Biochemical profiling of proteins and metabolites in wound exudate from chronic wound environments

The lack of fundamental knowledge on the biological processes associated with wound healing represents a significant challenge. Understanding the biochemical changes that occur within a chronic wound could provide insights into the wound environment and enable more effective wound management. We report on the stability of wound fluid samples under various conditions and describe a high-throughput approach to investigate the altered biochemical state within wound samples collected from various types of chronic, ulcerated wounds. Furthermore, we discuss the viability of this approach in the early stages of wound sample protein and metabolite profiling and subsequent biomarker discovery. This approach will facilitate the detection of factors that may correlate with wound severity and/or could be used to monitor the response to a particular treatment.

Constitutive production of IL-13 promotes early-life Chlamydia respiratory infection and allergic airway disease

Deleterious responses to pathogens during infancy may contribute to infection and associated asthma. Chlamydia respiratory infections in early life are common causes of pneumonia and lead to reduced lung function and asthma. We investigated the role of interleukin-13 (IL-13) in promoting early-life Chlamydia respiratory infection, infection-induced airway hyperresponsiveness (AHR), and severe allergic airway disease (AAD). Infected infant Il13−/− mice had reduced infection, inflammation, and mucus-secreting cell hyperplasia. Surprisingly, infection of wild-type (WT) mice did not increase IL-13 production but reduced IL-13Rα2 decoy receptor levels compared with sham-inoculated controls. Infection of WT but not Il13−/− mice induced persistent AHR. Infection and associated pathology were restored in infected Il13−/− mice by reconstitution with IL-13. Stat6−/− mice were also largely protected. Neutralization of IL-13 during infection prevented subsequent infection-induced severe AAD. Thus, early-life Chlamydia respiratory infection reduces IL-13Rα2 production, which may enhance the effects of constitutive IL-13 and promote more severe infection, persistent AHR, and AAD.

A rapid and sensitive UPLC-ESI MS method for analysis of isofraxidin, a natural antistress compound, and its metabolites in rat plasma

Isofraxidin is one of the main bioactive constituents in the root of Acanthopanax senticosus, which has antifatigue, antistress, and immuno-accommondating effects. In this study, an ultraperformance LC (UPLC)-ESI MS method was developed for analyzing isofraxidin and its metabolites in rat plasma. The analysis was performed on a UPLC coupled with ESI MS (quadropole MS tandem TOF MS). The lower LOD (LLOD) for isofraxidin was 0.25 ng/mL, the intraday precision was less than 10%, the interday precision was less than 10%, and the extraction recovery was more than 80%. Isofraxidin and two metabolites (M1 and M2) were detected in rat plasma after oral administration of isofraxidin, and the molecular polarities of M1 and M2 were both increased compared to isofraxidin. The metabolites were identified as 5,6-dihydroxyl-7-methoxycoumarin and 5-hydroxyl-6,7-dimethoxycoumarin when subjected to parent ion spectra, product ion spectra, and extract mass and element composition analyses.

Hyperelastic constitutive relationship for the strain-rate dependent behavior of shoulder and other joint cartilages

Non-linear finite deformations of articular cartilages under physiological loading conditions can be attributed to hyperelastic behavior. This paper contains experimental results of indentation tests in finite deformation and proposes an empirical based new generalized hyperelastic constitutive model to account for strain-rate dependency for humeral head cartilage tissues. The generalized model is based on existing hyperelastic constitutive relationships that are extensively used to represent biological tissues in biomechanical literature. The experimental results were obtained for three loading velocities, corresponding to low (1x10-3 s-1), moderate and high strain-rates (1x10-1 s-1), which represent physiological loading rates that are experienced in daily activities such as lifting, holding objects and sporting activities. Hyperelastic material parameters were identified by non linear curve fitting procedure. Analysis demonstrated that the material behavior of cartilage can be effectively decoupled into strain-rate independent(elastic) and dependent parts. Further, experiments conducted using different indenters indicated that the parameters obtained are significantly affected by the indenter size, potentially due to structural inhomogeneity of the tissue. The hyperelastic constitutive model developed in this paper opens a new avenue for the exploration of material properties of cartilage tissues.

Nedd4-2functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl– channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 μg/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 μg/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.

Determination of glutathione transferase (GSTT1-1) activities in different tissues based on formation of radioactive metabolites using 35S-glutathione

A new system has been developed to determine enzyme activities of glutathione transferase θ (GSTT1-1) based on radiometric product detection resulting from the enzymic reaction of methyl chloride with 35S-labelled glutathione. In principle, the method is universally applicable for determination of glutathione transferase activities towards a multiplicity of substrates. The method distinguishes between erythrocyte GSTT1-1 activities of human 'non-conjugators', 'low conjugators' and 'high conjugators'. Application to cytosol preparations of livers and kidneys of male and female Fischer 344 and B6C3F1 mice reveals differential GSTT1-1 activities in hepatic and renal tissues. These ought to be considered in species-specific modellings of organ toxicities of chlorinated hydrocarbons.

Collection and determination of nucleotide metabolites in neonatal and adult saliva by high performance liquid chromatography with tandem mass spectrometry

Saliva contains a number of biochemical components which may be useful for diagnosis/monitoring of metabolic disorders, and as markers of cancer or heart disease. Saliva collection is attractive as a non-invasive sampling method for infants and elderly patients. We present a method suitable for saliva collection from neonates. We have applied this technique for the determination of salivary nucleotide metabolites. Saliva was collected from 10 healthy neonates using washed cotton swabs, and directly from 10 adults. Two methods for saliva extraction from oral swabs were evaluated. The analytes were then separated using high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS). The limits of detection for 14 purine/pyrimidine metabolites were variable, ranging from 0.01 to 1.0 mu M. Recovery of hydrophobic purine/pyrimidine metabolites from cotton tips was consistently high using water/acetonitrile extraction (92.7-111%) compared with water extraction alone. The concentrations of these metabolites were significantly higher in neonatal saliva than in adults. Preliminary ranges for nucleotide metabolites in neonatal and adult saliva are reported. Hypoxanthine and xanthine were grossly raised in neonates (49.3 +/- 25.4; 30.9 +/- 19.5 mu M respectively) compared to adults (4.3 +/- 3.3; 4.6 +/- 4.5 mu M); nucleosides were also markedly raised in neonates. This study focuses on three essential details: contamination of oral swabs during manufacturing and how to overcome this; weighing swabs to accurately measure small saliva volumes; and methods for extracting saliva metabolites of interest from cotton swabs. A method is described for determining nucleotide metabolites using HPLC with photo-diode array or MS/MS. The advantages of utilising saliva are highlighted. Nucleotide metabolites were not simply in equilibrium with plasma, but may be actively secreted into saliva, and this process is more active in neonates than adults. (C) 2013 Elsevier B.V. All rights reserved.

Constitutive Regulation of the Firm: occupational health and safety, dismissal, discrimination and sexual harassment

As many other chapters in this book have noted, until recently labour lawyers have tended not to draaw on regulatory scholarship. In this chapter we look at certain areas of labour law through a particular kind of regulatory lens - regulation that requires firms to reconstitute their management processes and procedures, perhaps even their organisational cultures. In particular, we examine the kinds of regulatory demands made on firms by legal rules in four areas of labour law: (i) occupational health and safety (OHS)regulation; unfair dismissal law; equal opportunity (EO) and (iv) sexual harassment law.

A constitutive model for mechanical response characterization of pumpkin peel and flesh tissues under tensile and compressive loadings

Enhancing quality of food products and reducing volume of waste during mechanical operations of food industry requires a comprehensive knowledge of material response under loadings. While research has focused on mechanical response of food material, the volume of waste after harvesting and during processing stages is still considerably high in both developing and developed countries. This research aims to develop and evaluate a constitutive model of mechanical response of tough skinned vegetables under postharvest and processing operations. The model focuses on both tensile and compressive properties of pumpkin flesh and peel tissues where the behaviours of these tissues vary depending on various factors such as rheological response and cellular structure. Both elastic and plastic response of tissue were considered in the modelling process and finite elasticity combined with pseudo elasticity theory was applied to generate the model. The outcomes were then validated using the published results of experimental work on pumpkin flesh and peel under uniaxial tensile and compression. The constitutive coefficients for peel under tensile test was α = 25.66 and β = −18.48 Mpa and for flesh α = −5.29 and β = 5.27 Mpa. under compression the constitutive coefficients were α = 4.74 and β = −1.71 Mpa for peel and α = 0.76 and β = −1.86 Mpa for flesh samples. Constitutive curves predicted the values of force precisely and close to the experimental values. The curves were fit for whole stress versus strain curve as well as a section of curve up to bio yield point. The modelling outputs had presented good agreement with the empirical values and the constructive curves exhibited a very similar pattern to the experimental curves. The presented constitutive model can be applied next to other agricultural materials under loading in future.

Effect of constitutive material models on seismic response of two-story reinforced concrete frame

This paper focuses on the finite element (FE) response sensitivity and reliability analyses considering smooth constitutive material models. A reinforced concrete frame is modeled for FE sensitivity analysis followed by direct differentiation method under both static and dynamic load cases. Later, the reliability analysis is performed to predict the seismic behavior of the frame. Displacement sensitivity discontinuities are observed along the pseudo-time axis using non-smooth concrete and reinforcing steel model under quasi-static loading. However, the smooth materials show continuity in response sensitivity at elastic to plastic transition points. The normalized sensitivity results are also used to measure the relative importance of the material parameters on the structural responses. In FE reliability analysis, the influence of smoothness behavior of reinforcing steel is carefully noticed. More efficient and reasonable reliability estimation can be achieved by using smooth material model compare with bilinear material constitutive model.