Exogenous polyunsaturated fatty acids (PUFAs) influence permeability, antimicrobial peptide resistance, biofilm formation and membrane phospholipid structure in an A-layer and non-A-layer strain of Aeromonas salmonicida.

Aeromonas salmonicida is a Gram-negative bacterium that can infect a wide host range of fish populations, including salmonids and non-salmonids as well as freshwater and marine life. Some strains of A. salmonicida cause the disease furunculosis, which can cause lethargy, intestinal inflammation, ulcers, haemorrhaging and death. The infection is spread through fish-to-fish contact, and the presence of infection can have devastating effects on cultivated fish populations. The purpose of this study was to explore the ability of non-A-layer and A-layer A. salmonicida strains to incorporate polyunsaturated fatty acids (PUFAs) into their lipid profile and test the phenotypic effects thereof. Lipids were extracted from PUFA-exposed cultures and analysed for lipid modification by thin-layer chromatography and ultraperformance liquid chromatography-mass spectrometry, showing A. salmonicida, regardless of A-layer, capable of incorporating all seven of the PUFAs studied. Phenotypic effects were determined through the use of assays that tested for biofilm formation, membrane permeability and cyclic peptide susceptibility. Temperature-dependent effects on biofilm formation were observed, and PUFA exposure showed significant (p < .001) increases in membrane permeability as tested by the uptake of the hydrophobic compounds crystal violet and ethidium bromide. Additionally, some PUFAs elicited modest protection and vulnerability against the membrane-targeting cyclic peptides polymyxin B (PMB) and colistin. The diverse, strain-specific responses to exogenous PUFAs may allude to evolved adaptive strategies that enhance survival, persistence and virulence of non-pathogenic and pathogenic members of bacteria that oscillate between environmental and fish host niches.

Reliability and Validity of a Method for the Assessment of Sport Rock Climbers' Isometric Finger Strength.

ABSTRACT: Torr, O, Randall, T, Knowles, R, Giles, D, and Atkins, S. The reliability and validity of a method for the assessment of sport rock climbers' isometric finger strength. J Strength Cond Res 36(8): 2277-2282, 2022-Isometric strength of the finger flexors is considered to be one of the main physical determinants of sport rock climbing performance. We set out to determine the test-retest reliability and criterion validity of a low resource maximal isometric finger strength (MIFS) testing protocol that uses a pulley system to add or remove weight to/from a climber's body. To determine test-retest reliability, 15 subjects' MIFS was assessed on 2 occasions, separated by a minimum of 48 hours. Body mass and maximum load were recorded on both occasions. Intra-class correlation coefficients (ICCs) between visits for all variables were very good (ICC > 0.91), with small bias and effect sizes-particularly when expressed as a percentage of body mass (ICC = 0.98, 95% confidence interval 0.93-0.99). To determine the criterion validity of MIFS and climbing ability, data of 229 intermediate to higher elite climbers were compared. Pearson's product moment correlations demonstrated good agreement, again particularly between total load when expressed as a percentage of body mass and climbing performance ( r = 0.421-0.503). The results illustrate the sensitivity of a simple test for the determination of MIFS in intermediate to height elite climbers from an ecologically valid, climbing specific test that only requires equipment found at most climbing walls. This low resource test protocol for the assessment of isometric finger strength has wide-reaching utility, for instance when assessing strength before and after a training intervention or when prescribing load intensities for exercises aimed at improving maximal finger strength.

Isolated finger flexor vs. exhaustive whole-body climbing tests? How to assess endurance in sport climbers?

PURPOSE: Sport climbing requires high-intensity finger flexor contractions, along with a substantial whole-body systemic oxygen uptake ([Formula: see text]O2) contribution. Although fatigue is often localised to the finger flexors, the role of systemic ̇[Formula: see text]O2 and local aerobic mechanisms in climbing performance remains unclear. As such, the primary purpose of this study was to determine systemic and local muscle oxygen responses during both isolated finger flexion and incremental exhaustive whole-body climbing tests. The secondary aim was to determine the relationship of isolated and whole-body climbing endurance tests to climbing ability. METHODS: Twenty-two male sport climbers completed a series of isometric sustained and intermittent forearm flexor contractions, and an exhaustive climbing test with progressive steepening of the wall angle on a motorised climbing ergometer. Systemic [Formula: see text]O2 and flexor digitorum profundus oxygen saturation (StO2) were recorded using portable metabolic analyser and near-infra red spectroscopy, respectively. RESULTS: Muscle oxygenation breakpoint (MOB) was identifiable during an incremental exhaustive climbing test with progressive increases in angle (82 ± 8% and 88 ± 8% [Formula: see text]O2 and heart rate climbing peak). The peak angle from whole-body treadwall test and impulse from isolated hangboard endurance tests were interrelated (R2 = 0.58-0.64). Peak climbing angle together with mean [Formula: see text]O2 and StO2 from submaximal climbing explained 83% of variance in self-reported climbing ability. CONCLUSIONS: Both systemic and muscle oxygen kinetics determine climbing-specific endurance. Exhaustive climbing and isolated finger flexion endurance tests are interrelated and suitable to assess climbing-specific endurance. An exhaustive climbing test with progressive wall angle allows determination of the MOB.

Anthropometry and performance characteristics of recreational advanced to elite female rock climbers.

Despite climbing's popularity and an increasing number of female participants, there are limited anthropometric and performance data for this population. This study compares the characteristics of 55 experienced female climbers, divided into three categories (lower [ADV-L] and higher advanced [ADV-H] and elite [ELT]) based on self-reported ability. Data on climbing experience, body dimensions, body composition, flexibility, lower and upper-body power and finger strength were assessed. ELT climbers differed significantly from the ADV groups in age (Mean Difference [MD] = 8.8-9.8 yrs; despite smaller differences in years climbing MD = 1.6-2.4 yrs), greater climbing and hours training per week (MD = 3.0-3.7 h & MD = 0.9-1.6 h, respectively), and greater upper-body power (MD = 12.9-16.6 cm) and finger strength (MD = 51.6-65.4 N). Linear regression analysis showed finger strength and upper body power to be associated with ability, particularly when adjusting for descriptive and anthropometric variables (finger strength R2 = 53% and 45%; upper-body power R2 = 60% and 39% for boulder and sport, respectively). The findings support the importance of finger strength and upper-body power; changes in female anthropometric data over the last decade provide insight into the changing nature of the sport.

Influence of Artefact Correction and Recording Device Type on the Practical Application of a Non-Linear Heart Rate Variability Biomarker for Aerobic Threshold Determination.

Recent study points to the value of a non-linear heart rate variability (HRV) biomarker using detrended fluctuation analysis (DFA a1) for aerobic threshold determination (HRVT). Significance of recording artefact, correction methods and device bias on DFA a1 during exercise and HRVT is unclear. Gas exchange and HRV data were obtained from 17 participants during an incremental treadmill run using both ECG and Polar H7 as recording devices. First, artefacts were randomly placed in the ECG time series to equal 1, 3 and 6% missed beats with correction by Kubios software's automatic and medium threshold method. Based on linear regression, Bland Altman analysis and Wilcoxon paired testing, there was bias present with increasing artefact quantity. Regardless of artefact correction method, 1 to 3% missed beat artefact introduced small but discernible bias in raw DFA a1 measurements. At 6% artefact using medium correction, proportional bias was found (maximum 19%). Despite this bias, the mean HRVT determination was within 1 bpm across all artefact levels and correction modalities. Second, the HRVT ascertained from synchronous ECG vs. Polar H7 recordings did show an average bias of minus 4 bpm. Polar H7 results suggest that device related bias is possible but in the reverse direction as artefact related bias.

Exogenous polyunsaturated fatty acids (PUFAs) promote changes in growth, phospholipid composition, membrane permeability and virulence phenotypes in Escherichia coli.

BACKGROUND: The utilization of exogenous fatty acids by Gram-negative bacteria has been linked to many cellular processes, including fatty acid oxidation for metabolic gain, assimilation into membrane phospholipids, and control of phenotypes associated with virulence. The expanded fatty acid handling capabilities have been demonstrated in several bacteria of medical importance; however, a survey of the polyunsaturated fatty acid responses in the model organism Escherichia coli has not been performed. The current study examined the impacts of exogenous fatty acids on E. coli. RESULTS: All PUFAs elicited higher overall growth, with several fatty acids supporting growth as sole carbon sources. Most PUFAs were incorporated into membrane phospholipids as determined by Ultra performance liquid chromatography-mass spectrometry, whereas membrane permeability was variably affected as measured by two separate dye uptake assays. Biofilm formation, swimming motility and antimicrobial peptide resistance were altered in the presence of PUFAs, with arachidonic and docosahexaenoic acids eliciting strong alteration to these phenotypes. CONCLUSIONS: The findings herein add E. coli to the growing list of Gram-negative bacteria with broader capabilities for utilizing and responding to exogenous fatty acids. Understanding bacterial responses to PUFAs may lead to microbial behavioral control regimens for disease prevention.

Myeloid cell plasticity in the evolution of central nervous system autoimmunity.

OBJECTIVE: Myeloid cells, including macrophages and dendritic cells, are a prominent component of central nervous system (CNS) infiltrates during multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE). Although myeloid cells are generally thought to be proinflammatory, alternatively polarized subsets can serve noninflammatory and/or reparative functions. Here we investigate the heterogeneity and biological properties of myeloid cells during central nervous system autoimmunity. METHODS: Myeloid cell phenotypes in chronic active MS lesions were analyzed by immunohistochemistry. In addition, immune cells were isolated from the CNS during exacerbations and remissions of EAE and characterized by flow cytometric, genetic, and functional assays. RESULTS: Myeloid cells expressing inducible nitric oxide synthase (iNOS), indicative of a proinflammatory phenotype, were detected in the actively demyelinating rim of chronic active MS lesions, whereas macrophages expressing mannose receptor (CD206), a marker of alternatively polarized human myeloid cells, were enriched in the quiescent lesion core. During EAE, CNS-infiltrating myeloid cells, as well as microglia, shifted from expression of proinflammatory markers to expression of noninflammatory markers immediately prior to clinical remissions. Murine CNS myeloid cells expressing the alternative lineage marker arginase-1 (Arg1) were partially derived from iNOS+ precursors and were deficient in activating encephalitogenic T cells compared with their Arg1- counterparts. INTERPRETATION: These observations demonstrate the heterogeneity of CNS myeloid cells, their evolution during the course of autoimmune demyelinating disease, and their plasticity on the single cell level. Future therapeutic strategies for disease modification in individuals with MS may be focused on accelerating the transition of CNS myeloid cells from a proinflammatory to a noninflammatory phenotype. Ann Neurol 2018;83:131-141.

Signal Integration at Elongation Factor 2 Kinase: THE ROLES OF CALCIUM, CALMODULIN, AND SER-500 PHOSPHORYLATION.

Eukaryotic elongation factor 2 kinase (eEF-2K), the only calmodulin (CaM)-dependent member of the unique α-kinase family, impedes protein synthesis by phosphorylating eEF-2. We recently identified Thr-348 and Ser-500 as two key autophosphorylation sites within eEF-2K that regulate its activity. eEF-2K is regulated by Ca2+ ions and multiple upstream signaling pathways, but how it integrates these signals into a coherent output, i.e. phosphorylation of eEF-2, is unclear. This study focuses on understanding how the post-translational phosphorylation of Ser-500 integrates with Ca2+ and CaM to regulate eEF-2K. CaM is shown to be absolutely necessary for efficient activity of eEF-2K, and Ca2+ is shown to enhance the affinity of CaM toward eEF-2K. Ser-500 is found to undergo autophosphorylation in cells treated with ionomycin and is likely also targeted by PKA. In vitro, autophosphorylation of Ser-500 is found to require Ca2+ and CaM and is inhibited by mutations that compromise binding of phosphorylated Thr-348 to an allosteric binding pocket on the kinase domain. A phosphomimetic Ser-500 to aspartic acid mutation (eEF-2K S500D) enhances the rate of activation (Thr-348 autophosphorylation) by 6-fold and lowers the EC50 for Ca2+/CaM binding to activated eEF-2K (Thr-348 phosphorylated) by 20-fold. This is predicted to result in an elevation of the cellular fraction of active eEF-2K. In support of this mechanism, eEF-2K knock-out MCF10A cells reconstituted with eEF-2K S500D display relatively high levels of phospho-eEF-2 under basal conditions. This study reports how phosphorylation of a regulatory site (Ser-500) integrates with Ca2+ and CaM to influence eEF-2K activity.

An emerging role for eotaxins in neurodegenerative disease.

Eotaxins are C-C motif chemokines first identified as potent eosinophil chemoattractants. They facilitate eosinophil recruitment to sites of inflammation in response to parasitic infections as well as allergic and autoimmune diseases such as asthma, atopic dermatitis, and inflammatory bowel disease. The eotaxin family currently includes three members: eotaxin-1 (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26). Despite having only ~30% sequence homology to one another, each was identified based on its ability to bind the chemokine receptor, CCR3. Beyond their role in innate immunity, recent studies have shown that CCL11 and related molecules may directly contribute to degenerative processes in the central nervous system (CNS). CCL11 levels increase in the plasma and cerebrospinal fluid of both mice and humans as part of normal aging. In mice, these increases are associated with declining neurogenesis and impaired cognition and memory. In humans, elevated plasma levels of CCL11 have been observed in Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, and secondary progressive multiple sclerosis when compared to age-matched, healthy controls. Since CCL11 is capable of crossing the blood-brain barrier of normal mice, it is plausible that eotaxins generated in the periphery may exert physiological and pathological actions in the CNS. Here, we briefly review known functions of eotaxin family members during innate immunity, and then focus on whether and how these molecules might participate in the progression of neurodegenerative diseases.

An IFNγ/CXCL2 regulatory pathway determines lesion localization during EAE.

BACKGROUND: Myelin oligodendrocyte glycoprotein (MOG)-reactive T-helper (Th)1 cells induce conventional experimental autoimmune encephalomyelitis (cEAE), characterized by ascending paralysis and monocyte-predominant spinal cord infiltrates, in C57BL/6 wildtype (WT) hosts. The same T cells induce an atypical form of EAE (aEAE), characterized by ataxia and neutrophil-predominant brainstem infiltrates, in syngeneic IFNγ receptor (IFNγR)-deficient hosts. Production of ELR+ CXC chemokines within the CNS is required for the development of aEAE, but not cEAE. The cellular source(s) and localization of ELR+ CXC chemokines in the CNS and the IFNγ-dependent pathways that regulate their production remain to be elucidated. METHODS: The spatial distribution of inflammatory lesions and CNS expression of the ELR+ CXC chemokines, CXCL1 and CXCL2, were determined via immunohistochemistry and/or in situ hybridization. Levels of CXCL1 and CXCL2, and their cognate receptor CXCR2, were measured in/on leukocyte subsets by flow cytometric and quantitative PCR (qPCR) analysis. Bone marrow neutrophils and macrophages were cultured with inflammatory stimuli in vitro prior to measurement of CXCL2 and CXCR2 by qPCR or flow cytometry. RESULTS: CNS-infiltrating neutrophils and monocytes, and resident microglia, are a prominent source of CXCL2 in the brainstem of IFNγRKO adoptive transfer recipients during aEAE. In WT transfer recipients, IFNγ directly suppresses CXCL2 transcription in microglia and myeloid cells, and CXCR2 transcription in CNS-infiltrating neutrophils. Consequently, infiltration of the brainstem parenchyma from the adjacent meninges is blocked during cEAE. CXCL2 directly stimulates its own expression in cultured neutrophils, which is enhanced by IL-1 and suppressed by IFNγ. CONCLUSIONS: We provide evidence for an IFNγ-regulated CXCR2/CXCL2 autocrine/paracrine feedback loop in innate immune cells that determines the location of CNS infiltrates during Th1-mediated EAE. When IFNγ signaling is impaired, myeloid cell production of CXCL2 increases, which promotes brainstem inflammation and results in clinical ataxia. IFNγ, produced within the CNS of WT recipients, suppresses myeloid cell CXCR2 and CXCL2 production, thereby skewing the location of neuroinflammatory infiltrates to the spinal cord and the clinical phenotype to an ascending paralysis. These data reveal a novel mechanism by which IFNγ and CXCL2 interact to direct regional recruitment of leukocytes in the CNS, resulting in distinct clinical presentations.

Pseudomonas aeruginosa responds to exogenous polyunsaturated fatty acids (PUFAs) by modifying phospholipid composition, membrane permeability, and phenotypes associated with virulence.

BACKGROUND: Pseudomonas aeruginosa, a common opportunistic pathogen, is known to cause infections in a variety of compromised human tissues. An emerging mechanism for microbial survival is the incorporation of exogenous fatty acids to alter the cell's membrane phospholipid profile. With these findings, we show that exogenous fatty acid exposure leads to changes in bacterial membrane phospholipid structure, membrane permeability, virulence phenotypes and consequent stress responses that may influence survival and persistence of Pseudomonas aeruginosa. RESULTS: Thin-layer chromatography and ultra performance liquid chromatography / ESI-mass spectrometry indicated alteration of bacterial phospholipid profiles following growth in the presence of polyunsaturated fatty acids (PUFAs) (ranging in carbon length and unsaturation). The exogenously supplied fatty acids were incorporated into the major bacterial phospholipids phosphatidylethanolamine and phosphatidylglycerol. The incorporation of fatty acids increased membrane permeability as judged by both accumulation and exclusion of ethidium bromide. Individual fatty acids were identified as modifying resistance to the cyclic peptide antibiotics polymyxin B and colistin, but not the beta-lactam imipenem. Biofilm formation was increased by several PUFAs and significant fluctuations in swimming motility were observed. CONCLUSIONS: Our results emphasize the relevance and complexity of exogenous fatty acids in the membrane physiology and pathobiology of a medically important pathogen. P. aeruginosa exhibits versatility with regard to utilization of and response to exogenous fatty acids, perhaps revealing potential strategies for prevention and control of infection.

Heart Rate Variability During Exercise: A Comparison of Artefact Correction Methods.

Giles, DA and Draper, N. Heart rate variability during exercise: a comparison of artefact correction methods. J Strength Cond Res 32(3): 726-735, 2018-There is a need for standard practice in the collection and processing of RR interval data recorded using heart rate monitors (HRMs) in research. This article assessed the validity of RR intervals and heart rate variability (HRV) data obtained using an HRM during incremental exercise and artefact correction methods. Eighteen participants completed an active orthostatic test and incremental running V[Combining Dot Above]O2max test, while simultaneous recordings using a Polar V800 HRM and an electrocardiogram were made. Artefacts were corrected by deletion; degree zero, linear, cubic, and spline interpolation; and Kubios HRV software. Agreement was assessed using percentage bias, effect size (ES), intraclass correlation coefficients (ICC), and Bland-Altman limits of agreement (LoA). Artefacts increased relative to exercise intensity, to a peak of 4.46% during 80-100% V[Combining Dot Above]O2max. Correction of RR intervals was necessary with unacceptably increased bias, LoA, and ES and reduced ICC in all but resting recordings. All correction methods resulted in data with reduced percentage bias and ES for resting and <60% V[Combining Dot Above]O2max recordings. However, at >60% V[Combining Dot Above]O2max, even with correction, large amounts of variation were present in HRV measures of root mean square of the successive difference of intervals, low-to-high frequency ratio, Poincaré dispersion perpendicular to the axis (SD1), and sample entropy. Linear interpolation produced RR intervals with the lowest bias and ES. However, caution should be given to HRV parameters at high exercise intensities, as large amounts of variation were still present. Recommendations for minimizing artefacts are discussed, along with guidelines for their identification, correction, and reporting.

Hemodynamic and Cardiorespiratory Predictors of Sport Rock Climbing Performance.

Fryer, SM, Giles, D, Garrido Palomino, I, de la O Puerta, A, and España-Romero, V. Hemodynamic and cardiorespiratory predictors of sport rock climbing performance. J Strength Cond Res 32(12): 3543-3550, 2018-Rock climbing performance has been suggested to involve a notable contribution from aerobic metabolism. Previously, it has been shown that forearm oxygenation kinetics can be used to distinguish ability groups and predict red-point sport climbing performance. Currently, it is not known if forearm oxygenation kinetics or a sport-specific assessment of cardiorespiratory fitness best predicts sport rock climbing performance. The aim of the study was to determine whether forearm oxidative capacity index, maximal deoxygenation (Δ score) during a treadwall V[Combining Dot Above]O2peak test, treadwall V[Combining Dot Above]O2peak, or running V[Combining Dot Above]O2max best predicts self-reported sport climbing performance. Twenty-one male sport rock climbers completed a treadwall V[Combining Dot Above]O2peak, running V[Combining Dot Above]O2max, and an assessment of near-infrared spectroscopy-derived oxidative capacity index. Linear regression, adjusted for age and experience (years), revealed that forearm oxidative capacity index, treadwall maximal deoxygenation (Δ), and treadwall V[Combining Dot Above]O2peak all significantly predicted self-reported red-point sport climbing ability (Adj R = -0.398, -0.255, and 0.374, respectively), whereas treadmill running V[Combining Dot Above]O2max did not (Adj R = -0.052). Additionally, multiple regression suggested that the combined significant aerobic predictors accounted for 67% of the variance in red-point climbing ability. Findings suggest that training for sport rock climbing performance should look to incorporate modalities that focus on (a) improving local forearm aerobic capacity and (b) improving whole-body aerobic capacity using sport-specific apparatus, such as treadwalls.

Exogenous polyunsaturated fatty acids (PUFAs) alter phospholipid composition, membrane permeability, biofilm formation and motility in Acinetobacter baumannii.

Acinetobacter baumannii is a ubiquitous multidrug-resistant bacteria that is found on a variety of surfaces, including skin, hair and soil. During the past decade, A. baumannii has emerged as a significant cause of nosocomial infections in the United States. Recent studies have highlighted the ability of some bacteria to utilize a wide variety of fatty acids as a membrane remodelling strategy. Considering this, we hypothesized that fatty acids may have an effect on the emerging pathogen A. baumannii. Thin-layer chromatography indicated structural alterations to major phospholipids. Liquid chromatography/mass spectrometry confirmed the assimilation of numerous exogenous polyunsaturated fatty acids (PUFAs) into the phospholipid species of A. baumannii. The incorporation of fatty acids affected several bacterial phenotypes, including membrane permeability, biofilm formation, surface motility and antimicrobial peptide resistance.

Exogenous Polyunsaturated Fatty Acids Impact Membrane Remodeling and Affect Virulence Phenotypes among Pathogenic Vibrio Species.

The pathogenic Vibrio species (V. cholerae, V. parahaemolyticus, and V. vulnificus) represent a constant threat to human health, causing foodborne and skin wound infections as a result of ingestion of or exposure to contaminated water and seafood. Recent studies have highlighted Vibrio's ability to acquire fatty acids from environmental sources and assimilate them into cell membranes. The possession and conservation of such machinery provokes consideration of fatty acids as important factors in the pathogenic lifestyle of Vibrio species. The findings here link exogenous fatty acid exposure to changes in bacterial membrane phospholipid structure, permeability, phenotypes associated with virulence, and consequent stress responses that may impact survival and persistence of pathogenic Vibrio species. Polyunsaturated fatty acids (PUFAs) (ranging in carbon length and unsaturation) supplied in growth medium were assimilated into bacterial phospholipids, as determined by thin-layer chromatography and liquid chromatography-mass spectrometry. The incorporation of fatty acids variably affected membrane permeability, as judged by uptake of the hydrophobic compound crystal violet. For each species, certain fatty acids were identified as affecting resistance to antimicrobial peptide treatment. Significant fluctuations were observed with regard to both motility and biofilm formation following growth in the presence of individual PUFAs. Our results illustrate the important and complex roles of exogenous fatty acids in the membrane physiology and virulence of a bacterial genus that inhabits aquatic and host environments containing an abundance of diverse fatty acids.IMPORTANCE Bacterial responses to fatty acids include, but are not limited to, degradation for metabolic gain, modification of membrane lipids, alteration of protein function, and regulation of gene expression. Vibrio species exhibit significant diversity with regard to the machinery known to participate in the uptake and incorporation of fatty acids into their membranes. Both aquatic and host niches occupied by Vibrio are rife with various free fatty acids and fatty acid-containing lipids. The roles of fatty acids in the environmental survival and pathogenesis of bacteria have begun to emerge and are expected to expand significantly. The current study demonstrates the responsiveness of V. cholerae, V. parahaemolyticus, and V. vulnificus to exogenous PUFAs. In addition to phospholipid remodeling, PUFA assimilation impacts membrane permeability, motility, biofilm formation, and resistance to polymyxin B.

The effect of potential fall distance on hormonal response in rock climbing.

The aim of this study was to examine the effect of alterations in potential lead fall distance on the hormonal responses of rock climbers. Nine advanced female climbers completed two routes while clipping all (PRO-all) or half (PRO-½) of the fixed points of protection. Venous blood samples were analysed for total catecholamines, noradrenaline (norepinephrine), adrenaline (epinephrine), dopamine, lactate, cortisol and serotonin. Differences between the two conditions pre, immediately post and 15 min post climbing were assessed using a 2 × 3 repeated measures ANOVA. All hormones and blood lactate concentrations increased significantly (P < 0.05) immediately post climb, except for cortisol. Peak cortisol concentrations did not occur until 15 min post ascent. Further, significant interactions between climbing and clipping conditions were found for total catecholamines (890% of basal concentration in PRO-½ vs. 568% in PRO-all), noradrenaline (794% vs. 532%) and dopamine (500% vs. 210%). There were no significant interactions for adrenaline (1920% vs. 1045%), serotonin (150% vs. 127%) or lactate (329% vs. 279%). The study showed a greater catecholamine response with an increase in potential lead fall distance. The most pronounced increases seen in catecholamine concentration were reported for dopamine and noradrenaline.

Structure of the C-Terminal Helical Repeat Domain of Eukaryotic Elongation Factor 2 Kinase.

Eukaryotic elongation factor 2 kinase (eEF-2K) phosphorylates its only known physiological substrate, elongation factor 2 (eEF-2), which reduces the affinity of eEF-2 for the ribosome and results in an overall reduction in protein translation rates. The C-terminal region of eEF-2K, which is predicted to contain several SEL-1-like helical repeats (SLRs), is required for the phosphorylation of eEF-2. Using solution nuclear magnetic resonance methodology, we have determined the structure of a 99-residue fragment from the extreme C-terminus of eEF-2K (eEF-2K627-725) that encompasses a region previously suggested to be essential for eEF-2 phosphorylation. eEF-2K627-725 contains four helices, of which the first (αI) is flexible, and does not pack stably against the ordered helical core formed by the last three helices (αII-αIV). The helical core is structurally similar to members of the tetratricopeptide repeat (TPR) family that includes SLRs. The two penultimate helices, αII and αIII, comprise the TPR, and the last helix, αIV, appears to have a capping function. The eEF-2K627-725 structure illustrates that the C-terminal deletion that was shown to abolish eEF-2 phosphorylation does so by destabilizing αIV and, therefore, the helical core. Indeed, mutation of two conserved C-terminal tyrosines (Y712A/Y713A) in eEF-2K previously shown to abolish eEF-2 phosphorylation leads to the unfolding of eEF-2K627-725. Preliminary functional analyses indicate that neither a peptide encoding a region deemed crucial for eEF-2 binding nor isolated eEF-2K627-725 inhibits eEF-2 phosphorylation by full-length eEF-2K. Taken together, our data suggest that the extreme C-terminal region of eEF-2K, in isolation, does not provide a primary docking site for eEF-2.

IL-12-polarized Th1 cells produce GM-CSF and induce EAE independent of IL-23.

CD4(+) T-helper (Th) cells reactive against myelin antigens mediate the mouse model experimental autoimmune encephalomyelitis (EAE) and have been implicated in the pathogenesis of multiple sclerosis (MS). It is currently debated whether encephalitogenic Th cells are heterogeneous or arise from a single lineage. In the current study, we challenge the dogma that stimulation with the monokine IL-23 is universally required for the acquisition of pathogenic properties by myelin-reactive T cells. We show that IL-12-modulated Th1 cells readily produce IFN-γ and GM-CSF in the CNS of mice and induce a severe form of EAE via an IL-23-independent pathway. Th1-mediated EAE is characterized by monocyte-rich CNS infiltrates, elicits a strong proinflammatory cytokine response in the CNS, and is partially CCR2 dependent. Conversely, IL-23-modulated, stable Th17 cells induce EAE with a relatively mild course via an IL-12-independent pathway. These data provide definitive evidence that autoimmune disease can be driven by distinct CD4(+) T-helper-cell subsets and polarizing factors.

Validity of the Polar V800 heart rate monitor to measure RR intervals at rest.

PURPOSE: To assess the validity of RR intervals and short-term heart rate variability (HRV) data obtained from the Polar V800 heart rate monitor, in comparison to an electrocardiograph (ECG). METHOD: Twenty participants completed an active orthostatic test using the V800 and ECG. An improved method for the identification and correction of RR intervals was employed prior to HRV analysis. Agreement of the data was assessed using intra-class correlation coefficients (ICC), Bland-Altman limits of agreement (LoA), and effect size (ES). RESULTS: A small number of errors were detected between ECG and Polar RR signal, with a combined error rate of 0.086 %. The RR intervals from ECG to V800 were significantly different, but with small ES for both supine corrected and standing corrected data (ES <0.001). The bias (LoA) were 0.06 (-4.33 to 4.45 ms) and 0.59 (-1.70 to 2.87 ms) for supine and standing intervals, respectively. The ICC was >0.999 for both supine and standing corrected intervals. When analysed with the same HRV software no significant differences were observed in any HRV parameters, for either supine or standing; the data displayed small bias and tight LoA, strong ICC (>0.99) and small ES (≤0.029). CONCLUSIONS: The V800 improves over previous Polar models, with narrower LoA, stronger ICC and smaller ES for both the RR intervals and HRV parameters. The findings support the validity of the Polar V800 and its ability to produce RR interval recordings consistent with an ECG. In addition, HRV parameters derived from these recordings are also highly comparable.

Modulating the innate immune response by combinatorial engineering of endotoxin.

Despite its highly inflammatory nature, LPS is a molecule with remarkable therapeutic potential. Lipid A is a glycolipid that serves as the hydrophobic anchor of LPS and constitutes a potent ligand of the Toll-like receptor (TLR)4/myeloid differentiation factor 2 receptor of the innate immune system. A less toxic mixture of monophosphorylated lipid A species (MPL) recently became the first new Food and Drug Administration-approved adjuvant in over 70 y. Whereas wild-type Escherichia coli LPS provokes strong inflammatory MyD88 (myeloid differentiation primary response gene 88)-mediated TLR4 signaling, MPL preferentially induces less inflammatory TRIF (TIR-domain-containing adaptor-inducing IFN-ß)-mediated responses. Here, we developed a system for combinatorial structural diversification of E. coli lipid A, yielding a spectrum of bioactive variants that display distinct TLR4 agonist activities and cytokine induction. Mice immunized with engineered lipid A/antigen emulsions exhibited robust IgG titers, indicating the efficacy of these molecules as adjuvants. This approach demonstrates how combinatorial engineering of lipid A can be exploited to generate a spectrum of immunostimulatory molecules for vaccine and therapeutics development.