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The astigmatid mite Psoroptes ovis (Acari: Proroptidae) causes the highly contagious and debilitating ovine disease, sheep scab. This ectoparasitic infection has a high economic and animal welfare impact on British sheep farming. Following recent work demonstrating resistance of Psoroptes mites to moxidectin, a widely used macrocyclic lactone (ML) treatment for scab, the current study compared the toxicity of three of the commonly administered macrocylic lactone therapeutic treatments (moxidectin, ivermectin and doramectin) to P. ovis from outbreak populations that had appeared unresponsive to treatment. These outbreak populations were from Wales and south west England. The data presented demonstrate that there is resistance to all three available ML compounds in populations of Psoroptes mites. However, considerable variation in response suggested that resistance alone was not responsible for the reported lack of efficacy in all of the submitted cases; lack of response in others may be associated with inappropriate treatment application or management. These data highlight the importance of the appropriate use of these compounds to manage national scab incidence at levels that are consistent with acceptable animal welfare standards, while attempting to reduce the development and spread of resistance.

Objectives Rheumatoid arthritis (RA) is a common autoimmune disease where methotrexate is widely used as first-line therapy. The combination of RA and methotrexate are associated with lymphoproliferative disorders. RA patients with Epstein-Barr virus (EBV) have impaired T-lymphocyte function, thus allowing an overgrowth of EBV-positive lymphoblastoid cells. We examined the association of EBV with lymphoproliferative disorders in immunosuppressed RA patients, particularly those treated with methotrexate. Methods We reported two cases of RA patient with long-term methotrexate treatment who subsequently developed EBV-positive lymphoproliferative disorder, followed by a review of the relevant literature. Results Compared with normal population, RA patients have a higher risk of lymphoma with diffused large B-cell lymphoma the most common subtype. Methotrexate withdrawal can lead to lymphoma regression. Other biological therapies, such as abatacept and tocilizumab, are not associated with increased EBV-positive lymphoma diagnosis in RA patients. Conclusion The association between EBV, lymphoma and methotrexate highlight the need to consider reducing or stopping methotrexate in patients that have had stable RA for many years. This article is protected by copyright. All rights reserved.

New findings What is the topic of this hypothesis? Highland natives have undergone natural selection for genetic variants advantageous in adaptation to the hypobaric hypoxia experienced at high altitude. Why genes related to alcohol metabolism appear consistently selected for has not been greatly considered. We hypothesise that altitude-related changes in the gut microbiome offer one possible explanation. What advances does it highlight? Low intestinal oxygen tension may favour the production of ethanol through anaerobic fermentation by the gut microbiome. Subsequent increases in endogenous ethanol absorption could therefore be a selection pressure for gene variants favouring its increased degradation - or reduced degradation if endogenously synthesised ethanol acts as a metabolic signalling molecule. Abstract Reduced tissue availability of oxygen results from ascent to high altitude, where atmospheric pressure, and thus partial pressure of inspired oxygen, fall (hypobaric hypoxia). In humans, adaptation to such hypoxia is necessary for survival. These functional changes remain incompletely characterized, although metabolic adaptation (rather than simple increases in convective oxygen delivery) appear to play a fundamental role. Those populations which have remained native to high altitude have undergone natural selection for genetic variants associated with advantageous phenotypic traits. Interestingly, a consistent genetic signal has implicated alcohol metabolism in the human adaptive response to hypobaric hypoxia. The reasons for this remain unclear. One possibility is that increased alcohol synthesis occurs through fermentation by gut bacteria in response to enteric hypoxia. There is growing evidence that anaerobes capable of producing ethanol become increasingly prevalent with high altitude exposure. We thus hypothesise that (i) ascent to high-altitude renders the gut luminal environmental increasingly hypoxic, favouring (ii) an increase in the population of enteric fermenting anaerobes and thus (iii) the synthesis of alcohol which, through systemic absorption, leads to (iv) selection pressure on genes relating to alcohol metabolism. In theory, alcohol could be viewed as a toxic product, leading to selection of gene variants favouring its metabolism. On the other hand, alcohol is a metabolic substrate which might be beneficial. This mechanism could also account for some of the interindividual differences in lowlander acclimatisation to altitude. Future research should be aimed at determining any shifts to favour ethanol-producing anaerobes following ascent to altitude. This article is protected by copyright. All rights reserved.

Increases in cannabis use among young people has heightened concern about the potential interactive health effects of cannabis with other drugs. We examined the longitudinal association between concurrent and simultaneous (SAM) co-use of alcohol and cannabis in young adulthood on mental health symptoms, substance use behaviors, and substance-related harms two years later. Data were drawn from Time 5 (T5, n = 464, 46% male) and 6 (T6, n = 478, 45% male) of the Victoria Healthy Youth Survey. At T5, 42% of participants used alcohol-only, 13% used concurrently, 41% used SAM, 1% were cannabis only users, and 3% abstained from cannabis and alcohol. Boys were more likely to use SAM. Higher T5 SAM use frequency was associated with heavier use of substances, more substance-related harms, and symptoms of psychosis and externalizing problems at T6. T5 Concurrent use was associated with conduct symptoms, illicit drug use, and alcohol use disorders at T6 relative to alcohol-only use. Cannabis is commonly used with alcohol and the findings suggest that any co-use (concurrent or simultaneous) may be problematic in young adulthood. Public health messages need to explicitly inform consumers about the possible consequences of using both alcohol and marijuana and the addictive pharmacological impact of using them together.

Objective Serum potassium levels frequently are maintained at high levels (≥4.5 mEq/L) to prevent atrial fibrillation after cardiac surgery (AFACS), with limited evidence. Before undertaking a noninferiority randomized controlled trial to investigate the noninferiority of maintaining levels ≥3.6 mEq/L compared with this strategy, the authors wanted to assess the feasibility, acceptability, and safety of recruiting for such a trial. Design Pilot and feasibility study of full trial protocol. Setting Two university tertiary-care hospitals. Participants A total of 160 individuals undergoing first-time elective isolated coronary artery bypass grafting. Interventions Randomization (1:1) to protocols aiming to maintain serum potassium at either ≥3.6 mEq/L or ≥4.5 mEq/L after arrival in the postoperative care facility and for 120 hours or until discharge from the hospital or AFACS occurred, whichever happened first. Measurements and Main Results Primary outcomes: (1) whether it was possible to recruit and randomize 160 patients for six months (estimated 20% of those eligible); (2) maintaining supplementation protocol violation rate ≤10% (defined as potassium supplementation being inappropriately administered or withheld according to treatment allocation after a serum potassium measurement); and (3) retaining 28-day follow-up rates ≥90% after surgery. Between August 2017 and April 2018, 723 patients were screened and 160 (22%) were recruited. Potassium protocol violation rate = 9.8%. Follow-up rate at 28 days = 94.3%. Data on planned outcomes for the full trial also were collected. Conclusions It is feasible to recruit and randomize patients to a study assessing the impact of maintaining serum potassium concentrations at either ≥3.6 mEq/L or ≥4.5 mEq/L on the incidence of AFACS.

Focal adhesions are force sensitive structures that dynamically alter their composition, protein-protein interactions, and signaling in response to external mechanical stimuli. These dynamic changes are critical for focal adhesion function and are required for cellular mechanosensing. Here, we describe a simple protocol that allows for isolation of the focal adhesion complex from adherent cells in culture in response to different mechanical stimuli applied at adhesion sites. By combining this assay with approaches such as proteomics or western blot analysis, one can study the force-dependent changes in focal adhesion composition, protein-protein interactions and signaling.

Purpose: The purpose of this study is to investigate the potential interplay between opioid analgesia and tumor metastasis through modulation of μ-opioid receptor (MOR), Toll-like receptor 4 (TLR4) activation, and matrix degradation potential. Experimental Design: Plasma samples were collected from 60 patients undergoing elective lower limb joint replacement preoperatively and at 3, 6, and 24 hours after surgery; pain scores were documented at the same time points. Opioid administration was recorded and converted into morphine IV equivalents. Plasma samples were also collected from 10 healthy volunteers. Alphascreen cyclic AMP assay and MOR-overexpressing cells were employed to quantify MOR activation. HEK-Blue hTLR4 were utilized to measure TLR4 activation. Circulating matrix metalloprotease and tissue inhibitor of matrix protease activities were assessed by gelatin zymography and reverse zymography, respectively. Results: Postoperative plasma samples displayed the ability to activate MOR and to inhibit lipopolysaccharide (LPS)-induced TLR4 activation. Linear mixed model analysis revealed that MOR activation had a significant effect on inhibition of LPS-induced TLR4 activation. Furthermore, TLR4 had a significant effect to explain pain scores. Postoperative samples also displayed altered circulating matrix-degrading enzymes activity potential, but this was correlated neither to opioid administration nor to MOR activation potential. Conclusions: Our results show for the first time that (i) opioids administered to surgery patients result in modulation of ligand-induced TLR4 activation and (ii) postoperative pain is associated with increased circulating TLR4 activation potential. Our study further promotes the use of MOR activation potential rather than opioid intake in clinical studies measuring opioid exposure at a given time point. Clin Cancer Res; 24(10); 2319–27. ©2018 AACR.

Β2-Glycoprotein I (β2GPI) is an important anti-thrombotic protein and is the major auto-antigen in the antiphospholipid syndrome (APS). The clinical relevance of nitrosative stress in post translational modification of β2GPI was examined.The effects of nitrated (n)β2GPI on its anti-thrombotic properties and its plasma levels in primary and secondary APS were determined with appropriate clinical control groups. β2-glycoprotein I was nitrated at tyrosines 218, 275 and 309. β2-glycoprotein I binds to lipid peroxidation modified products through Domains IV and V. Nitrated β2GPI loses this binding (p

A series of polymers capable of self-assembling into infinite networks via supramolecular interactions have been designed, synthesized, and characterized for use in 3D printing applications. The biocompatible polymers and their composites with silica nanoparticles were successfully utilized to deposit both simple cubic structures, as well as a more complex twisted pyramidal feature. The polymers were found to be not toxic to a chondrogenic cell line, according to ISO 10993-5 and 10993-12 standard tests and the cells attached to the supramolecular polymers as demonstrated by confocal microscopy. Silica nanoparticles were then dispersed within the polymer matrix, yielding a composite material which was optimized for inkjet printing. The hybrid material showed promise in preliminary tests to facilitate the 3D deposition of a more complex structure.

An efficient and step-economic new approach to alkyl citrate natural products from a cyclobutene diester is presented. The key sequence involves a formal [2 + 2]-cycloaddition of a silylketene acetal with dimethylacetylene dicarboxylate to provide the cyclobutene diester 14 with 4.5:1 stereoselectivity. Exposure of diester 14 in acidic methanol effected a hydrolysis, intramolecular oxy-Michael reaction, and cyclobutanone methanolysis cascade to give the triester 15. Iodination and elimination then afforded a key alkyl citrate alkene intermediate, which was converted into the natural products (−)-CJ-13,982 (1), (−)-CJ-13,981 (2), and (−)-L-731,120 (3) via a cross-metathesis and subsequent reduction.

© 2017 The Royal Society of Chemistry. Material jetting is a process whereby liquid material can be deposited onto a substrate to solidify. Through a process of progressive additional layers, this deposition can then be used to produce 3D structures. However, the current material jetting catalogue is limited owing to the constraints on the viscosity of inks that can be deposited. Most inks currently being used are either solvent or photocuring based, with the latter becoming increasingly popular due to increased throughput. Full Reactive Inkjet Printing (FRIJP) is an alternative processing method currently being investigated as a route to widen the material catalogue. FRIJP is the combination, on the substrate, of two reactive components which then react together in contact on the substrate. In this work a two-part polydimethylsiloxane (PDMS) ink has been developed, printed individually, and cured. The successful printing of PDMS has been used to fabricate complex 3D geometry for the first time using FRIJP. Through the use of a prepared substrate feature resolutions up to 48 ± 2 μm (X, Y) were possible. Curing analysis has been conducted. It was found that not only does the reaction occur to a similar degree to conventional processes, but that there is no variation in the cured sample when printed at elevated substrate temperatures.

Crossbred steers (n=l,370) purchased from auction markets in Texas were received into a commercial feedlot near Hereford, Texas. Steers were recently weaned and considered at high risk of developing bovine respiratory disease (BRO). Upon arrival, steers were randomly assigned (within arrival blocks) to 1 of 3 treatment groups that received: 1) tilmicosin (TLM; 4.5 mg/lb (10.0 mg/kg)), 2) tildipirosin (TLD; 1.82 mg/lb ( 4.0 mg/kg)), or 3) tulathromycin (TUL; 1.14 mg/lb (2.5 mg/kg)). Steers were penned by treatment into 36 pens with 12 pens per treatment. Data were analyzed with linear mixed models for a randomized complete block design. Steers administered TUL had significantly lower BRO morbidity than both TLD and TLM (P< 0.01) treatments, and TLD had lower BRO morbidity than TLM (P < 0.05). Cattle receiving TUL also had a lower percentage of chronic illness than both TLD and TLM treatments (P < 0.05). Steers in the TUL treatment group had lower BRO and overall mortality at closeout than TLD (P < 0.05), but the TLM-treated steers did not differ from the other groups. Deads-in average daily gain was 0.55 lb (0.25 kg) greater in TUL-treated steers than steers administered TLM, and 0.56 lb (0.25 kg) greater than steers receiving TLD (P < 0.05). Cattle receiving TUL had 1.33 lb (0.6 kg) lower deads-in feed-to-gain ratio at closeout than those receiving TLM, and 1.51 lb (0.68 kg) lower than those receiving TLD (P < 0.05). During the first 30 days-onfeed, TUL-treated steers had greater daily dry-matter intake compared with TLD-treated steers (P < 0.01), but no other differences among treatment groups. By closeout, there were no differences in cumulative mean dry-matter intake between treatment groups. Overall, TUL treatment on arrival resulted in improved health and performance as compared to TLM and TLD treatments., The Bovine Practitioner, Vol. 51, No. 1 (2017 Spring)

OBJECTIVES To measure the percentage of the stated amount of S-adenosyl methionine present in a range of commercially available S-adenosyl methionine supplements for veterinary use. METHOD Sixty-four samples of products containing S-adenosyl methionine marketed to support liver function were obtained from five manufacturers via three commercial wholesalers. The amount of S-adenosyl methionine in each product was measured using high-pressure liquid chromatography. RESULTS There were greater than threefold variation in the percentage of measured S-adenosyl methionine compared to the stated amount on the packaging which was significantly (P

The pharmacokinetics and neurotoxicity of paraquat dichloride (PQ) were assessed following once weekly administration to C57BL/6J male mice by intraperitoneal injection for 1, 2 or 3 weeks at doses of 10, 15 or 25 mg/kg/week. Approximately 0.3% of the administered dose was taken up by the brain and was slowly eliminated, with a half-life of approximately 3 weeks. PQ did not alter the concentration of dopamine (DA), homovanillic acid (HVA) or 3,4-dihydroxyphenylacetic acid (DOPAC), or increase dopamine turnover in the striatum. There was inconsistent stereological evidence of a loss of DA neurons, as identified by chromogenic or fluorescent-tagged antibodies to tyrosine hydroxylase in the substantia nigra pars compacta (SNpc). There was no evidence that PQ induced neuronal degeneration in the SNpc or degenerating neuronal processes in the striatum, as indicated by the absence of uptake of silver stain or reduced immunolabeling of tyrosine-hydroxylase-positive (TH(+)) neurons. There was no evidence of apoptotic cell death, which was evaluated using TUNEL or caspase 3 assays. Microglia (IBA-1 immunoreactivity) and astrocytes (GFAP immunoreactivity) were not activated in PQ-treated mice 4, 8, 16, 24, 48, 96 or 168 h after 1, 2 or 3 doses of PQ. In contrast, mice dosed with the positive control substance, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 10mg/kg/dose×4 doses, 2 h apart), displayed significantly reduced DA and DOPAC concentrations and increased DA turnover in the striatum 7 days after dosing. The number of TH(+) neurons in the SNpc was reduced, and there were increased numbers of degenerating neurons and neuronal processes in the SNpc and striatum. MPTP-mediated cell death was not attributed to apoptosis. MPTP activated microglia and astrocytes within 4 h of the last dose, reaching a peak within 48 h. The microglial response ended by 96 h in the SNpc, but the astrocytic response continued through 168 h in the striatum. These results bring into question previous published stereological studies that report loss of TH(+) neurons in the SNpc of PQ-treated mice. This study also suggests that even if the reduction in TH(+) neurons reported by others occurs in PQ-treated mice, this apparent phenotypic change is unaccompanied by neuronal cell death or by modification of dopamine levels in the striatum.

In this study, we quantified the ability of opioids present in biological samples to activate the μ-opioid receptor and TLR4 using cell-based assays. Each assay was standardised, in the presence of plasma, using morphine, its μ receptor-active metabolite morphine-6 glucuronide (M6G) and its μ receptor-inactive, but TLR4-active metabolite morphine-3 glucuronide (M3G). Specificity was verified using antagonists. Morphine- and M6G-spiked plasma samples exhibited μ receptor activation, which M3G-spiked plasma lacked. In contrast, M3G showed moderate but consistent activation of TLR-4. Plasma samples were collected at a number of time points from mice administered morphine (1 or 10 mg/kg every 12 h for 3 days) or saline. Morphine administration led to intermittent μ receptor activation, reversed by μ receptor antagonists, and to TRL4 activation at time points where M3G is measured in plasma. Interestingly, this protocol of morphine administration also led to TLR4-independent NF-κB activation, at time points where M3G was not detected, presumably via elevation of circulating cytokines including, but not limited to, TNFα. Circulating TNFα was increased after three days of morphine administration, and TNFα mRNA elevated in the spleen of morphine-treated mice.

The neurotoxicity of paraquat dichloride (PQ) was assessed in two inbred strains of 9- or 16-week old male C57BL/6 mice housed in two different laboratories and compared to the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). PQ was administered by intraperitoneal injections; either once (20 mg/kg) or twice (10 mg/kg) weekly for 3 weeks, while MPTP-HCl was injected 4 times on a single day (20 mg/kg/dose). Brains were collected 8, 16, 24, 48, 96 or 168 hours after the last PQ treatment, and 48 or 168 hours after MPTP treatment. Dopamine neurons in the substantia nigra pars compacta (SNpc) were identified by antibodies to tyrosine hydroxylase (TH+) and microglia were identified using Iba-1 immunoreactivity. The total number of TH+ neurons and the number of resting and activated microglia in the SNpc at 168 hours after the last dose were estimated using model- or design-based stereology, with investigators blinded to treatment. In a further analysis, a pathologist, also blinded to treatment, evaluated the SNpc and/or striatum for loss of TH+ neurons (SNpc) or terminals (striatum), cell death (as indicated by amino cupric silver uptake, TUNEL and/or caspase 3 staining) and neuroinflammation (as indicated by Iba-1 and/or GFAP staining). PQ, administered either once or twice weekly to 9- or 16-week old mice from two suppliers, had no effect on the number of TH+ neurons or microglia in the SNpc, as assessed by two groups, each blinded to treatment, using different stereological methods. PQ did not induce neuronal cell loss or degeneration in the SNpc or striatum. Additionally, there was no evidence of apoptosis, microgliosis or astrogliosis. In MPTP-treated mice, the number of TH+ neurons in the SNpc was significantly decreased and the number of activated microglia increased. Histopathological assessment found degenerating neurons/terminals in the SNpc and striatum but no evidence of apoptotic cell death. MPTP activated microglia in the SNpc and increased the number of astrocytes in the SNpc and striatum.

Caffeine is widely consumed throughout the world, but little is known about the mechanisms underlying its rewarding and aversive properties. We show that pharmacological antagonism of dopamine not only blocks conditioned place aversion to caffeine, but also reveals dopamine blockade-induced conditioned place preferences. These aversive effects are mediated by the dopamine D(2) receptor, as knockout mice showed conditioned place preferences in response to doses of caffeine that C57Bl/6 mice found aversive. Furthermore, these aversive responses appear to be centrally mediated, as a quaternary analog of caffeine failed to produce conditioned place aversion. Although the adenosine A(2A) receptor is important for caffeine's physiological effects, this receptor seems only to modulate the appetitive and aversive effects of caffeine. A(2A) receptor knockout mice showed stronger dopamine-dependent aversive responses to caffeine than did C57Bl/6 mice, which partially obscured the dopamine-independent and A(2A) receptor-independent preferences. Additionally, the A(1) receptor, alone or in combination with the A(2A) receptor, does not seem to be important for caffeine's rewarding or aversive effects. Finally, excitotoxic lesions of the tegmental pedunculopontine nucleus revealed that this brain region is not involved in dopamine blockade-induced caffeine reward. These data provide surprising new information on the mechanism of action of caffeine, indicating that adenosine receptors do not mediate caffeine's appetitive and aversive effects. We show that caffeine has an atypical reward mechanism, independent of the dopaminergic system and the tegmental pedunculopontine nucleus, and provide additional evidence in support of a role for the dopaminergic system in aversive learning.

Neurotoxicity and mechanistic data were collected for six alpha-cyano pyrethroids (beta-cyfluthrin, cypermethrin, deltamethrin, esfenvalerate, fenpropathrin and lambda-cyhalothrin) and up to six non-cyano containing pyrethroids (bifenthrin, S-bioallethrin [or allethrin], permethrin, pyrethrins, resmethrin [or its cis-isomer, cismethrin] and tefluthrin under standard conditions. Factor analysis and multivariate dissimilarity analysis were employed to evaluate four independent data sets comprised of (1) fifty-six behavioral and physiological parameters from an acute neurotoxicity functional observatory battery (FOB), (2) eight electrophysiological parameters from voltage clamp experiments conducted on the Na(v)1.8 sodium channel expressed in Xenopus oocytes, (3) indices of efficacy, potency and binding calculated for calcium ion influx across neuronal membranes, membrane depolarization and glutamate released from rat brain synaptosomes and (4) changes in chloride channel open state probability using a patch voltage clamp technique for membranes isolated from mouse neuroblastoma cells. The pyrethroids segregated into Type I (T--syndrome-tremors) and Type II (CS syndrome--choreoathetosis with salivation) groups based on FOB data. Of the alpha-cyano pyrethroids, deltamethrin, lambda-cyhalothrin, cyfluthrin and cypermethrin arrayed themselves strongly in a dose-dependent manner along two factors that characterize the CS syndrome. Esfenvalerate and fenpropathrin displayed weaker response profiles compared to the non-cyano pyrethroids. Visual clustering on multidimensional scaling (MDS) maps based upon sodium ion channel and calcium influx and glutamate release dissimilarities gave similar groupings. The non-cyano containing pyrethroids were arrayed in a dose-dependent manner along two different factors that characterize the T-syndrome. Bifenthrin was an outlier when MDS maps of the non-cyano pyrethroids were based on sodium ion channel characteristics and permethrin was an outlier when the MDS maps were based on calcium influx/glutamate release potency. Four of six alpha-cyano pyrethroids (lambda-cyfluthrin, cypermethrin, deltamethrin and fenpropathrin) reduced open chloride channel probability. The R-isomers of lambda-l-cyhalothrin reduced open channel probability whereas the S-isomers, antagonized the action of the R-isomers. None of the non-cyano pyrethroids reduced open channel probability, except bioallethrin, which gave a weak response. Overall, based upon neurotoxicity data and the effect of pyrethroids on sodium, calcium and chloride ion channels, it is proposed that bioallethrin, cismethrin, tefluthrin, bifenthrin and permethrin belong to one common mechanism group and deltamethrin, lambda-cyhalothrin, cyfluthrin and cypermethrin belong to a second. Fenpropathrin and esfenvalerate occupy an intermediate position between these two groups.

ALTHOUGH a number of different types of urolith have been reported in dogs, including those associated with drugs and toxins such as sulphonamides, struvite (magnesium ammonium phosphate hexahydrate) accounts for approximately 40 per cent of canine urolithiasis cases and can be successfully treated with diet alone. Diet is also a central component in the treatment and management of calcium oxalate, urate and cystine uroliths, as well as playing a key role in preventing or reducing the risk of recurrence of virtually all types of urolith encountered. This article discusses some of the general current concepts regarding urolith formation, describes some dietary strategies that may be appropriate to try to prevent their recurrence, and briefly outlines possible predisposing factors.

Serum protein oxidation levels in people with the autoimmune disease systemic lupus erythematosus (SLE) have previously been shown to (a) be elevated at a single time point and (b) correlate with disease activity. This study investigates whether this elevation is a chronic phenomenon, by analysis of multiple serum samples collected from 21 SLE patients and nine controls over a period of up to 38 months. Protein thiols were chronically decreased in SLE patients with stable or variable disease activity compared to controls, whilst protein-bound carbonyls and glycine were chronically increased. 2D-gel analysis of carbonyl distribution showed albumin and immunoglobulins to be particularly affected. In SLE patients with stable disease activity, higher long-term protein oxidation correlated with higher long-term disease activity. SLE patients with variable disease activity exhibited varying correlations between protein oxidation and disease activity markers. These results further support a role for oxidative stress in the pathogenesis of SLE.

The work presented here had two main aims: first, it investigated the relationship between in-cylinder combustion performance and particulate size distribution and number count; second, it examined the possibility of reconstructing transient events using steady state test data relating to particulate size distribution and number count. It identified a strong correlation between the geometric mean diameter (GMD) of the engine-out particulate matter and the rate of burning during the early stage of combustion. In addition, it identified correlations between the coefficient of variance for 50 per cent mass fraction burned and the coefficient of variance for particulate GMD. The tests relating to transient events identified that steady state data can be used to reconstruct acceleration events, but not deceleration events. The methodology followed to obtain these findings is detailed in this paper.

Assessment of renal function by means of plasma clearance of a suitable marker has become standard procedure for estimation of glomerular filtration rate (GFR). Sinistrin, a polyfructan solely cleared by the kidney, is often used for this purpose. Pharmacokinetic modeling using adequate software is necessary to calculate disappearance rate and half-life of sinistrin. The purpose of this study was to describe the use of a Microsoft excel based add-in program to calculate plasma sinistrin clearance, as well as additional pharmacokinetic parameters such as transfer rates (k), half-life (t1/2) and volume of distribution (Vss) for sinistrin in dogs with varying degrees of renal function.

PedvaxHIB is a pediatric vaccine that protects children from severe disease caused by the gram-negative bacterium Haemophilus influenzae type b (Hib). The vaccine is made by chemically conjugating Hib capsular polysaccharide to the outer membrane protein complex of Neisseria meningitidis. The protein-conjugated vaccine has proven to be extremely effective in preventing invasive Hib disease in infants and young children. This paper presents the nuclear magnetic resonance (NMR) methodology for the quantitative characterization of derivatized polysaccharide and its validation closely following ICH guidelines. The assay has been shown to be precise and accurate (relative standard deviation [RSD]

Purified capsular polysaccharide preparations from Streptococcus pneumoniae that are used for vaccine production typically contain residual levels of C-polysaccharide (C-Ps). Residual C-Ps is typically found in one of two forms, either chemically linked to the capsular polysaccharide (bound) or present by itself (free). Two analytical methods have been developed and applied to determine the relative percentages of the two C-Ps forms present in various capsular polysaccharide preparations. Both methods differentiate the two forms of C-Ps according to the difference of their hydrodynamic sizes. One method is based on labeling C-Ps with a fluorescent tag and separating the two forms of C-Ps by high-performance size exclusion chromatography with on-line refractive index and fluorescence detection, and the other method is based on measuring self-diffusion rates of the two forms of C-Ps by nuclear magnetic resonance (NMR) and quantifying each form with deconvolution. Both methods were evaluated for relative accuracy, precision, and ease of application, and they were found to provide comparable results for a large number of pneumococcal polysaccharide preparations. These analyses, combined with other quantitative NMR measurement of total C-Ps in the polysaccharide powder, provide a more refined means of evaluating the amount of each form of C-Ps in polysaccharide preparations targeted for vaccine production.

Objective To examine protein oxidation in systemic lupus erythematosus (SLE) and to correlate levels of protein oxidation products with disease activity. Methods Serum was collected from SLE patients and healthy control subjects. Protein-bound carbonyls and the pro-oxidant enzyme myeloperoxidase (MPO) were quantified by enzyme-linked immunosorbent assay. Protein thiols were quantified using 5,5′-dithionitrobenzoic acid. Protein-bound amino acids and methionine, tyrosine, and phenylalanine oxidation products were quantified by acid hydrolysis and high-performance liquid chromatography. Disease activity was assessed by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Levels of anti–double-stranded DNA (anti-dsDNA) antibodies were measured by radioimmunoassay. Results Compared with control subjects, SLE patients exhibited elevated levels of protein carbonyls (0.108 ± 0.078 versus 0.064 ± 0.028 nmoles/mg of protein; P = 0.046), decreased levels of protein thiols (3.9 ± 1.1 versus 4.9 ± 0.7 nmoles/mg of protein; P = 0.003), decreased levels of protein-bound methionine (P = 0.0007), and increased levels of protein-bound methionine sulfoxide (P = 0.0043) and 3-nitrotyrosine (P = 0.0477). SLE patients with high SLEDAI scores or elevated anti-dsDNA antibody levels exhibited increased oxidation compared with patients with low SLEDAI scores or low antibody levels. Serum MPO levels were decreased in SLE patients (P = 0.03), suggesting that this enzyme is not responsible for the enhanced protein oxidation. Conclusion We found elevated levels of multiple markers of protein oxidation in sera from SLE patients compared with controls, and these levels correlated with disease activity. The findings suggest that protein oxidation may play a role in the pathogenesis of chronic organ damage in SLE.

Grinding tests were performed using a laboratory batch ball mill to investigate the grindability of pure sulphur and examine the influence of selected grinding parameters (slurry concentration, grinding time, wetting agent solution strength, and mass of solid) on the process. Experiments were conducted in accordance with the Box–Wilson experimental design method, and the grinding data obtained were used to establish mathematical models and set up a computer simulation program. Results have indicated that the highest grinding rates would be achieved with a slurry concentration of 70 wt.% solid and a wetting agent [sodium lignosulphonate (SL)] solution strength of 7.5%, but the right combination of solid mass and grinding time would need to be selected based on the product size required. Particle size distributions were represented by a modified Rosin–Rammler–Bennett (RRB) function, which provided an excellent fit to the measured size analysis data.

Continuous wave (CW) and equivalently powered, pulsed radio frequency plasmas were used to deposit thin films of titanium dioxide on to Si(1 0 0) substrates. Plasmas were created by feeding oxygen gas through the coil region, TIP was introduced downstream from the coil region. These films have been characterized using XPS, SEM and AES. Results show high quality amorphous films were produced with some carbon incorporation. Sputtering the TiO2 films to the Si interface results in the formation of TiSi2. Annealing the films in vacuum at 900 °C results in the reduction of TiO2 to Ti2O3 at the interfacial region, while Si is oxidized to SiOx. Upon annealing at 950 °C, further reduction of the TiO2 film was noted to include TiO. The 950 °C film is partially reoxidized upon exposure to atmosphere. SEM of the TiO2 film surfaces showed the as deposited films were smooth and structureless and remained so even after being annealed at 850 °C. Annealing at 950 °C, however, caused the surface to become very rough, resulting in the rupturing of the TiO2 surface, thus exposing areas of the underlying Si/SiO2 substrate.

Neurotoxicity of paraquat and paraquat-induced mechanisms of developing Parkinson’s disease

We have developed a novel method to study the interactions of nucleic acids with cationic species. The method, called phosphorus relaxation enhancement (PhoRE), uses (1)H-detected (31)P NMR of exogenous probe ions to monitor changes in the equilibrium between free Mn(2+) and Mn(2+) bound to the RNA. To demonstrate the technique, we describe the interactions of four RNA molecules with metal ions (K(+) and Mg(2+)), a small molecule drug (neomycin b), and a cationic peptide (RSG1.2). In each case, cationic ligand binding caused Mn(2+) to be displaced from the RNA. Free Mn(2+) was determined from its effect on the T(2) NMR relaxation rate of either phosphite (HPO(3)(2-)) or methyl phosphite (MeOPH, CH(3)OP(H)O(2-)). Using this method, the effects of [RNA] as low as 1 microM could be measured in 20 min of accumulation using a low field (200 MHz) instrument without pulsed field gradients. Cation association behavior was sequence and [RNA] dependent. At low [K(+)], Mn(2+) association with each of the RNAs decreased with increasing [K(+)] until approximately 40 mM, where saturation was reached. While saturating K(+) displaced all the bound Mn(2+) from a 31-nucleotide poly-uridine (U(31)), Mn(2+) remained bound to each of three hairpin-forming sequences (A-site, RRE1, and RRE2), even at 150 mM K(+). Bound Mn(2+) was displaced from each of the hairpins by Mg(2+), allowing determination of Mg(2+) dissociation constants (K(d,Mg)) ranging from 50 to 500 microM, depending on the RNA sequence and [K(+)]. Both neomycin b and RSG1.2 displaced Mn(2+) upon binding the hairpins. At [RNA] approximately 3 microM, RRE1 bound a single equivalent of RSG1.2, whereas neither RRE2 nor A-site bound the peptide. These behaviors were confirmed by fluorescence polarization using TAMRA-labeled peptide. At 2.7 microM RNA, the A-site hairpin bound a single neomycin b molecule. The selectivity of RSG1.2 binding was greatly diminished at higher [RNA]. Similarly, each hairpin bound multiple equivalents of neomycin at the higher [RNA]. These results demonstrate the utility of the PhoRE method for characterizing metal binding behaviors of nucleic acids and for studying RNA/ligand interactions.

This review highlights the synthesis of members of the alkyl citrate family of natural products. The focus is on the stereoselective construction of the alkyl citrate moiety common to these compounds.

This review highlights the synthesis of members of the alkyl citrate family of natural products. The focus is on the stereoselective construction of the alkyl citrate moiety common to these compounds.

SUMMARYThe diagnosis of the antiphospholipid syndrome (APS) requires both a typical clinical event plus a persistently positive test in an assay for either anticardiolipin (aCL) antibodies or a lupus anticoagulant (LA). Enzyme linked immunosorbent assays (ELISA) specific for autoantibodies against β2-glycoprotein I (β2GPI) or prothrombin are also used, but none of the tests are adequately sensitive or specific. A chromogenic assay was developed that measures the effect of test antibody or plasma samples on in vitro thrombin formation. It is able to detect both LA and β2GPI-dependent aCL antibodies and may have greater specificity for APS than currently available tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with β2GPI, mice with a spontaneous animal model of APS, and from three humans with APS. Plasma and affinity purified antibodies from patients with APS and control groups were also examined. Thrombin inhibition was more sensitive to perturbation by MoAbs than a combination of tests for LA (P

The phenomenon of shell wiping in Calliostoma zizyphinum (L.) was investigated with regard to its potential function as a feeding adaptation. It was determined that C. zizyphinum will, on average, wipe the surface of its shell with its foot twice every 24 h, in the process consuming any mat- ter that has adhered to the pedal mucus present on the surface of its shell. Examination of the impor- tance of shell wiping to C. zizyphinum, both as a mechanism to reduce shell fouling and as a source of nutrition, revealed that the prevention of shell wiping resulted in a reduction in shell growth by a factor of 0.67 and an increase in shell fouling by a factor of 9. Measurement of the metabolic rate of C. zizyphinum ascertained that an average sized adult had a metabolic requirement of ~117 J 24 h -1 . Examination of the ability of the pedal mucus produced by C. zizyphinum to act as a provendering agent showed that the pedal mucus produced by C. zizyphinum could increase the entrapment of particulate matter by a factor of 8, and increase the chlorophyll a content of a substratum by a factor of 3. Calculation of the potential contribution of the matter trapped by pedal mucus, which is ingested through shell wiping, to the daily energetic requirements of C. zizyphinum revealed that shell wip- ing can contribute to approximately one-fifth of the daily energetic requirement of C. zizyphinum. Shell wiping therefore has an important role for both feeding and antifouling in C. zizyphinum.

SUMMARYIL-17 is a T cell cytokine with a complex and important role in the immune system. It has been detected in rheumatoid arthritis (RA) synovial membrane and found to stimulate the production of the proinflammatory cytokines IL-6, IL-8, tumour necrosis factor-alpha (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. To date, there are few data available on the agents that stimulate IL-17 production. We therefore investigated the in vitro IL-17 response to a variety of mitogens and antigens, and compared the IL-17 response to interferon-gamma (IFN-γ), IL-4, IL-10 and TNF-α. In this study we used a type-0 antigen, tetanus toxoid (TT), a type-1 antigen, PPD from Mycobacterium tuberculosis, a potential type-2 rye grass (RG) antigen (Lol I) and an autoantigen SS.B (La), to stimulate PBMC from healthy controls. Cytokine mRNA was measured using semiquantitative reverse transcriptase-polymerase chain reaction and cytokine protein measured using specific ELISA techniques, while the frequency of IL-17-producing T cells was determined by flow cytometry. The mitogens concanavalin A, phytohaemagglutinin and phorbol myristate acetate/ionomycin induced a significant increase in IL-17, with the highest levels being produced by anti-CD3/anti-CD28 stimulation. The antigens TT and PPD significantly increased IL-17 mRNA expression over time, but failed to have such an effect at the protein level. IL-17 protein was also detectable in both antigen-specific (TT, SS.B) and non-specific T cell clones, but at levels lower than IFN-γ. IL-17 production did not correlate with either the type-1 cytokine IFN-γ or TNF-α or the type-2 cytokine IL-4 or IL-10 at either the mRNA or protein level.

The monohalomethane methyl iodide (MeI) is toxic to a number of organ systems including the central nervous system. Clinical symptoms of neurotoxicity suggest that the cerebellum is the target within the brain, and we have now modelled the toxicity of MeI in cultured rat cerebellar granule cells. Cytotoxicity is maximal 24 h after a 5 min exposure to MeI, and the EC50 for MeI under these conditions was calculated to be 1.6 mM. The glutathione S-transferase (GST) dependent metabolism of MeI was investigated in these cultures. There was a marked decrease in intracellular glutathione (GSH) 15 min after exposure to MeI, and GSH concentrations then increased, reaching 130% of control levels 7 h after exposure. To investigate the role of conjugation with GSH in the toxicity of MeI, GSH levels were modulated prior to exposure. Depletion of GSH exacerbated the cytotoxicity of MeI while provision of a bioavailable source of GSH was protective. Inclusion of antioxidants [vitamin E, butylated hydroxytoluene (BHT) or desferrioxamine mesylate (DF)] also protected against the cytotoxicity of MeI. Our in vitro data suggest that MeI is conjugated with GSH in the cerebellum, and the resulting extensive depletion of GSH may be the first step en route to toxicity, rendering the tissue susceptible to methylation and/or oxidative stress.

The glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor, given its potential as pharmacological target, has been thoroughly studied by structure-activity relationships, which has made possible its description in terms of spatial limits and interactions of various types. A structural model, based on mutational analysis and sequence alignements, has been proposed. Yet, the amino acid residues responsible for the interactions with the ligand have not been unambiguously characterized. To evidence nucleophilic pocket-lining residues, we have designed and synthesized reactive glycine-site ligands derived from 3-substituted 4-hydroxy-quinolin-2(1H)-ones by introducing various electrophilic groups at different positions of the molecule. These ligands were found to have high affinity at the glycine site and to be functional antagonists by inhibiting glycine/glutamate-induced currents in transfected oocytes. The correlation between their potency and their substitution pattern was strictly consistent with previously established structure-activity relationships. Most ligands displayed intrinsic reactivity toward cysteine, but none inactivated wild-type receptors. This is consistent with the model since it indicates the absence of exposed cysteine in the glycine-binding site. A strategy of cysteine incorporation by point mutations at selected polypeptide positions will create unambiguously localized targets for our reactive probes.

An experiment was carried out to examine the effects of 40 mg of caffeine given in different drinks (coffee, water, tea, cola) on mood and performance. One hundred and forty-four volunteers were randomly assigned to one of the groups formed by combining the caffeine/placebo and drinks conditions. Following a baseline session measuring mood and different aspects of performance, the volunteers were given their drink and then carried out another test session 1 h later. Administration of the caffeine/placebo was double-blind. The results showed that those given caffeine reported greater alertness and anxiety at the end of the test session, as well as improved performance on choice reaction time tasks involving focused attention and categoric search, a semantic memory task and a delayed recognition memory task. The effect of the caffeine was not modified by the nature of the drink in which it was given. Overall, these results show that a dose of caffeine typical of the level found in commercial products can improve alertness and performance efficiency.

The design and development of low-emission, lean premixed aeroand industrial gas-turbine combustors is challenging because of a need to satisfy conflicting requirements of operability, combustion performance, and low emissions. A toroidal well-stirred reactor (WSR) is a laboratory idealization of a compact primary zone of a gas-turbine combustor. Such a WSR was designed, built, and operated at atmospheric pressure to study lean blowout and emissions of CO, unburned hydrocarbons, and NOV over a range of residence time and fuel-air ratios. A variety of normal and cyclic alkanes, aromatics, and a blend of pure hydrocarbon fuels were tested. Results showed that fuel type affects lean blowout limits, combustion efficiency, and pollutant emissions. These observations have important practical implications to the design of gas-turbine combustors.

The glycine co-agonist binding site of the NMDA receptor is a target for the prevention and treatment of neurotoxic and neurodegenerative conditions. Until now, the interactions taking place at this site, and its structure, have been investigated by ligand structure-activity relationships and by site-directed mutagenesis. On the basis of a structural model which is currently proposed for this site, we have designed and synthesized six affinity markers by substituting electrophilic reactive groups in the 4, the 7 and the 3' positions of L 701,324, a high-affinity glycine site antagonist. These compounds compete with 3H-DCKA binding to rat brain membranes at equilibrium with nanomolar to low-micromolar affinities, and antagonize glycine-evoked currents in oocytes transfected with wild-type NR1-NR2B. However, they do not induce a time-shift in binding equilibria, and do not inactivate irreversibly the glycine evoked currents. Since they react only with cysteine at physiological pH, we conclude that there is no such residue in the site, in agreement with the model. Our affinity markers therefore represent potential topological probes for NMDA receptors with sequence positions related to the glycine-binding site mutated into cysteine.

We have measured the binding affinity (KA) and electron transfer (ET) rate constants (k) for the complex of hemoglobin (Hb) and cytochrome b5 (b5), using triplet quenching titrations of mixed-metal [ZnM, Fe3+(N3-)] Hb hybrids and of fully substituted Zn−mesoporphyrin (ZnM)Hb by b5 (trypsin-solubilized, bovine) (pH values 6.0 and 7.0). The use of the mixed-metal Hb hybrids with Zn in one chain type allows us to selectively monitor the 3ZnP → Fe3+P ET reaction of Fe3+b5 with either the α-chains or the β-chains. The self-consistent analysis of the results for the mixed-metal hybrids and those for the (ZnM)Hb allows us to determine the reactivity and affinity constants for the interactions of b5 with the individual subunits of T-state Hb. The results confirm that ET occurs within a complex between b5 and Hb, not through a simple bimolecular collision process. At pH 6.0, the binding affinity constant of the α-chains (Kα ≈ 2.0 × 104 M-1) is ∼4-fold larger than that of the β-chains (Kβ = 4.9 × 103 M-1); the intr...

A homology model of the AMPA receptor has been employed to construct a QSAR model of the AMPA receptor antagonist binding site.

NMDA receptors require both L-glutamate and the coagonist glycine for efficient channel activation. The glycine binding site of these heteromeric receptor proteins is formed by regions of the NMDAR1 (NR1) subunit that display sequence similarity to bacterial amino acid binding proteins. Here, we demonstrate that the glutamate binding site is located on the homologous regions of the NR2B subunit. Mutation of residues within the N-terminal domain and the loop region between membrane segments M3 and M4 significantly reduced the efficacy of glutamate, but not glycine, in channel gating. Some of the mutations also decreased inhibition by the glutamate antagonists, D-AP5 and R-CPP. Homology-based molecular modeling of the glutamate and glycine binding domains indicates that the NR2 and NR1 subunits use similar residues to ligate the agonists' α-aminocarboxylic acid groups, whereas differences in side chain interactions and size of aromatic residues determine ligand selectivity.

Several investigations have reported that mice administered paraquat dichloride (PQ·Cl2) by intraperitoneal injection exhibit a loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). In this study, male and female C57BL/6J mice were administered PQ·Cl2 in the diet at concentrations of 0 (control), 10, and 50ppm for a duration of 13weeks. A separate group of mice were administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) during week 12 as positive controls to produce a loss of dopaminergic neurons in the SNpc. The comparative effects of PQ and MPTP on the SNpc and/or striatum were assessed using neurochemical, neuropathological, and stereological endpoints. Morphological and stereological assessments were performed by investigators ‘blinded’ to the origin of the tissue. Neither dose of PQ·Cl2 (10 or 50 ppm in the diet) caused a loss of striatal dopamine or dopamine metabolite concentrations in the brains of mice. Pathological assessments of the SNpc and striatum showed no evidence of neuronal degeneration or astrocytic/microglial activation. Furthermore, the number of tyrosine hydroxylase-positive (TH+) neurons in the SNpc was not reduced in PQ-treated mice. In contrast, MPTP caused a decrease in striatal dopamine concentration, a reduction in TH+ neurons in the SNpc, and significant pathological changes including astrocytic and microglial activation in the striatum and SNpc. The MPTP-induced effects were greater in males than in females. It is concluded that 13weeks of continuous dietary exposure of C57BL/6J mice to 50ppm PQ·Cl2 (equivalent to 10.2 and 15.6mg PQ ion/kg body weight/day for males and females, respectively) does not result in the loss of, or damage to, dopaminergic neurons in the SNpc.