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Increased inflammasome responses are strongly implicated in inflammatory diseases; however, their specific roles are incompletely understood. Therefore, we sought to examine the roles of nucleotide-binding oligomerization domain-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) and absent in melanoma-2 (AIM2) inflammasomes in cigarette smoke-induced inflammation in a model of experimental chronic obstructive pulmonary disease (COPD). We targeted NLRP3 with the inhibitor MCC950 given prophylactically or therapeutically and examined Aim2 -/- mice in cigarette smoke-induced experimental COPD. MCC950 treatment had minimal effects on disease development and/or progression. Aim2 -/- mice had increased airway neutrophils with decreased caspase-1 levels, independent of changes in lung neutrophil chemokines. Suppressing neutrophils with anti-Ly6G in experimental COPD in wild-type mice reduced neutrophils in bone marrow, blood and lung. By contrast, anti-Ly6G treatment in Aim2 -/- mice with experimental COPD had no effect on neutrophils in bone marrow, partially reduced neutrophils in the blood and had no effect on neutrophils or neutrophil caspase-1 levels in the lungs. These findings identify that following cigarette smoke exposure, Aim2 is important for anti-Ly6G-mediated depletion of neutrophils, suppression of neutrophil recruitment and mediates activation of caspase-1 in neutrophils., (© 2022 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Sphingosine-1-phosphate (S1P) lyase is a vitamin B6-dependent enzyme that degrades sphingosine-1-phosphate in the final step of sphingolipid metabolism. In 2017, a new inherited disorder was described caused by mutations in SGPL1, which encodes sphingosine phosphate lyase (SPL). This condition is referred to as SPL insufficiency syndrome (SPLIS) or alternatively as nephrotic syndrome type 14 (NPHS14). Patients with SPLIS exhibit lymphopenia, nephrosis, adrenal insufficiency, and/or neurological defects. No targeted therapy for SPLIS has been reported. Vitamin B6 supplementation has therapeutic activity in some genetic diseases involving B6-dependent enzymes, a finding ascribed largely to the vitamin's chaperone function. We investigated whether B6 supplementation might have activity in SPLIS patients. We retrospectively monitored responses of disease biomarkers in patients supplemented with B6 and measured SPL activity and sphingolipids in B6-treated patient-derived fibroblasts. In two patients, disease biomarkers responded to B6 supplementation. S1P abundance and activity levels increased and sphingolipids decreased in response to B6. One responsive patient is homozygous for an SPL R222Q variant present in almost 30% of SPLIS patients. Molecular modeling suggests the variant distorts the dimer interface which could be overcome by cofactor supplementation. We demonstrate the first potential targeted therapy for SPLIS and suggest that 30% of SPLIS patients might respond to cofactor supplementation., (© 2020 SSIEM.)

The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production. We achieved recombinant protein production (∼600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell-free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag-free self-cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose-level production of therapeutic proteins at the point-of-care., (© 2017 Wiley Periodicals, Inc.)

Dietary-associated diseases have increased tremendously in our current population, yet key molecular changes associated with high-fat diets that cause clinical pre-diabetes, obesity, hyperglycemia, and peripheral neuropathy remain unclear. This study examines molecular and metabolic aspects altered by voluntary exercise and a high-fat diet in the mouse dorsal root ganglion. Mice were examined for changes in mRNA and proteins encoding anti-inflammatory mediators, metabolic-associated molecules, and pain-associated ion channels. Proteins involved in the synaptosomal complex and pain-associated TRP ion channels decrease in the dorsal root ganglion of high-fat exercise animals relative to their sedentary controls. Exercise reversed high-fat diet induced mechanical allodynia without affecting weight gain, elevated blood glucose, and utilization of fat as a fuel source. Independent of weight or fat mass changes, high-fat exercised mice display reduced inflammation-associated mRNAs. The benefits of exercise on abnormal peripheral nerve function appear to occur independent of systemic metabolic changes, suggesting that the utilization of fats and inflammation in the peripheral nervous system may be key for diet-induced peripheral nerve dysfunction and the response to exercise., (© 2016 Peripheral Nerve Society.)

The complement cascade is comprised of a highly sophisticated network of innate immune proteins that are activated in response to invading pathogens or tissue injury. The complement activation peptide, C5a, binds two seven transmembrane receptors, namely the C5a receptor 1 (C5aR1) and C5a receptor 2 (C5aR2, or C5L2). C5aR2 is a non-G-protein-signalling receptor whose biological role remains controversial. Some of this controversy arises owing to the lack of selective ligands for C5aR2. In this study, a library of 61 peptides based on the C-terminus of C5a was assayed for the ability to selectively modulate C5aR2 function. Two ligands (P32 and P59) were identified as functionally selective C5aR2 ligands, exhibiting selective recruitment of β-arrestin 2 via C5aR2, partial inhibition of C5a-induced ERK1/2 activation and lipopolysaccharide-stimulated interleukin-6 release from human monocyte-derived macrophages. Importantly, neither ligand could induce ERK1/2 activation or inhibit C5a-induced ERK1/2 activation via C5aR1 directly. Finally, P32 inhibited C5a-mediated neutrophil mobilisation in wild-type, but not C5aR2(-/-) mice. These functionally selective ligands for C5aR2 are novel tools that can selectively modulate C5a activity in vitro and in vivo, and thus will be valuable tools to interrogate C5aR2 function.

Background and Purpose: Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1β and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1β and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis., Experimental Approach: Wild-type and inflammasome-deficient ASC(-/-) mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry., Key Results: 1K/DOCA/salt-induced hypertension in mice was associated with increased renal mRNA expression of inflammasome subunits NLRP3, ASC and pro-caspase-1, and the cytokine, pro-IL-1β, as well as protein levels of active caspase-1 and mature IL-1β. Following treatment with 1K/DOCA/salt, ASC(-/-) mice displayed blunted pressor responses and were also protected from increases in renal expression of IL-6, IL-17A, CCL2, ICAM-1 and VCAM-1, and accumulation of macrophages and collagen. Finally, treatment with a novel inflammasome inhibitor, MCC950, reversed hypertension in 1K/DOCA/salt-treated mice., Conclusions and Implications: Renal inflammation, fibrosis and elevated BP induced by 1K/DOCA/salt treatment are dependent on inflammasome activity, highlighting the inflammasome/IL-1β pathway as a potential therapeutic target in hypertension., (© 2015 The British Pharmacological Society.)

Purpose: High spatial-temporal four-dimensional imaging with large volume coverage is necessary to accurately capture and characterize liver lesions. Traditionally, parallel imaging and adapted sampling are used toward this goal, but they typically result in a loss of signal to noise. Furthermore, residual under-sampling artifacts can be temporally varying and complicate the quantitative analysis of contrast enhancement curves needed for pharmacokinetic modeling. We propose to overcome these problems using a novel patch-based regularization approach called Patch-based Reconstruction Of Under-sampled Data (PROUD)., Theory and Methods: PROUD produces high frame rate image reconstructions by exploiting the strong similarities in spatial patches between successive time frames to overcome the severe k-space under-sampling. To validate PROUD, a numerical liver perfusion phantom was developed to characterize contrast-to-noise ratio (CNR) performance compared with a previously proposed method, TRACER. A second numerical phantom was constructed to evaluate the temporal footprint and lag of PROUD and TRACER reconstructions. Finally, PROUD and TRACER were evaluated in a cohort of five liver donors., Results: In the CNR phantom, PROUD, compared with TRACER, improved peak CNR by 3.66 times while maintaining or improving temporal fidelity. In vivo, PROUD demonstrated an average increase in CNR of 60% compared with TRACER., Conclusion: The results presented in this work demonstrate the feasibility of using a combination of patch based image constraints with temporal regularization to provide high SNR, high temporal frame rate and spatial resolution four dimensional imaging., (© 2014 Wiley Periodicals, Inc.)

Purpose: To develop a fast and accurate monoexponential fitting algorithm based on Auto-Regression on Linear Operations (ARLO) of data, and to validate its accuracy and computational speed by comparing it with the conventional Levenberg-Marquardt (LM) and Log-Linear (LL) algorithms., Methods: ARLO, LM, and LL performances for T2* mapping were evaluated in simulation and in vivo imaging of liver (n=15) and myocardial (n=1) iron overload patients and the brain (two healthy volunteers)., Results: In simulations, ARLO consistently delivered accuracy similar to LM and significantly superior to LL. In in vivo mapping of T2 * values, ARLO showed excellent agreement with LM, while LL showed only limited agreements with ARLO and LM. Compared with LM and LL in the liver, ARLO was 125 and 8 times faster using our Matlab implementations, and 156 and 13 times faster using our C++ implementations. In C++ implementations, ARLO reduced the online whole-brain processing time from 9 min 15 s of LM and 35 s of LL to 2.7 s, providing T2 * maps approximately in real time., Conclusion: Due to comparable accuracy and significantly higher speed, ARLO can be considered as a valid alternative to the conventional LM algorithm for online T2 * mapping., (© 2014 Wiley Periodicals, Inc.)

Background and Purpose: Proteinase activated receptor 2 (PAR2) is a GPCR associated with inflammation, metabolism and disease. Clues to understanding how to block PAR2 signalling associated with disease without inhibiting PAR2 activation in normal physiology could be provided by studies of biased signalling., Experimental Approach: PAR2 ligand GB88 was profiled for PAR2 agonist and antagonist properties by several functional assays associated with intracellular G-protein-coupled signalling in vitro in three cell types and with PAR2-induced rat paw oedema in vivo., Key Results: In HT29 cells, GB88 was a PAR2 antagonist in terms of Ca(2+) mobilization and PKC phosphorylation, but a PAR2 agonist in attenuating forskolin-induced cAMP accumulation, increasing ERK1/2 phosphorylation, RhoA activation, myosin phosphatase phosphorylation and actin filament rearrangement. In CHO-hPAR2 cells, GB88 inhibited Ca(2+) release, but activated G(i/o) and increased ERK1/2 phosphorylation. In human kidney tubule cells, GB88 inhibited cytokine secretion (IL6, IL8, GM-CSF, TNF-α) mediated by PAR2. A rat paw oedema induced by PAR2 agonists was also inhibited by orally administered GB88 and compared with effects of locally administered inhibitors of G-protein coupled pathways., Conclusions and Implications: GB88 is a biased antagonist of PAR2 that selectively inhibits PAR2/G(q/11)/Ca(2+)/PKC signalling, leading to anti-inflammatory activity in vivo, while being an agonist in activating three other PAR2-activated pathways (cAMP, ERK, Rho) in human cells. These findings highlight opportunities to design drugs to block specific PAR2-linked signalling pathways in disease, without blocking beneficial PAR2 signalling in normal physiology, and to dissect PAR2-associated mechanisms of disease in vivo., (© 2014 The British Pharmacological Society.)

The complement system is a major component of our innate immune system, in which the complement proteins C5a and C5a-des Arg bind to two G-protein-coupled receptors: namely, the C5a receptor (C5a1) and C5a receptor like-2 receptor (C5a2, formerly called C5L2). Recently, it has been demonstrated that C5a, but not C5a-des Arg, upregulates heteromer formation between C5a1 and C5a2, leading to an increase in IL-10 release from human monocyte-derived macrophages (HMDMs). A bioluminescence resonance energy transfer (BRET) assay was used to assess the recruitment of β-arrestins by C5a and C5a-des Arg at the C5a1 and C5a2 receptors. C5a demonstrated elevated β-arrestin 2 recruitment levels in comparison with C5a-des Arg, whereas no significant difference was observed at C5a2. A constitutive complex that formed between β-arrestin 2 and C5a2 accounted for half of the BRET signal observed. Interestingly, both C5a and C5a-des Arg exhibited higher potency for β-arrestin 2 recruitment via C5a2, indicating preference for C5a2 over C5a1. When C5a was tested in a functional ERK1/2 assay in HMDMs, inhibition of ERK1/2 was observed only at concentrations at or above the EC50 for heteromer formation. This suggested that increased recruitment of the β-arrestin-C5a2 complex at these C5a concentrations might have an inhibitory role on C5a1 signaling through ERK1/2. An improved understanding of C5a2 modulation of signaling in acute inflammation could be of benefit in the development of ligands for conditions such as sepsis.

Purpose: Direct 2D tracking of cardiac motion may provide superior respiratory navigator gating for coronary magnetic resonance angiography compared to conventional liver-diaphragm navigators. However, additional 2D processing for motion extraction is unsuitable for real-time prospective gating. In this work, a 2D fat-selective image navigator, which delineates the epicardial fat surrounding coronary arteries, is developed to directly monitor epicardial fat motion at every heartbeat in real-time for prospective gating., Methods: The proposed navigator is incorporated into a real-time interactive software that allows rapid setup and efficient motion extraction, and runs on standard clinical hardware without any additional dedicated components for processing. The proposed 2D cardiac fat image navigator was compared with the conventional 1D diaphragm navigator in free-breathing b-SSFP coronary MRAs in 12 healthy volunteers at 1.5T., Results: Real-time motion extraction from 2D cardiac fat navigator images was feasible within 20 ms, enabling successful prospectively gated coronary magnetic resonance angiographies in all subjects. Compared to 1D diaphragmatic navigator, 2D fat image navigator reduced scan time by 38% (P < 0.0005), and significantly improved vessel sharpness, myocardial suppression, and image quality (P < 0.05)., Conclusion: This demonstrates the feasibility of a 3D SSFP coronary magnetic resonance angiography sequence using a 2D epicardial fat image as a navigator for real-time prospective motion tracking., (Copyright © 2013 Wiley Periodicals, Inc.)

Receptors for C5a have an important role in innate immunity and inflammation where their expression and activation is tightly regulated. There are two known receptors for C5a: the C5a receptor (C5aR) and the C5a receptor like-2 (C5L2) receptor. Here we hypothesized that activation of C5aR might lead to heteromer formation with C5L2, as a downregulatory mechanism for C5aR signaling. To investigate this experimentally, bioluminescent resonance energy transfer (BRET) was implemented and supported by wide-field microscopy to analyze receptor localization in transfected HEK293 cells and human monocyte-derived macrophages (HMDM). BRET experiments indicated the presence of constitutive C5aR-C5L2 heteromers, where C5a, but not C5a-des Arg, was able to induce further heteromer formation, which was inhibited by a C5aR-specific antagonist. The data obtained suggest that C5aR-C5L2 can form heteromers in a process enhanced by C5a, but not by C5a-des Arg. There was also a significant difference in the levels of the anti-inflammatory cytokine IL-10 detected in HMDM following exposure to C5a compared with that seen for C5a-des Arg but no differences in the pro-inflammatory cytokines TNFα and IL-6. These subtle differences in C5a and C5a-des Arg induced receptor function may be of benefit in understanding the regulation of C5a in acute inflammation.

Fast methods using balanced steady-state free precession have been developed to reduce the scan time of T₁ and T₂ mapping. However, flip angle (FA) profiles created by the short radiofrequency pulses used in steady-state free precession deviate substantially from the ideal rectangular profile, causing T₁ and T₂ mapping errors. The purpose of this study was to develop a FA profile correction for T₁ and T₂ mapping with Look-Locker 2D inversion recovery steady-state free precession and to validate this method using 2D spin echo as a reference standard. Phantom studies showed consistent improvement in T₁ and T₂ accuracy using profile correction at multiple FAs. Over six human calves, profile correction provided muscle T₁ estimates with mean error ranging from excellent (-0.6%) at repetition time/FA = 18 ms/60° to acceptable (6.8%) at repetition time/FA = 4.9 ms/30°, while muscle T₂ estimates were less accurate with mean errors of 31.2% and 47.9%, respectively., (Copyright © 2012 Wiley Periodicals, Inc.)

Quantitative assessment of myelination is important for characterizing tissue damage and evaluating response to therapy in white matter diseases such as multiple sclerosis. Conventional multicomponent T(2) relaxometry based on the two-dimensional (2D) multiecho spin echo sequence is a promising method to measure myelin water fraction, but its clinical utility is impeded by the prohibitively long data acquisition and limited brain coverage. The objective of this study was to develop a signal-to-noise ratio efficient 3D T(2) prep spiral gradient echo (3D SPIRAL) sequence for full brain T(2) relaxometry and to validate this sequence using 3D multiecho spin echo as reference standard in healthy brains at 1.5 T. 3D SPIRAL was found to provide similar myelin water fraction in six selected white and gray matter areas using region-of-interest signal averaging analysis (N = 7, P > 0.05). While 3D multiecho spin echo only provided partial brain coverage, 3D SPIRAL enabled whole brain coverage with a fivefold higher acquisition speed per imaging slice and similar signal-to-noise ratio efficiency. Both 3D sequences provided superior signal-to-noise ratio efficiency when compared to the conventional 2D multiecho spin echo approach., (Copyright © 2011 Wiley Periodicals, Inc.)

Citropin 1.1, maculatin 1.1, and caerin 1.1 are short antibacterial cationic peptides from the skin glands of the Australian tree frog Litoria species. Several analogues have been synthesized to give a better insight into the relationship between the structure of the peptides and their antibacterial and haemolytic activity. Binding studies using a surface plasmon resonance (SPR) biosensor together with a vesicle-capture sensor chip have been used to investigate selectivity of the peptides and their analogues for 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles, as well as for vesicles made from lipid extracts from Escherichia coli and bovine brain. Data obtained for membrane selectivity using natural lipid extracts show better correlation with minimum inhibitory concentration (MIC) values against Gram-positive bacteria and haemolytic activity than that obtained using synthetic DMPG and DMPC. Electron microscopy and membrane leakage studies using Gram-positive bacteria gave further insight into the membrane disruption properties of the peptides. For maculatin 1.1, it was found that the central proline residue, which is responsible for a bend in the alpha-helical structure, is essential not only for the antibacterial activity but also for binding, and perturbation of membranes. The caerin analogues showed only small variations in their MIC values and membrane binding. In contrast, for citropin 1.1, the analogue replacing the aspartate with a lysine showed the lowest MIC against Gram-positive bacteria and best membrane binding to E. coli lipid extracts, coinciding with an increased hydrophobic moment of the peptide. These data give further insight into these antimicrobial natural products, toward the development and evaluation of these and other analogues as potential antibiotics.

Background: Increased bone marrow angiogenesis and vascular endothelial growth factor (VEGF) levels are of adverse prognostic significance in patients with myeloproliferative disorders (MPD), including agnogenic myeloid metaplasia (AMM), chronic myeloid leukemia in blastic phase (CML-BP), and chronic myelomonocytic leukemia (CMML). VEGF is a soluble, circulating, angiogenic molecule that acts through receptor tyrosine kinases (RTK), including VEGF receptor 2 (VEGFR-2). SU5416 is a small-molecule RTK inhibitor (RTKI) that targets VEGFR-2, c-kit, and fms-related tyrosine kinase Flk2., Methods: Adult patients with advanced CMML, AMM, CML-BP, or other BCR-ABL negative MPD were entered on a multicenter, Phase II study., Results: Thirty-two patients (19 patients with BCR-ABL negative MPD, 6 patients with CMML, 4 patients with CML-BP, and 3 patients with AMM) with a median age of 66 years (range, 29-85 years) received SU5416 145 mg/m(2) twice weekly intravenously for a median of three 4-week cycles (maximum, 12 cycles). Drug-related Grade 3-4 toxicities included acute abdominal pain (13%), bone pain (9%), infusion-related dyspnea (9%) or headache (6%), fatigue (6%), diarrhea (3%), and catheter site reactions (3%). Eleven patients (34%) did not receive a second cycle of therapy (6 patients had progressive disease, 3 because of adverse events; 2 patients withdrew due to lack of response). One patient with AMM achieved a partial response. Eight patients received more than 6 months of therapy., Conclusions: SU5416 had minimal clinical activity in patients with MPD. Long-term administration of a twice-weekly, hyperosmolar, intravenous solution containing polyoxyl 35 castor oil was difficult. More tolerable RTKI may be worthy of further investigation in patients with MPD., (Copyright 2003 American Cancer Society.)

The four classic electrical mechanisms of lightning injury cannot account for all injuries. A fifth mechanism, injury by a weak upward streamer that does not become part of a completed lightning channel, has long been postulated in the engineering literature by lightning researchers. This paper reports a case of death where injury from a weak upward streamer is strongly suspected following forensic investigation. Neither high voltage nor any of the previously accepted mechanisms of lightning injury can explain this incident.

Objective: To define sources of job satisfaction and stress among emergency physicians and assess self-projected career longevity., Design: A survey containing questions regarding emergency medicine (EM) practice satisfaction was mailed to 1,317 diplomates of the American Board of Emergency Medicine (ABEM). Specific sources of practice satisfaction and dissatisfaction, self-reported burnout or impairment, and plans for remaining in the specialty were assessed. Data were compared between two groups of physicians, namely, those residency-trained in EM and those attaining certification through the practice or special category tracts., Results: Of the physicians returning the survey, 25.2% stated that they felt burned out or impaired and 23.1% planned to leave the practice of EM within five years. Perceptions of burnout/impairment and plans to stop practice were associated with less overall practice satisfaction but were not significantly different between the two groups of physicians. Burnout/impairment was linked with psychiatric, drug, or alcohol problems and the feeling that EM had contributed to that problem., Conclusion: This study confirms the relatively high levels of projected attrition in EM and supports the perception that stress and burnout are associated with the specialty. Differences in job satisfaction and stress between those ABEM diplomates who were residency-trained in EM and those who became eligible for the board examination through practice or special-category eligibility appear minor.

Educators often find themselves confronted with the need to address health-related problems of chronically impaired students. Specific needs often are demonstrated by chronically impaired individuals. The roles and responsibilities of the classroom teacher with respect to meeting these needs are discussed. In addition, suggestions about how these concerns can be addressed through existing classroom health instruction are offered. Teachers can use the health curriculum to compliment the efforts of parents and health-care providers in informing, supplementing, and reinforcing disabled students' knowledge, attitudes, and skills. In this way, chronically impaired children can be assisted to meet present and future health needs in a manner conducive to optimal growth and development.