a gold-standard in the anti-tubercular drug discovery field. The fungal-SPOTi assay correlated with the disc-diffusion method, and is validated using mycelial plugs. We propose the fungal-SPOTi as a high-throughput alternative to the disc-diffusion and broth micro-dilution anti-fungal assays to screen novel anti-fungals

Beyond RPE: The Perception of Exercise Under Normal and Ketotic Conditions

AimSubjective perceptions of exercising exertion are integral to maintaining homeostasis. Traditional methods have utilized scores of ‘rating of perceived exertion’ (RPE) to quantify these subjective perceptions, and here we aimed to test whether RPE may encompass identifiable localized perceptions from the lungs (breathlessness) and legs (leg discomfort), as well as their corresponding measures of anxiety. We utilized the intervention of ketoacidosis (via consumption of an exogenous ketone ester drink) to independently perturb exercise-related metabolites and humoral signals, thus allowing us to additionally identify the possible contributing physiological signals to each of these perceptions.MethodsTwelve trained volunteers underwent two incremental bicycle ergometer tests to exhaustion, following ingestion of either an exogenous ketone ester or a taste-matched placebo drink. Cardiorespiratory measures, blood samples and perceived exertion scales were taken throughout. Firstly, two-way repeated-measures ANOVAs were employed to identify the overall effects of ketoacidosis, followed by generalized linear mixed model regression to isolate physiological predictors contributing to each perception.ResultsRating of perceived exertion was found to contain contributions from localized perceptions of breathlessness and leg discomfort, with no measurable effect of ketoacidosis on overall exertion. Leg discomfort, anxiety of breathing and anxiety of leg discomfort were increased during ketoacidosis, and correspondingly contained pH within their prediction models. Anxiety of leg discomfort also encompassed additional humoral signals of blood glucose and ketone concentrations.ConclusionThese results indicate the presence of localized components of RPE in the form of breathlessness and leg discomfort. Furthermore, subjective perceptions of anxiety appear to result from a complex interplay of humoral signals, which may be evolutionarily important when monitoring exertion under times of metabolic stress, such as during starvation

Nutritional Ketoacidosis During Incremental Exercise in Healthy Athletes

Purpose: Ketosis, achieved through ingestion of ketone esters, may influence endurance exercise capacity by altering substrate metabolism. However, the effects of ketone consumption on acid-base status and subsequent metabolic and respiratory compensations are poorly described.Methods: Twelve athletically trained individuals completed an incremental bicycle ergometer exercise test to exhaustion following the consumption of either a ketone ester [(R)-3-hydroxybutyrate-(R)-1,3-butanediol] or a taste-matched control drink (bitter flavoured water) in a blinded, cross-over study. Respiratory gases and arterialised blood gas samples were taken at rest and at regular intervals during exercise.Results: Ketone ester consumption increased blood D-β-hydroxybutyrate concentration from 0.2 to 3.7 mM/L (p < 0.01), causing significant falls versus control in blood pH to 7.37 and bicarbonate to 18.5 mM/L before exercise. To compensate for ketoacidosis, minute ventilation was modestly increased (p < 0.05) with non-linearity in the ventilatory response to exercise (ventilatory threshold) occurring at a 22 W lower workload (p < 0.05). Blood pH and bicarbonate concentrations were the same at maximal exercise intensities. There was no difference in exercise performance having consumed the ketone ester or control drink.Conclusion: Athletes compensated for the greater acid load caused by ketone ester ingestion by elevating minute ventilation and earlier hyperventilation during incremental exercise

Attentional focus and performance anxiety: Effects on simulated race-driving performance and heart rate variability

Previous studies have demonstrated that an external focus can enhance motor learning compared to an internal focus. The benefits of adopting an external focus are attributed to the use of less effortful automatic control processes, while an internal focus relies upon more effort-intensive consciously controlled processes.The aim of this study was to compare the effectiveness of a distal external focus with an internal focus in the acquisition of a simulated driving task and subsequent performance in a competitive condition designed to increase state anxiety.To provide further evidence for the automatic nature of externally controlled movements, the study included heart rate variability (HRV) as an index of mental effort. Sixteen participants completed eight blocks of four laps in either a distal external or internal focus condition, followed by two blocks of four laps in the competitive condition. During acquisition, the performance of both groups improved; however, the distal external focus group outperformed the internal focus group.The poorer performance of the internal focus group was accompanied by a larger reduction in HRV, indicating a greater investment of mental effort. In the competition condition, state anxiety increased, and for both groups, performance improved as a function of the increased anxiety. Increased heart rate and self-reported mental effort accompanied the performance improvement. The distal external focus group also outperformed the internal focus group across both neutral and competitive conditions and this more effective performance was again associated with lower levels of HRV. Overall, the results offer support for the suggestion that an external focus promotes a more automatic mode of functioning. In the competitive condition, both foci enhanced performance and while the improved performance may have been achieved at the expense of greater compensatory mental effort, this was not reflected in HRV scores. © 2012 Mullen, Faull, Jones and Kingston

Treating breathlessness via the brain: changes in brain activity over a course of pulmonary rehabilitation

Breathlessness in chronic obstructive pulmonary disease (COPD) is often discordant with airway pathophysiology ("over-perception"). Pulmonary rehabilitation profoundly affects breathlessness, without influencing lung function. Learned associations influence brain mechanisms of sensory perception. We hypothesised that improvements in breathlessness with pulmonary rehabilitation may be explained by changing neural representations of learned associations.In 31 patients with COPD, we tested how pulmonary rehabilitation altered the relationship between brain activity during a breathlessness-related word-cue task (using functional magnetic resonance imaging), and clinical and psychological measures of breathlessness.Changes in ratings of breathlessness word cues positively correlated with changes in activity in the insula and anterior cingulate cortex. Changes in ratings of breathlessness-anxiety negatively correlated with activations in attention regulation and motor networks. Baseline activity in the insula, anterior cingulate cortex and prefrontal cortex correlated with improvements in breathlessness and breathlessness-anxiety.Pulmonary rehabilitation is associated with altered neural responses related to learned breathlessness associations, which can ultimately influence breathlessness perception. These findings highlight the importance of targeting learned associations within treatments for COPD, demonstrating how neuroimaging may contribute to patient stratification and more successful personalised therapy

Tracking extra-celullar enzyme production by Trichoderma harzianum in the rhizosphere.

The exact role of extracellular enzyme production in the activities of Trichoderma harzianum as a biological control agent are much debated. Production of such enzymes as cellulase, proteinase, endoglucanase, (glucosidase and chitinase has been linked to biological control in vitro experiments (Elad et al, 1982, Ahmad & Baker, 1988 a,b) bur it has been difficult to prove that these enzymes are important in vivo. Baker (1991) demonstrated that mutant strains of Trichoderma with improved rhizosphere competence had higher extracellular cellulase production than wild types strains, and he speculated that enhanced cellulase production might lead to better utilisation of rhizosphere substrates and thus improved colonisation of the resource. However, it has been recently shown that rhizosphere competence of Trichoderma strains is not always associated with improved in vitro production of extracellular enzymes (de Melo, Faull, J, L, and Graeme-Cook, 1997). In order to extend this work to the soil environment we have developed a sensitive, quantifiable assay for extracellular enzymes that uses a fluorescent substrate to detect enzyme activity in soil. Using a number of different strains of Trichoderma with known in vitro extra-cellular enzyme production patterns (both high and low) we will demonstrate differences in the production of enzymes in the rhizosphere with varying soil depth. We will attempt to relate these differences to the root colonisation ability of the Trichoderma strain

Integrin activation - the importance of a positive feedback

Integrins mediate cell adhesion and are essential receptors for the development and functioning of multicellular organisms. Integrin activation is known to require both ligand and talin binding and to correlate with cluster formation but the activation mechanism and precise roles of these processes are not yet resolved. Here mathematical modeling, with known experimental parameters, is used to show that the binding of a stabilizing factor, such as talin, is alone insufficient to enable ligand-dependent integrin activation for all observed conditions; an additional positive feedback is required.Comment: in press in Bulletin of Mathematical Biolog