The developmental and genetic basis of 'clubfoot' in the peroneal muscular atrophy mutant mouse.

Genetic factors underlying the human limb abnormality congenital talipes equinovarus ('clubfoot') remain incompletely understood. The spontaneous autosomal recessive mouse 'peroneal muscular atrophy' mutant (PMA) is a faithful morphological model of human clubfoot. In PMA mice, the dorsal (peroneal) branches of the sciatic nerves are absent. In this study, the primary developmental defect was identified as a reduced growth of sciatic nerve lateral motor column (LMC) neurons leading to failure to project to dorsal (peroneal) lower limb muscle blocks. The pma mutation was mapped and a candidate gene encoding LIM-domain kinase 1 (Limk1) identified, which is upregulated in mutant lateral LMC motor neurons. Genetic and molecular analyses showed that the mutation acts in the EphA4-Limk1-Cfl1/cofilin-actin pathway to modulate growth cone extension/collapse. In the chicken, both experimental upregulation of Limk1 by electroporation and pharmacological inhibition of actin turnover led to defects in hindlimb spinal motor neuron growth and pathfinding, and mimicked the clubfoot phenotype. The data support a neuromuscular aetiology for clubfoot and provide a mechanistic framework to understand clubfoot in humans.

Ndufs2, a Core Subunit of Mitochondrial Complex I, Is Essential for Acute Oxygen-Sensing and Hypoxic Pulmonary Vasoconstriction.

RATIONALE: Hypoxic pulmonary vasoconstriction (HPV) optimizes systemic oxygen delivery by matching ventilation to perfusion. HPV is intrinsic to pulmonary artery smooth muscle cells (PASMCs). Hypoxia dilates systemic arteries, including renal arteries. Hypoxia is sensed by changes in mitochondrial-derived reactive oxygen species, notably hydrogen peroxide (H2O2) ([H2O2]mito). Decreases in [H2O2]mito elevate pulmonary vascular tone by increasing intracellular calcium ([Ca2+]i) through reduction-oxidation regulation of ion channels. Although HPV is mimicked by the Complex I inhibitor, rotenone, the molecular identity of the O2 sensor is unknown. OBJECTIVE: To determine the role of Ndufs2 (NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 2), Complex I's rotenone binding site, in pulmonary vascular oxygen-sensing. METHODS AND RESULTS: Mitochondria-conditioned media from pulmonary and renal mitochondria isolated from normoxic and chronically hypoxic rats were infused into an isolated lung bioassay. Mitochondria-conditioned media from normoxic lungs contained more H2O2 than mitochondria-conditioned media from chronic hypoxic lungs or kidneys and uniquely attenuated HPV via a catalase-dependent mechanism. In PASMC, acute hypoxia decreased H2O2 within 112±7 seconds, followed, within 205±34 seconds, by increased intracellular calcium concentration, [Ca2+]i. Hypoxia had no effects on [Ca2+]i in renal artery SMC. Hypoxia decreases both cytosolic and mitochondrial H2O2 in PASMC while increasing cytosolic H2O2 in renal artery SMC. Ndufs2 expression was greater in PASMC versus renal artery SMC. Lung Ndufs2 cysteine residues became reduced during acute hypoxia and both hypoxia and reducing agents caused functional inhibition of Complex I. In PASMC, siNdufs2 (cells/tissue treated with Ndufs2 siRNA) decreased normoxic H2O2, prevented hypoxic increases in [Ca2+]i, and mimicked aspects of chronic hypoxia, including decreasing Complex I activity, elevating the nicotinamide adenine dinucleotide (NADH/NAD+) ratio and decreasing expression of the O2-sensitive ion channel, Kv1.5. Knocking down another Fe-S center within Complex I (Ndufs1, NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 1) or other mitochondrial subunits proposed as putative oxygen sensors (Complex III's Rieske Fe-S center and COX4i2 [cytochrome c oxidase subunit 4 isoform 2] in Complex IV) had no effect on hypoxic increases in [Ca2+]i. In vivo, siNdufs2 significantly decreased hypoxia- and rotenone-induced constriction while enhancing phenylephrine-induced constriction. CONCLUSIONS: Ndufs2 is essential for oxygen-sensing and HPV.

Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain.

The human soluble Epoxide Hydrolase (hsEH) is an enzyme involved in the hydrolysis of endogenous anti-inflammatory and cardio-protective signalling mediators known as epoxyeicosatrienoic acids (EETs). EETs' conversion into the corresponding diols by hsEH generates non-bioactive molecules, thereby the enzyme inhibition would be expected to enhance the EETs bioavailability, and their beneficial properties. Numerous inhibitors have been developed to target the enzyme, some of which are showing promising antihypertensive and anti-inflammatory properties in vivo. Thus far, the preparation of the recombinant enzyme for enzymatic and structural in vitro studies has been performed mainly using a baculovirus expression system. More recently, it was reported that the enzyme could be exogenously expressed and isolated from E. coli, although limited amounts of active protein were obtained. We herein describe two novel methods to yield pure recombinant enzyme. The first describes the expression and purification of the full-length enzyme from eukaryotic cells HEK293-F, whilst the second concerns the C-terminal domain of hsEH obtained from the cost-effective and rapid E. coli prokaryotic system. The two methods successfully generated satisfactory amounts of functional enzyme, with virtually identical enzymatic activity. Overall, the protocols described in this paper can be employed for the recombinant expression and purification of active hsEH, to be used in future biomedical investigations and for high-throughput screening of inhibitors for potential use in the treatment of cardiovascular disease.

Redox-dependent dimerization of p38α mitogen-activated protein kinase with mitogen-activated protein kinase kinase 3.

The kinase p38α MAPK (p38α) plays a pivotal role in many biological processes. p38α is activated by canonical upstream kinases that phosphorylate the activation region. The purpose of our study was to determine whether such activation may depend on redox-sensing cysteines within p38α. p38α was activated and formed a disulfide-bound heterodimer with MAP2K3 (MKK3) in rat cardiomyocytes and isolated hearts exposed to H2O2 This disulfide heterodimer was sensitive to reduction by mercaptoethanol and was enhanced by the thioredoxin-reductase inhibitor auranofin. We predicted that Cys-119 or Cys-162 of p38α, close to the known MKK3 docking domain, were relevant for these redox characteristics. The C119S mutation decreased whereas the C162S mutation increased the dimer formation, suggesting that these two Cys residues act as vicinal thiols, consistent with C119S/C162S being incapable of sensing H2O2 Similarly, disulfide heterodimer formation was abolished in H9C2 cells expressing both MKK3 and p38α C119S/C162S and subjected to simulated ischemia and reperfusion. However, the p38α C119S/C162S mutants did not exhibit appreciable alteration in activating dual phosphorylation. In contrast, the anti-inflammatory agent 10-nitro-oleic acid (NO2-OA), a component of the Mediterranean diet, reduced p38α activation and covalently modified Cys-119/Cys-162, probably obstructing MKK3 access. Moreover, NO2-OA reduced the dephosphorylation of p38α by hematopoietic tyrosine phosphatase (HePTP). Furthermore, steric obstruction of Cys-119/Cys-162 by NO2-OA pretreatment in Langendorff-perfused murine hearts prevented the p38-MKK3 disulfide dimer formation and attenuated H2O2-induced contractile dysfunction. Our findings suggest that cysteine residues within p38α act as redox sensors that can dynamically regulate the association between p38 and MKK3.

Protection from hypertension in mice by the Mediterranean diet is mediated by nitro fatty acid inhibition of soluble epoxide hydrolase.

Soluble epoxide hydrolase (sEH) is inhibited by electrophilic lipids by their adduction to Cys521 proximal to its catalytic center. This inhibition prevents hydrolysis of the enzymes' epoxyeicosatrienoic acid (EET) substrates, so they accumulate inducing vasodilation to lower blood pressure (BP). We generated a Cys521Ser sEH redox-dead knockin (KI) mouse model that was resistant to this mode of inhibition. The electrophilic lipid 10-nitro-oleic acid (NO2-OA) inhibited hydrolase activity and also lowered BP in an angiotensin II-induced hypertension model in wild-type (WT) but not KI mice. Furthermore, EET/dihydroxy-epoxyeicosatrienoic acid isomer ratios were elevated in plasma from WT but not KI mice following NO2-OA treatment, consistent with the redox-dead mutant being resistant to inhibition by lipid electrophiles. sEH was inhibited in WT mice fed linoleic acid and nitrite, key constituents of the Mediterranean diet that elevates electrophilic nitro fatty acid levels, whereas KIs were unaffected. These observations reveal that lipid electrophiles such as NO2-OA mediate antihypertensive signaling actions by inhibiting sEH and suggest a mechanism accounting for protection from hypertension afforded by the Mediterranean diet.

Gel-based methods in redox proteomics.

BACKGROUND: The key to understanding the full significance of oxidants in health and disease is the development of tools and methods that allow the study of proteins that sense and transduce changes in cellular redox. Oxidant-reactive deprotonated thiols commonly operate as redox sensors in proteins and a variety of methods have been developed that allow us to monitor their oxidative modification. SCOPE OF THE REVIEW: This outline review specifically focuses on gel-based methods used to detect, quantify and identify protein thiol oxidative modifications. The techniques we discuss fall into one of two broad categories. Firstly, methods that allow oxidation of thiols in specific proteins or the global cellular pool to be monitored are discussed. These typically utilise thiol-labelling reagents that add a reporter moiety (e.g. affinity tag, fluorophore, chromophore), in which loss of labelling signifies oxidation. Secondly, we outline methods that allow specific thiol oxidation states of proteins (e.g. S-sulfenylation, S-nitrosylation, S-thionylation and interprotein disulfide bond formation) to be investigated. MAJOR CONCLUSIONS: A variety of different gel-based methods for identifying thiol proteins that are sensitive to oxidative modifications have been developed. These methods can aid the detection and quantification of thiol redox state, as well as identifying the sensor protein. GENERAL SIGNIFICANCE: By understanding how cellular redox is sensed and transduced to a functional effect by protein thiol redox sensors, this will help us better appreciate the role of oxidants in health and disease. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

CE: Breaking Through the Bottleneck: Acuity Adaptability in Noncritical Trauma Care.

BACKGROUND: Achieving efficient throughput of patients is a challenge faced by many hospital systems. Factors that can impede efficient throughput include increased ED use, high surgical volumes, lack of available beds, and the complexities of coordinating multiple patient transfers in response to changing care needs. Traditionally, many hospital inpatient units operate via a fixed acuity model, relying on multiple intrahospital transfers to move patients along the care continuum. In contrast, the acuity-adaptable model allows care to occur in the same room despite fluctuations in clinical condition, removing the need for transfer. This model has been shown to be a safe and cost-effective approach to improving throughput in populations with predictable courses of hospitalization, but has been minimally evaluated in other populations, such as patients hospitalized for traumatic injury. PURPOSE: This quality improvement project aimed to evaluate implementation of an acuity-adaptable model on a 20-bed noncritical trauma unit. Specifically, we sought to examine and compare the pre- and postimplementation metrics for throughput efficiency, resource utilization, and nursing quality indicators; and to determine the model's impact on patient transfers for changes in level of care. METHODS: This was a retrospective, comparative analysis of 1,371 noncritical trauma patients admitted to a level 1 trauma center before and after the implementation of an acuity-adaptable model. Outcomes of interest included throughput efficiency, resource utilization, and quality of nursing care. Inferential statistics were used to compare patients pre- and postimplementation, and logistic regression analyses were performed to determine the impact of the acuity-adaptable model on patient transfers. RESULTS: Postimplementation, the median ED boarding time was reduced by 6.2 hours, patients more often remained in their assigned room following a change in level of care, more progressive care patient days occurred, fall and hospital-acquired pressure injury index rates decreased respectively by 0.9 and 0.3 occurrences per 1,000 patient days, and patients were more often discharged to home. Logistic regression analyses revealed that under the new model, patients were more than nine times more likely to remain in the same room for care after a change in acuity and 81.6% less likely to change rooms after a change in acuity. An increase of over $11,000 in average daily bed charges occurred postimplementation as a result of increased progressive care-level bed capacity. CONCLUSIONS: The implementation of an acuity-adaptable model on a dedicated noncritical trauma unit improved throughput efficiency and resource utilization without sacrificing quality of care. As hospitals continue to face increasing demand for services as well as numerous barriers to meeting such demand, leaders remain challenged to find innovative ways to optimize operational efficiency and resource utilization while ensuring delivery of high-quality care. The findings of this study demonstrate the value of the acuity-adaptable model in achieving these goals in a noncritical trauma care population.

Original Research: Breaking Through the Bottleneck: Acuity Adaptability in Noncritical Trauma Care.

BACKGROUND: Achieving efficient throughput of patients is a challenge faced by many hospital systems. Factors that can impede efficient throughput include increased ED use, high surgical volumes, lack of available beds, and the complexities of coordinating multiple patient transfers in response to changing care needs. Traditionally, many hospital inpatient units operate via a fixed acuity model, relying on multiple intrahospital transfers to move patients along the care continuum. In contrast, the acuity-adaptable model allows care to occur in the same room despite fluctuations in clinical condition, removing the need for transfer. This model has been shown to be a safe and cost-effective approach to improving throughput in populations with predictable courses of hospitalization, but has been minimally evaluated in other populations, such as patients hospitalized for traumatic injury. PURPOSE: This quality improvement project aimed to evaluate implementation of an acuity-adaptable model on a 20-bed noncritical trauma unit. Specifically, we sought to examine and compare the pre- and postimplementation metrics for throughput efficiency, resource utilization, and nursing quality indicators; and to determine the model's impact on patient transfers for changes in level of care. METHODS: This was a retrospective, comparative analysis of 1,371 noncritical trauma patients admitted to a level 1 trauma center before and after the implementation of an acuity-adaptable model. Outcomes of interest included throughput efficiency, resource utilization, and quality of nursing care. Inferential statistics were used to compare patients pre- and postimplementation, and logistic regression analyses were performed to determine the impact of the acuity-adaptable model on patient transfers. RESULTS: Postimplementation, the median ED boarding time was reduced by 6.2 hours, patients more often remained in their assigned room following a change in level of care, more progressive care patient days occurred, fall and hospital-acquired pressure injury index rates decreased respectively by 0.9 and 0.3 occurrences per 1,000 patient days, and patients were more often discharged to home. Logistic regression analyses revealed that under the new model, patients were more than nine times more likely to remain in the same room for care after a change in acuity and 81.6% less likely to change rooms after a change in acuity. An increase of over $11,000 in average daily bed charges occurred postimplementation as a result of increased progressive care-level bed capacity. CONCLUSIONS: The implementation of an acuity-adaptable model on a dedicated noncritical trauma unit improved throughput efficiency and resource utilization without sacrificing quality of care. As hospitals continue to face increasing demand for services as well as numerous barriers to meeting such demand, leaders remain challenged to find innovative ways to optimize operational efficiency and resource utilization while ensuring delivery of high-quality care. The findings of this study demonstrate the value of the acuity-adaptable model in achieving these goals in a noncritical trauma care population.

Naked mole-rats have distinctive cardiometabolic and genetic adaptations to their underground low-oxygen lifestyles.

The naked mole-rat Heterocephalus glaber is a eusocial mammal exhibiting extreme longevity (37-year lifespan), extraordinary resistance to hypoxia and absence of cardiovascular disease. To identify the mechanisms behind these exceptional traits, metabolomics and RNAseq of cardiac tissue from naked mole-rats was compared to other African mole-rat genera (Cape, Cape dune, Common, Natal, Mahali, Highveld and Damaraland mole-rats) and evolutionarily divergent mammals (Hottentot golden mole and C57/BL6 mouse). We identify metabolic and genetic adaptations unique to naked mole-rats including elevated glycogen, thus enabling glycolytic ATP generation during cardiac ischemia. Elevated normoxic expression of HIF-1α is observed while downstream hypoxia responsive-genes are down-regulated, suggesting adaptation to low oxygen environments. Naked mole-rat hearts show reduced succinate levels during ischemia compared to C57/BL6 mouse and negligible tissue damage following ischemia-reperfusion injury. These evolutionary traits reflect adaptation to a unique hypoxic and eusocial lifestyle that collectively may contribute to their longevity and health span.

Redox regulation of soluble epoxide hydrolase by 15-deoxy-delta-prostaglandin J2 controls coronary hypoxic vasodilation.

RATIONALE: 15-Deoxy-Δ-prostaglandin (15d-PG)J(2) is an electrophilic oxidant that dilates the coronary vasculature. This lipid can adduct to redox active protein thiols to induce oxidative posttranslational modifications that modulate protein and tissue function. OBJECTIVE: To investigate the role of oxidative protein modifications in 15d-PGJ(2)-mediated coronary vasodilation and define the distal signaling pathways leading to enhanced perfusion. METHODS AND RESULTS: Proteomic screening with biotinylated 15d-PGJ(2) identified novel vascular targets to which it adducts, most notably soluble epoxide hydrolase (sEH). 15d-PGJ(2) inhibited sEH by specifically adducting to a highly conserved thiol (Cys521) adjacent to the catalytic center of the hydrolase. Indeed a Cys521Ser sEH "redox-dead" mutant was resistant to 15d-PGJ(2)-induced hydrolase inhibition. 15d-PGJ(2) dilated coronary vessels and a role for hydrolase inhibition was supported by 2 structurally different sEH antagonists each independently inducing vasorelaxation. Furthermore, 15d-PGJ(2) and sEH antagonists also increased coronary effluent epoxyeicosatrienoic acids consistent with their vasodilatory actions. Indeed 14,15-EET alone induced relaxation and 15d-PGJ(2)-mediated vasodilation was blocked by the EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE). Additionally, the coronary vasculature of sEH-null mice was basally dilated compared to wild-type controls and failed to vasodilate in response to 15d-PGJ(2). Coronary vasodilation to hypoxia in wild-types was accompanied by 15d-PGJ(2) adduction to and inhibition of sEH. Consistent with the importance of hydrolase inhibition, sEH-null mice failed to vasodilate during hypoxia. CONCLUSION: This represents a new paradigm for the regulation of sEH by an endogenous lipid, which is integral to the fundamental physiological response of coronary hypoxic vasodilation.

Case Report: Response to ipilimumab and nivolumab in a patient with adrenocortical carcinoma.

Background: Adrenocortical carcinoma (ACC) is a rare malignancy with limited treatment options. The evidence for the use of immunotherapy in ACC has been conflicting, with overall response rates ranging from 6 - 33%. Case presentation: We describe the case of a 32 year old patient who was initially thought to have an inoperable clear cell renal cell carcinoma and was treated with immunotherapy with ipilimumab and nivolumab. The patient had an excellent partial response to treatment. Further work-up prior to consideration of surgery demonstrated that the tumour was an ACC, rather than a renal cancer. She had a right adrenalectomy and right hepatectomy, achieving an R0 resection and remains disease-free one year after surgery. Conclusion: This case illustrates the challenge of diagnosing ACC, and that doublet immunotherapy with ipilimumab and nivolumab can have significant clinical efficacy in ACC.

Endothelial Nox4 NADPH oxidase enhances vasodilatation and reduces blood pressure in vivo.

OBJECTIVE: Increased reactive oxygen species (ROS) production is involved in the pathophysiology of endothelial dysfunction. NADPH oxidase-4 (Nox4) is a ROS-generating enzyme expressed in the endothelium, levels of which increase in pathological settings. Recent studies indicate that it generates predominantly hydrogen peroxide (H(2)O(2)), but its role in vivo remains unclear. METHODS AND RESULTS: We generated transgenic mice with endothelium-targeted Nox4 overexpression (Tg) to study the in vivo role of Nox4. Tg demonstrated significantly greater acetylcholine- or histamine-induced vasodilatation than wild-type littermates. This resulted from increased H(2)O(2) production and H(2)O(2)-induced hyperpolarization but not altered nitric oxide bioactivity. Tg had lower systemic blood pressure than wild-type littermates, which was normalized by antioxidants. CONCLUSION: Endothelial Nox4 exerts potentially beneficial effects on vasodilator function and blood pressure that are attributable to H(2)O(2) production. These effects contrast markedly with those reported for Nox1 and Nox2, which involve superoxide-mediated inactivation of nitric oxide. Our results suggest that therapeutic strategies to modulate ROS production in vascular disease may need to separately target individual Nox isoforms.

Redox Regulation of Soluble Epoxide Hydrolase-Implications for Cardiovascular Health and Disease.

Cell responses to changes in their redox state are significantly mediated by reversible oxido-reductive post-translational modifications of proteins, potentially altering their activities or interactions. These modifications are important for the homeostatic responses of cells to environmental changes that alter their redox state. Such redox regulatory mechanisms not only operate to maintain health, but can become dysregulated and contribute to pathophysiology. In this review, we focus on the redox control of soluble epoxide hydrolase (sEH), which is widely expressed, including in blood vessels and cardiomyocytes. We review the different types of oxidative modifications that regulate sEH and how they may alter cardiovascular physiology and affect disease progression during stress.

Why are children with asthma bullied? A risk factor analysis.

The links between bullying and asthma have not been explored in children. We wanted to determine the child/parent factors and attitudes associated with asthma-related bullying. Individual child/parent responses of children with asthma (N=943) from the Room to Breathe survey were analysed. 1 in 10 children reported asthma-related bullying/teasing (n=93). Children with well-controlled asthma were less likely to report being a victim of asthma-related bullying/teasing (OR 0.51, 95% CI 0.23 to 0.84, p=0.006). Being a victim of bullying/teasing was more common in children reporting activity restriction (OR 1.74, 95% CI 1.11 to 2.75, p=0.010), who described their asthma as 'bad' (OR 3.02, 95% CI 1.86 to 4.85, p<0.001) and those whose parents reported ongoing asthma-related health worries (OR 1.64, 95% CI 1.04 to 2.58, p=0.024). Asthma consultations should incorporate specific questions about bullying and be child-focused in order to gain a representative appreciation of asthma control and its impact on the child's life.

Paraglider Reserve Parachute Deployment Under Radial Acceleration.

INTRODUCTION: The paragliding reserve parachute system is safety-critical but underused, unstandardized, and known to fail. This study aimed to characterize reserve parachute deployment under radial acceleration to make recommendations for system design and paraglider pilot training.METHODS: There were 88 licensed amateur paraglider pilots who were filmed deploying their reserve parachutes from a centrifuge. Of those, 43 traveled forward at 4 G simulating a spiral dive, and 45 traveled backward at 3 G simulating a rotational maneuver known as SAT. Tests incorporated ecologically valid body, hand, and gaze positions, and cognitive loading and switching akin to real deployment. The footage was reviewed by subject matter experts and compared to previous work in linear acceleration.RESULTS: Of the pilots, 2.3 failed to extract the reserve container from the harness. SAT appeared more cognitively demanding than spiral, despite lower G. Participants located the reserve handle by touch not sight. The direction of travel influenced their initial contact with the harness: 82.9 searched first on their hip in spiral, 63.4 searched first on their thigh in SAT. Search patterns followed skeletal landmarks. Participants had little directional control over their throw.CONCLUSIONS: Paraglider pilots are part of the reserve system. Maladaptive behaviors observed under stress highlighted that components must work in harmony with pilots natural responses, with minimal cognitive demands or need for innovation or problem-solving. Recommendations include positioning prominent, tactile reserve handles overlying the pilots hip; deployment bags extractable with any angle of pull; deployment in a single sweeping backward action; and significantly increasing reserve deployment drills.Wilkes M, Long G, Charles R, Massey H, Eglin C, Tipton MJ. Paraglider reserve parachute deployment under radial acceleration. Aerosp Med Hum Perform. 2021; 92(7):579587.

A thiol redox sensor in soluble epoxide hydrolase enables oxidative activation by intra-protein disulfide bond formation.

Soluble epoxide hydrolase (sEH), an enzyme that broadly regulates the cardiovascular system, hydrolyses epoxyeicosatrienoic acids (EETs) to their corresponding dihydroxyeicosatrienoic acids (DHETs). We previously showed that endogenous lipid electrophiles adduct within the catalytic domain, inhibiting sEH to lower blood pressure in angiotensin II-induced hypertensive mice. As angiotensin II increases vascular H2O2, we explored sEH redox regulation by this oxidant and how this integrates with inhibition by lipid electrophiles to regulate vasotone. Kinetics analyses revealed that H2O2 not only increased the specific activity of sEH but increased its affinity for substrate and increased its catalytic efficiency. This oxidative activation was mediated by formation of an intra-disulfide bond between C262 and C264, as determined by mass spectrometry and substantiated by biotin-phenylarsinate and thioredoxin-trapping mutant assays. C262S/264S sEH mutants were resistant to peroxide-induced activation, corroborating the disulfide-activation mechanism. The physiological impact of sEH redox state was determined in isolated arteries and the effect of the pro-oxidant vasopressor angiotensin II on arterial sEH redox state and vasodilatory EETs indexed in mice. Angiotensin II induced the activating intra-disulfide in sEH, causing a decrease in plasma EET/DHET ratios that is consistent with the pressor response to this hormone. Although sEH C262-C264 disulfide formation enhances hydrolysis of vasodilatory EETs, this modification also sensitized sEH to inhibition by lipid electrophiles. This explains why angiotensin II decreases EETs and increases blood pressure, but when lipid electrophiles are also present, that EETs are increased and blood pressure lowered.

Phospholemman Ser69 phosphorylation contributes to sildenafil-induced cardioprotection against reperfusion injury.

The phosphodiesterase type-5 inhibitor sildenafil has powerful cardioprotective effects against ischemia-reperfusion injury. PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated. In this study we assessed the role of phospholemman (PLM) phosphorylation, which activates the Na(+)/K(+)-ATPase, in cardioprotection afforded by sildenafil administered during reperfusion. Isolated perfused mouse hearts were optimally protected against infarction (indexed by tetrazolium staining) by 0.1 muM sildenafil treatment during the first 10 min of reperfusion. Extended sildenafil treatment (30, 60, or 120 min at reperfusion) did not alter the degree of protection provided. This protection was PKG dependent, since it was blocked by KT-5823. Western blot analysis using phosphospecific antibodies to PLM showed that sildenafil at reperfusion did not modulate PLM Ser63 or Ser68 phosphorylation but significantly increased Ser69 phosphorylation. The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive. Patch-clamp studies showed that sildenafil treatment also activated the Na(+)/K(+)-ATPase, which is anticipated in light of PLM Ser69 phosphorylation. Na(+)/K(+)-ATPase activation during reperfusion would attenuate Na(+) overload at this time, providing a molecular explanation of how sildenafil guards against injury at this time. Indeed, using flame photometry and rubidium uptake into isolated mouse hearts, we found that sildenafil enhanced Na(+)/K(+)-ATPase activity during reperfusion. In this study we provide a molecular explanation of how sildenafil guards against myocardial injury during postischemic reperfusion.

Complex interrelationships between nitro-alkene-dependent inhibition of soluble epoxide hydrolase, inflammation and tumor growth.

Nitro-oleate (10-nitro-octadec-9-enoic acid), which inhibits soluble epoxide hydrolase (sEH) by covalently adducting to C521, increases the abundance of epoxyeicosatrienoic acids (EETs) that can be health promoting, for example by lowering blood pressure or their anti-inflammatory actions. However, perhaps consistent with their impact on angiogenesis, increases in EETs may exacerbate progression of some cancers. To assess this, Lewis lung carcinoma (LLc1) cells were exposed to oleate or nitro-oleate, with the latter inhibiting the hydrolase and increasing their proliferation and migration in vitro. The enhanced proliferation induced by nitro-oleate was EET-dependent, being attenuated by the ETT-receptor antagonist 14,15-EE-5(Z)-E. LLc1 cells were engineered to stably overexpress wild-type or C521S sEH, with the latter exhibiting resistance to nitro-oleate-dependent hydrolase inhibition and the associated stimulation of tumor growth in vitro or in vivo. Nitro-oleate also increased migration in endothelial cells isolated from wild-type (WT) mice, but not those from C521S sEH knock-in (KI) transgenic mice genetically modified to render the hydrolase electrophile-resistant. These observations were consistent with nitro-oleate promoting cancer progression, and so the impact of this electrophile was examined in vivo again, but this time comparing growth of LLc1 cells expressing constitutive levels of wild-type hydrolase when implanted into WT or KI mice. Nitro-oleate inhibited tumor sEH (P < 0.05), with a trend for elevated plasma 11(12)-EET/DHET and 8(9)EET/DHET (dihydroxyeicosatrienoic acid) ratios when administered to WT, but not KI, mice. Although in vitro studies with LLc1 cells supported a role for nitro-oleate in cancer cell proliferation, it failed to significantly stimulate tumor growth in WT mice implanted with the same LLc1 cells in vivo, perhaps due to its well-established anti-inflammatory actions. Indeed, pro-inflammatory cytokines were significantly down-regulated in nitro-oleate treated WT mice, potentially countering any impact of the concomitant inhibition of sEH.

Measuring mental workload using physiological measures: A systematic review.

Technological advances have led to physiological measurement being increasingly used to measure and predict operator states. Mental workload (MWL) in particular has been characterised using a variety of physiological sensor data. This systematic review contributes a synthesis of the literature summarising key findings to assist practitioners to select measures for use in evaluation of MWL. We also describe limitations of the methods to assist selection when being deployed in applied or laboratory settings. We detail fifty-eight peer reviewed journal articles which present original data using physiological measures to include electrocardiographic, respiratory, dermal, blood pressure and ocular. Electroencephalographic measures have been included if they are presented with another measure to constrain scope. The literature reviewed covers a range of applied and experimental studies across various domains, safety-critical applications being highly represented in the sample of applied literature reviewed. We present a summary of the six measures and provide an evidence base which includes how to deploy each measure, and characteristics that can affect or preclude the use of a measure in research. Measures can be used to discriminate differences in MWL caused by task type, task load, and in some cases task difficulty. Varying ranges of sensitivity to sudden or gradual changes in taskload are also evident across the six measures. We conclude that there is no single measure that clearly discriminates mental workload but there is a growing empirical basis with which to inform both science and practice.

Notes on the Nest Architecture and Colony Composition in Winter of the Yellow-Legged Asian Hornet, Vespa velutina Lepeletier 1836 (Hym.: Vespidae), in Its Introduced Habitat in Galicia (NW Spain).

Fifteen years ago, at least one multimated female yellow-legged Asian hornet (Vespa velutina Lepeletier 1836) arrived in France, giving rise to a pan-European invasion, altering the environment, affecting ecosystem processes, and impacting society. During winter, V. velutina nests (n = 3) were collected in Galicia and data on internal and external aspects of the nests and the colony as a whole were collected. The whole colony population (WCPN; adult insects, larvae, and pupae in percentages) was as follows: nest A: n = 176 (49%, 3%, and 48%); nest B: n = 1979 (52%, 36%, and 12%); and nest C: n = 662 (5%, 27%, and 8%). The adult insect population (IAPN; males, workers, and gynes in percentages) was as follows: nest A: n = 87 (11%, 66%, and 23%); nest B: n = 1021 (3%, 62%, and 35%); and nest C: n = 430 (20%, 73%, and 7%). As a small number of queens is sufficient for a population to develop, it is necessary to avoid continued spread by deactivating and removing all nests, even those detected in winter. This practice can be of greatest importance in border areas where V. velutina is expanding into new territory.