Quantitative charge-tags for sterol and oxysterol analysis.

BACKGROUND: Global sterol analysis is challenging owing to the extreme diversity of sterol natural products, the tendency of cholesterol to dominate in abundance over all other sterols, and the structural lack of a strong chromophore or readily ionized functional group. We developed a method to overcome these challenges by using different isotope-labeled versions of the Girard P reagent (GP) as quantitative charge-tags for the LC-MS analysis of sterols including oxysterols. METHODS: Sterols/oxysterols in plasma were extracted in ethanol containing deuterated internal standards, separated by C18 solid-phase extraction, and derivatized with GP, with or without prior oxidation of 3ß-hydroxy to 3-oxo groups. RESULTS: By use of different isotope-labeled GPs, it was possible to analyze in a single LC-MS analysis both sterols/oxysterols that naturally possess a 3-oxo group and those with a 3ß-hydroxy group. Intra- and interassay CVs were <15%, and recoveries for representative oxysterols and cholestenoic acids were 85%-108%. By adopting a multiplex approach to isotope labeling, we analyzed up to 4 different samples in a single run. Using plasma samples, we could demonstrate the diagnosis of inborn errors of metabolism and also the export of oxysterols from brain via the jugular vein. CONCLUSIONS: This method allows the profiling of the widest range of sterols/oxysterols in a single analytical run and can be used to identify inborn errors of cholesterol synthesis and metabolism.

Revised sample preparation for the analysis of oxysterols by enzyme-assisted derivatisation for sterol analysis (EADSA).

Sterols, and specifically oxysterols, play important roles in the biosynthesis of bile acids and steroid hormones as well as possessing biological activities in their own right. Analysis of oxysterols is complicated due to their low abundance in biological systems and poor ionisation characteristics in mass spectrometry. Over the past decade, we have developed a liquid chromatography-mass spectrometry method termed enzyme-assisted derivatisation for sterol analysis (EADSA). Our derivatisation procedure relies on two solid-phase extraction steps to (i) separate cholesterol from oxysterols and (ii) remove excess derivatisation reagents. Recent inter-batch variation in C18 reversed-phase cartridges has led us to experiment with alternative columns. Here, we present our findings and report an improved sample preparation procedure using polymeric hydrophilic-lipophilic balanced reversed-phase cartridges.

Calculated third order rate constants for interpreting the mechanisms of hydrolyses of chloroformates, carboxylic Acid halides, sulfonyl chlorides and phosphorochloridates.

Hydrolyses of acid derivatives (e.g., carboxylic acid chlorides and fluorides, fluoro- and chloroformates, sulfonyl chlorides, phosphorochloridates, anhydrides) exhibit pseudo-first order kinetics. Reaction mechanisms vary from those involving a cationic intermediate (SN1) to concerted SN2 processes, and further to third order reactions, in which one solvent molecule acts as the attacking nucleophile and a second molecule acts as a general base catalyst. A unified framework is discussed, in which there are two reaction channels-an SN1-SN2 spectrum and an SN2-SN3 spectrum. Third order rate constants (k3) are calculated for solvolytic reactions in a wide range of compositions of acetone-water mixtures, and are shown to be either approximately constant or correlated with the Grunwald-Winstein Y parameter. These data and kinetic solvent isotope effects, provide the experimental evidence for the SN2-SN3 spectrum (e.g., for chloro- and fluoroformates, chloroacetyl chloride, p-nitrobenzoyl p-toluenesulfonate, sulfonyl chlorides). Deviations from linearity lead to U- or V-shaped plots, which assist in the identification of the point at which the reaction channel changes from SN2-SN3 to SN1-SN2 (e.g., for benzoyl chloride).

Evaluation of novel derivatisation reagents for the analysis of oxysterols.

Oxysterols are oxidised forms of cholesterol that are intermediates in the synthesis of bile acids and steroid hormones. They are also ligands to nuclear and G protein-coupled receptors. Analysis of oxysterols in biological systems is challenging due to their low abundance coupled with their lack of a strong chromophore and poor ionisation characteristics in mass spectrometry (MS). We have previously used enzyme-assisted derivatisation for sterol analysis (EADSA) to identify and quantitate oxysterols in biological samples. This technique relies on tagging sterols with the Girard P reagent to introduce a charged quaternary ammonium group. Here, we have compared several modified Girard-like reagents and show that the permanent charge is vital for efficient MS(n) fragmentation. However, we find that the reagent can be extended to include sites for potential stable isotope labels without a loss of performance.

Nucleophilicity parameters for amines, amino acids and peptides in water. Variations in selectivities for quinone methides.

Second order rate constants for reactions of 4,4'-dimethylaminobenzhydrylium cations with amines and other nucleophiles in water define a scale of nucleophilicity (N(+)'' = log k + 2.63). The N(+)'' scale can be extended by linking directly to an established N(+) scale based on reactions of methyl vinyl pyridinium cations with amine nucleophiles. Logarithms of rate constants for other benzhydrylium ions and quinone methides (QMs) are correlated by the equation: log k = s(E)N(+)'' + constant, having a nucleophilicity parameter (N(+)'' defined as in the Ritchie N(+) equation with N(+)'' = 4.75 for hydroxide ion), and an electrophile's response (selectivity) parameter (s(E), as in the Swain-Scott equation). Correlations for other benzhydrylium cations require only one slope and one intercept per cation, and fit data for up to 54 amines, amino acids and peptide nucleophiles; the slope s(E) increases as the reactivity of the cation decreases. Contrary to recent reports, s(E) is significantly less than unity for reactions of o- and p-benzoquinone methides. As the reactivities of QMs decrease, s(E) increases and the response of s(E) to changes in reactivity is larger for QMs than for cations.

Methods for improving efficiency in quality measurement: the example of pain screening.

OBJECTIVE: Collecting unnecessary data when assessing quality of care wastes valuable resources. We evaluated three approaches for estimating quality-measure adherence and determined minimum visit data required to achieve accurate estimates. DESIGN: We abstracted medical records for calculating physician-level pain screening rates as: visit-specific, using single-visit data for each patient; visit-level average, using data for all patients and visits; and patient-level average, using data from a subset of patients and visits. SETTING: VA Greater Los Angeles Health-care System, 2006. PARTICIPANTS: One hundred and six patients with Stage IV solid tumors. INTERVENTION: Pain screening at every medical encounter, measured by a 0-10 numeric rating scale and reported to the national Medicare insurance program under a 'pay-for-reporting' program. MAIN OUTCOME MEASURES: Amount of visit data needed to reach the smallest 95% confidence interval (CI) and stable pain screening estimates. RESULTS: Pain screening occurred at 22% (23/106; 95% CI: 14-30%) of initial visits and 50% (8/16; 95% CI: 25-75%) of single visits. Across all visits, screening adherence averaged 34% when estimated at the visit-level precision and 30% at the patient level. Maximum patient-level precision was reached at visit 4 (95% CI: ± 8%) and visit level at visit 14 (95% CI: ± 6%). Using patient-level and visit-level approaches, estimates stabilized at visits 8 and 11, respectively, and reached within 1 percentage point of the steady-state value at visits 4 and 9. CONCLUSION: To address low-pain screening among cancer patients, an oncology pain screening measure may be most efficiently evaluated with data from a sample of patients and visits. This approach may be valid for visit-level quality measures in other settings.

Structural effects on the solvolytic reactivity of carboxylic and sulfonic acid chlorides. Comparisons with gas-phase data for cation formation.

Kinetic data for solvolyses of 28 acid chlorides in 97% w/w trifluoroethanol (TFE)-water spanning over 10 (9) in rate constant at 25 degrees C are obtained directly or by short extrapolation from published values. G3 calculations of the energy required for cation formation in the gas phase are validated from proton affinities and from other experimental data. G3 calculations of heterolytic bond dissociation enthalpies (HBDEs) for formation of cations from acid chlorides in the gas phase show the following trends when compared with the solvolysis rate constants: (i) electron-rich sulfonyl chlorides and most carboxylic acid chlorides, including thione derivatives, give a satisfactory linear correlation with a significant negative slope; (ii) most sulfonyl chlorides and some chloroformates and thio derivatives have higher HBDEs and fit another correlation with a small, negative slope. A significant deviation is observed for the acyl series (RCOCl), for which both solvolysis rates and HBDEs increase in the order R = Bu ( t ) < Pr ( i ) < Et < Me. The deviation may be explained either by a prior hydration mechanism or preferably by electrostatic effects on the formation of small cations. The above results of structural effects support independent evidence from solvent effects that cationic ionization reaction pathways (with nucleophilic solvent assistance or S N2 character) are involved in the solvolyses of acid chlorides.

Characterization of a resistance locus (Pfs-1) to the spinach downy mildew pathogen (Peronospora farinosa f. sp. spinaciae) and development of a molecular marker linked to Pfs-1.

Downy mildew is a destructive disease of spinach worldwide. There have been 10 races described since 1824, six of which have been identified in the past 10 years. Race identification is based on qualitative disease reactions on a set of diverse host differentials which include open-pollinated cultivars, contemporary hybrid cultivars, and older hybrid cultivars that are no longer produced. The development of a set of near-isogenic open-pollinated spinach lines (NILs), having different resistance loci in a susceptible and otherwise common genetic background, would facilitate identification of races of the downy mildew pathogen, provide a tool to better understand the genetics of resistance, and expedite the development of molecular markers linked to these disease resistance loci. To achieve this objective, the spinach cv. Viroflay, susceptible to race 6 of Peronospora farinosa f. sp. spinaciae, was used as the recurrent susceptible parent in crosses with the hybrid spinach cv. Lion, resistant to race 6. Resistant F(1) progeny were subsequently backcrossed to Viroflay four times with selection for race 6 resistance each time. Analysis of the segregation data showed that resistance was controlled by a single dominant gene, and the resistance locus was designated Pfs-1. By bulk segregant analysis, an amplified fragment length polymorphism (AFLP) marker (E-ACT/M-CTG) linked to Pfs-1 was identified and used to develop a co-dominant Sequence characterized amplified region (SCAR) marker. This SCAR marker, designated Dm-1, was closely linked ( approximately 1.7 cM) to the Pfs-1 locus and could discriminate among spinach genotypes that were homozygous resistant (Pfs-1Pfs-1), heterozygous resistant (Pfs-1pfs-1), or homozygous susceptible (pfs-1pfs-1) to race 6 within the original mapping population. Evaluation of a wide range of commercial spinach lines outside of the mapping population indicated that Dm-1 could effectively identify Pfs-1 resistant genotypes; the Dm-1 marker correctly predicted the disease resistance phenotype in 120 out of 123 lines tested. In addition, the NIL containing the Pfs-1 locus (Pfs-1Pfs-1) was resistant to multiple races of the downy mildew pathogen indicating Pfs-1 locus may contain a cluster of resistance genes.

Cost Effectiveness of Metal Stents in Relieving Obstructive Jaundice in Patients with Pancreatic Cancer.

BACKGROUND: ASGE and ESGE guidelines recommend endoscopic metal stent placement for pancreatic carcinoma patients with biliary obstruction, and whose estimated life expectancy is greater than 6 months. Because median overall survival (OS) of metastatic pancreatic adenocarcinoma until recently has been less than 6 months, plastic biliary stents were preferentially placed rather than metal due to the greater upfront cost of the latter. Recent advances in the treatment of metastatic pancreatic cancer have extended median OS beyond the 6-month range. Given this improvement in OS, we performed a cost-effectiveness analysis of initial metal biliary versus plastic stent placement in metastatic pancreatic cancer patients with biliary obstruction. METHODS: A Markov model was developed to predict lifetime costs, quality-adjusted life years (QALYs), and cost effectiveness of metal compared with plastic stents. Adult patients entered the model with locally advanced cancer and underwent endoscopic retrograde cholangiopancreatography (ERCP) with placement of metal or plastic stents. A targeted literature search was conducted to identify published sources, which were used to estimate clinical, cost, utility, and event rate inputs to the model. Results were estimated from the third-party payer perspective in 2012 US dollars per QALY. One-way and probabilistic sensitivity analyses were conducted to assess the impact on model outcomes resulting from uncertainty among inputs. RESULTS: Our analysis found that initial placement of metal stents was more cost effective than plastic biliary stents with lower overall costs due to lower restenting rates while at the same time associated with a better quality of life. Based on model projections, placement of metal stents could save approximately $1450 per patient over a lifetime, while simultaneously improving quality of life. These findings were robust in sensitivity analyses. CONCLUSIONS: Placement of metal biliary stents at initial onset of obstructive jaundice in adult patients with metastatic pancreatic carcinoma with an expected OS greater than 6 months was found to be a more cost-effective strategy than plastic stents. These results reinforce guidelines' suggestions for metal stent placement.

The application of an industry level participatory ergonomics approach in developing MSD interventions.

Participatory ergonomics projects are traditionally applied within one organisation. In this study, a participative approach was applied across the New Zealand meat processing industry, involving multiple organisations and geographical regions. The purpose was to develop interventions to reduce musculoskeletal disorder (MSD) risk. This paper considers the value of an industry level participatory ergonomics approach in achieving this. The main rationale for a participative approach included the need for industry credibility, and to generate MSD interventions that address industry level MSD risk factors. An industry key stakeholder group became the primary vehicle for formal participation. The study resulted in an intervention plan that included the wider work system and industry practices. These interventions were championed across the industry by the key stakeholder group and have extended beyond the life of the study. While this approach helped to meet the study aim, the existence of an industry-supported key stakeholder group and a mandate for the initiative are important prerequisites for success.

The role of organisational support in teleworker wellbeing: a socio-technical systems approach.

The prevalence of telework and other forms of mobile working enabled by digital technology is increasing markedly. Following a socio-technical systems approach, this study aims to examine the role of organisational social support and specific support for teleworkers in influencing teleworker wellbeing, the mediating role of social isolation, potentially resulting from a person-environment mismatch in these relationships, and possible differences in these relationships between low-intensity and hybrid teleworkers. Teleworkers' (n = 804) perceptions of support and telework outcomes (psychological strain, job satisfaction, and social isolation) were collected using an on-line survey of teleworking employees distributed within 28 New Zealand organisations where knowledge work was undertaken. Organisational social support and teleworker support was associated with increased job satisfaction and reduced psychological strain. Social isolation mediated the relationship between organisational social support and the two outcome variables, and some differences were observed in the structural relationships for hybrid and low-intensity teleworker sub-samples. These findings suggest that providing the necessary organisational and teleworker support is important for enhancing the teleworker-environment fit and thereby ensuring desirable telework outcomes.

Multiple binding sites on the CH2 domain of IgG for mouse Fc gamma R11.

Important mammalian defensive functions such as phagocytosis are triggered in leukocytes by the interaction of the Fc region of IgG with cell surface receptors (Fc gamma R). The CH2 domain of IgG has been implicated previously as the site of interaction with human and mouse Fc gamma R. This domain was mapped for interaction with mouse Fc gamma R11 expressed by the macrophage-like cell line P388D1, using two panels of a total of 32 site-directed mutants of mouse IgG2b and chimeric human IgG3 monoclonal antibodies. Two potential binding sites have been identified: one in or within the vicinity of the lower hinge site on IgG for human Fc gamma R1, and one within the binding site on IgG for Clq. The three mutant IgGs (Gly 237----Ala, Asn 297----Ala, and Glu 318----Ala) which do not interact in complexed form also fail to bind as monomers. A 1H NMR study of the three non-binding monomeric mutants suggests that the mutations are largely site-specific, indicating that IgG interacts with mouse Fc gamma R11 at two regions within the CH2 domain. This interaction dictates phagocytosis mediated by Fc gamma R11 of the P388D1 cell line.

Control of IgG/Fc glycosylation: a comparison of oligosaccharides from chimeric human/mouse and mouse subclass immunoglobulin Gs.

Oligosaccharide profiles were obtained for chimeric mouse-human antibodies corresponding to each of the human IgG subclasses 1-4, and mouse IgG2b antibodies each expressed in the mouse J558L cell line. These antibodies have specificity for the NIP hapten and form a matched set of IgGs. An IgG4 chimeric antibody (B72.3) produced in the chinese hamster ovary (CHO-K1) cell line was also analysed for carbohydrate. Additionally aglycosylated mutants of this IgG4 (B72.3) and anti-NIP mouse IgG2b were analysed. The total lack of carbohydrate found in the aglycosylated site-directed mutants human chimeric IgG4 B72.3 (Asn 297-->Gln) and mouse IgG2b (Asn 297-->Ala) demonstrates that there are no N-glycosylation sites other than Asn 297. Therefore glycosylation profiles for all the IgGs analysed reflect carbohydrate attached to this site. Factors such as cell type (A), template direction by the IgG heavy chains (B) and culture conditions (C) are shown to influence IgG glycosylation profiles. (A) The anti-NIP IgG antibodies expressed by the J558L cell line may have one or two Gal (alpha 1-->3) Gal residues per oligosaccharide unit, indicative of the presence of (alpha 1-->3) galactosyl transferase in the J558L mouse cell line. (B) The galactosylation profiles obtained for the IgG heavy chains, in particular the preference for galactosylation of the Man (alpha 1-->6) arm rather than the Man (alpha 1-->3) arm, contrary to the beta-galactosyltransferase specificity, suggest that the polypeptide chain may act as a template to influence the extent of galactosylation and hence the proportions of each oligosaccharide incorporated. The IgG2 antibody does not display this galactosylation preference. (C) The extent of galactosylation appears to be influenced by the growth conditions, with the highest levels of galactosylation being found for IgG produced by cells grown in still cultures, rather than cells grown as ascites or in hollow fibre bioreactors. It is concluded that though the profile of glycosylation is controlled predominantly by the glycosylation activity of the cell in which the IgG is expressed, differences between the IgG heavy chain templates of the various subclasses and culture conditions can also influence glycosylation.

An implementation evaluation of a qualitative culture assessment tool.

Safety culture has been identified as a critical element of healthy and safe workplaces and as such warrants the attention of ergonomists involved in occupational health and safety (OHS). This study sought to evaluate a tool for assessing organisational safety culture as it impacts a common OHS problem: musculoskeletal disorders (MSD). The level of advancement across nine cultural aspects was assessed in two implementation site organisations. These organisations, in residential healthcare and timber processing, enabled evaluation of the tool in contrasting settings, with reported MSD rates also high in both sectors. Interviews were conducted with 39 managers and workers across the two organisations. Interview responses and company documentation were compared by two researchers to the descriptor items for each MSD culture aspect. An assignment of the level of advancement, using a five stage framework, was made for each aspect. The tool was readily adapted to each implementation site context and provided sufficient evidence to assess their levels of advancement. Assessments for most MSD culture aspects were in the mid to upper levels of advancement, although the levels differed within each organisation, indicating that different aspects of MSD culture, as with safety culture, develop at a different pace within organisations. Areas for MSD culture improvement were identified for each organisation. Reflections are made on the use and merits of the tool by ergonomists for addressing MSD risk.

Descriptive data on cardiovascular and metabolic risk factors in ambulatory and non-ambulatory adults with cerebral palsy.

Forty-two participants with cerebral palsy were recruited for a study examining traditional and novel indicators of cardiovascular risk (McPhee et al., 2015 [1]). Data pertaining to the prevalence of obesity, smoking, hypertension, and metabolic risk are provided. These data are presented along with the scoring methods used in evaluation of the study participants. Percentages are included for comparative purposes with the existing literature.

Evaluation of monoclonal antibodies having specificity for human IgG subclasses: results of the 2nd IUIS/WHO collaborative study.

Following the 1st IUIS/WHO Collaborative Study of monoclonal anti-IgG subclass antibodies, a panel of WHO Specificity Reference Reagents (SRR) was established [Jefferis, R., et al. (1985) Immunol. Lett., 10, 223]. At the time, the hope was expressed that further reagents particularly for IgG2, and other allotypic specificities would become available which could be applied in a wide range of assay protocols. The 2nd study reports the evaluation of nineteen anti-subclass and seven anti-allotype monoclonal antibodies. The anti-IgG1 antibody HP6187 was equivalent in performance to the SRR. Others, that were not of the mouse IgG1 isotype, may be useful for particular applications. The anti-IgG2 antibody HP6200 could be a valuable addition to the WHO SRR; it is specific for an epitope in the Fab region but does not have the light chain bias of HP6014. Antibodies of putative allotype specificity exhibited the claimed specificity when used within protocols similar to those employed by the originating laboratory. It appears to be inherent in the nature of the epitopes (allotopes) recognized that it will take several years before reagents applicable to a wide range of techniques will become available.

Cell-mediated immune response of Chelonia mydas.

Blastogenic and cytotoxic responsiveness of peripheral blood leukocytes from the green turtle, Chelonia mydas were examined. Blastogenic responses were low level and showed considerable variation between animals. Blastogenesis in response to phytohemagglutinin and concanavalin A was observed through out all seasons. Responses to pokeweed mitogen and lipopolysaccharide were seasonal, appearing in spring. No blastogenic response to protein A or allogeneic leukocytes was observed. Cytotoxic responses to phytohemagglutinin and antibody-dependent cell-mediated cytotoxicity were of significant magnitude and, all animals responded in a similar manner. No spontaneous cytotoxic reactivity was observed.

Distribution and role of lipopolysaccharide in the pathogenesis of acute renal proximal tubule injury.

Endotoxins (lipopolysaccharides; LPS) are known to cause multiple organ failure, including renal dysfunction. The present report elucidates LPS distribution and effect on renal proximal tubules in an attempt to gain a better understanding of the cellular mechanism underlying the pathogenesis of renal dysfunction in endotoxemia and sepsis. Rats were intravenously treated with biotin-linked or regular Escherichia coli (0111:B4) LPS (3 mg/kg) and sacrificed at different times. Kidneys were retrieved and examined for LPS localization, tubular permeability, ultracytochemical alterations, leukocyte sequestration, and ICAM-1 expression. The functional impact of endotoxemia was also assessed by monitoring the changes in urine levels of glucose in timed collections up to 6 h. LPS was localized on the plasma membranes of the apical microvilli, the labyrinth of the lateral intercellular spaces, in various organelles of epithelial cells, and in the endothelial cells of the peritubular capillaries. LPS caused structural damage and calcium accumulation in the mitochondria, leakage of tight junctions, widening of the basolateral intercellular spaces, intracellular and extracellular edema, leukocyte margination and accumulation, vascular expression of ICAM-1, and decrease of plasma membrane and mitochondrial Ca2(+)-ATPase. Physiological study showed that both urine volume and glucose were greatly increased after LPS infusion. The pathological alterations in the proximal tubules may directly contribute to the reduction in the reabsorption ability of the proximal tubules.

Myoglobin content of hamster skeletal muscles.

Myoglobin (Mb) may facilitate O2 diffusion in muscle tissue, yet models of O2 transport are often simplified by ignoring the role of Mb. A recent analysis of O2 transport in hamster retractor muscle revealed a large discrepancy between the observed O2 diffusion from arterioles and that predicted by a mathematical model that did not include Mb. To establish whether this simplification was justified, we measured Mb content ([Mb]) in three hamster muscles that vary markedly in histochemical fiber type composition. [Mb] was determined spectrophotometrically using freshly excised tissues from hamsters of different ages (5-34 wk). [Mb] increased rapidly up to 15 wk of age and then rose more slowly. [Mb] in hamster muscles paralleled oxidative capacity. Our measurements of rat and dog muscles also show that [Mb] varies greatly among species and among muscles of a given species. The results indicate that in the hamster the variability in [Mb] with age and muscle should be taken into account when the potential role of Mb in studies on O2 transport is interpreted.