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Human skin has the capacity to metabolise foreign chemicals (xenobiotics), but knowledge of the various enzymes involved is incomplete. A broad-based unbiased proteomics approach was used to describe the profile of xenobiotic metabolising enzymes present in human skin and hence indicate principal routes of metabolism of xenobiotic compounds. Several in vitro models of human skin have been developed for the purpose of safety assessment of chemicals. The suitability of these epidermal models for studies involving biotransformation was assessed by comparing their profiles of xenobiotic metabolising enzymes with those of human skin. Label-free proteomic analysis of whole human skin (10 donors) was applied and analysed using custom-built PROTSIFT software. The results showed the presence of enzymes with a capacity for the metabolism of alcohols through dehydrogenation, aldehydes through dehydrogenation and oxidation, amines through oxidation, carbonyls through reduction, epoxides and carboxylesters through hydrolysis and, of many compounds, by conjugation to glutathione. Whereas protein levels of these enzymes in skin were mostly just 4-10 fold lower than those in liver and sufficient to support metabolism, the levels of cytochrome P450 enzymes were at least 300-fold lower indicating they play no significant role. Four epidermal models of human skin had profiles very similar to one another and these overlapped substantially with that of whole skin. The proteomics profiling approach was successful in producing a comprehensive analysis of the biotransformation characteristics of whole human skin and various in vitro skin models. The results show that skin contains a range of defined enzymes capable of metabolising different classes of chemicals. The degree of similarity of the profiles of the in vitro models indicates their suitability for epidermal toxicity testing. Overall, these results provide a rational basis for explaining the fate of xenobiotics in skin and will aid chemical safety testing programmes.

Group A streptococcal isolates of serotype M18 are historically associated with epidemic waves of pharyngitis and the non-suppurative immune sequela rheumatic fever. The serotype is defined by a unique, highly encapsulated phenotype, yet the molecular basis for this unusual colony morphology is unknown. Here we identify a truncation in the regulatory protein RocA, unique to and conserved within our serotype M18 GAS collection, and demonstrate that it underlies the characteristic M18 capsule phenotype. Reciprocal allelic exchange mutagenesis of rocA between M18 GAS and M89 GAS demonstrated that truncation of RocA was both necessary and sufficient for hyper-encapsulation via up-regulation of both precursors required for hyaluronic acid synthesis. Although RocA was shown to positively enhance covR transcription, quantitative proteomics revealed RocA to be a metabolic regulator with activity beyond the CovR/S regulon. M18 GAS demonstrated a uniquely protuberant chain formation following culture on agar that was dependent on excess capsule and the RocA mutation. Correction of the M18 rocA mutation reduced GAS survival in human blood, and in vivo naso-pharyngeal carriage longevity in a murine model, with an associated drop in bacterial airborne transmission during infection. In summary, a naturally occurring truncation in a regulator explains the encapsulation phenotype, carriage longevity and transmissibility of M18 GAS, highlighting the close interrelation of metabolism, capsule and virulence.

A study into the effects of amorphous nano-SiO2 particles on A549 lung epithelial cells was undertaken using proteomics to understand the interactions that occur and the biological consequences of exposure of lung to nanoparticles. Suitable conditions for treatment, where A549 cells remained viable for the exposure period, were established by following changes in cell morphology, flow cytometry, and MTT reduction. Label-free proteomics was used to estimate the relative level of proteins from their component tryptic peptides detected by mass spectrometry. It was found that A549 cells tolerated treatment with 100 µg/ml nano-SiO2 in the presence of 1.25% serum for at least 4 h. After this time detrimental changes in cell morphology, flow cytometry, and MTT reduction were evident. Proteomics performed after 4 h indicated changes in the expression of 47 proteins. Most of the proteins affected fell into four functional groups, indicating that the most prominent cellular changes were those that affected apoptosis regulation (e.g. UCP2 and calpain-12), structural reorganisation and regulation of actin cytoskeleton (e.g. PHACTR1), the unfolded protein response (e.g. HSP 90), and proteins involved in protein synthesis (e.g. ribosomal proteins). Treatment with just 10 µg/ml nano-SiO2 particles in serum-free medium resulted in a rapid deterioration of the cells and in medium containing 10% serum the cells were resistant to up to 1000 µg/ml nano-SiO2 particles, suggesting interaction of serum components with the nanoparticles. A variety of serum proteins were found which bound to nano-SiO2 particles, the most prominent of which were albumin, apolipoprotein A-I, hemoglobin, vitronectin and fibronectin. The use of a proteomics platform, with appropriately designed experimental conditions, enabled the early biological perturbations induced by nano-SiO2 in a model target cell system to be identified. The approach facilitates the design of more focused test systems for use in tiered evaluations of nanomaterials.

Immunity to common bacteria requires the generation of antibodies that promote opsonophagocytosis and neutralise toxins. Pooled human immunoglobulin is widely advocated as an adjunctive treatment for clinical Streptococcus pyogenes infection however, the protein targets of the reagent remain ill defined. Affinity purification of the anti-streptococcal antibodies present within pooled immunoglobulin resulted in the generation of an IgG preparation that promoted opsonophagocytic killing of S. pyogenes in vitro and provided passive immunity in vivo . Isolation of the streptococcal surface proteins recognised by pooled human immunoglobulin permitted identification and ranking of 94 protein antigens, ten of which were reproducibly identified across four contemporary invasive S. pyogenes serotypes (M1, M3, M12 and M89). The data provide novel insight into the action of pooled human immunoglobulin during invasive S. pyogenes infection and demonstrate a potential route to enhance the efficacy of antibody based therapies.

Invasive Streptococcus pyogenes infections are rare, with often-unexplained severity. Prompt diagnosis is desirable, as deaths can occur rapidly following onset and there is an increased, but preventable, risk to contacts. Here, proteomic analyses of clinical samples from invasive human S . pyogenes infections were undertaken to determine if novel diagnostic targets could be detected, and to augment our understanding of disease pathogenesis. Fluid samples from 17 patients with confirmed invasive S . pyogenes infection (empyema, septic arthritis, necrotising fasciitis) were analysed by proteomics for streptococcal and human proteins; 16/17 samples had detectable S . pyogenes DNA. Nineteen unique S . pyogenes proteins were identified in just 6/17 samples, and 15 of these were found in a single pleural fluid sample including streptococcal inhibitor of complement, trigger factor, and phosphoglycerate kinase. In contrast, 469 human proteins were detected in patient fluids, 177 (38%) of which could be identified as neutrophil proteins, including alpha enolase and lactotransferrin which, together, were found in all 17 samples. Our data suggest that streptococcal proteins are difficult to detect in infected fluid samples. A vast array of human proteins associated with leukocyte activity are, however, present in samples that deserve further evaluation as potential biomarkers of infection.

Accurate differentiation between benign and malignant causes of biliary obstruction remains challenging and reliable biomarkers are urgently needed. Bile is a potential source of such biomarkers. Our aim was to apply a proteomic approach to identify a potential biomarker in bile that differentiates between malignant and benign disease, and to assess its diagnostic accuracy. Neutrophil gelatinase-associated lipocalin (NGAL) is multi-functional protein, released from activated neutrophils, with roles in inflammation, immune function, and carcinogenesis. It has not previously been described in bile. Bile, urine, and serum were collected prospectively from 38 patients undergoing endoscopic retrograde cholangiopancreatography (\"discovery\" cohort); 22 had benign and 16 had malignant pancreatobiliary disease. Initially, label-free proteomics and immunoblotting were performed in samples from a subset of these patients. Enzyme-linked immunosorbent assay was then performed for NGAL as a potential biomarker on all samples in this cohort. The diagnostic performance of biliary NGAL was then validated in a second, independent group (\"validation\" cohort) of 21 patients with pancreatobiliary disease (benign n=14, malignant n=7). NGAL levels were significantly raised in bile from the malignant disease group, compared with bile from the benign disease group in the discovery cohort (median 1,556 vs. 480 ng/ml, P=0.007). Biliary NGAL levels had a receiver operating characteristic area under curve of 0.76, sensitivity 94%, specificity 55%, positive predictive value 60%, and negative predictive value 92% for distinguishing malignant from benign causes. Biliary NGAL was independent of serum biochemistry and carbohydrate antigen 19-9 (CA 19-9) in differentiating between underlying benign and malignant disease. No significant differences in serum and urine NGAL levels were found between benign and malignant disease. Combining biliary NGAL and serum CA 19-9 improved diagnostic accuracy for malignancy (sensitivity 85%, specificity 82%, positive predictive value 79%, and negative predictive value 87%). The diagnostic accuracy of biliary NGAL was confirmed in the second independent validation cohort. NGAL in bile is a novel potential biomarker to help distinguish benign from malignant biliary obstruction.

Group A streptococcal isolates of serotype M18 are historically associated with epidemic waves of pharyngitis and the non-suppurative immune sequela rheumatic fever. The serotype is defined by a unique, highly encapsulated phenotype, yet the molecular basis for this unusual colony morphology is unknown. Here we identify a truncation in the regulatory protein RocA, unique to and conserved within our serotype M18 GAS collection, and demonstrate that it underlies the characteristic M18 capsule phenotype. Reciprocal allelic exchange mutagenesis of rocA between M18 GAS and M89 GAS demonstrated that truncation of RocA was both necessary and sufficient for hyper-encapsulation via up-regulation of both precursors required for hyaluronic acid synthesis. Although RocA was shown to positively enhance covR transcription, quantitative proteomics revealed RocA to be a metabolic regulator with activity beyond the CovR/S regulon. M18 GAS demonstrated a uniquely protuberant chain formation following culture on agar that was dependent on excess capsule and the RocA mutation. Correction of the M18 rocA mutation reduced GAS survival in human blood, and in vivo naso-pharyngeal carriage longevity in a murine model, with an associated drop in bacterial airborne transmission during infection. In summary, a naturally occurring truncation in a regulator explains the encapsulation phenotype, carriage longevity and transmissibility of M18 GAS, highlighting the close interrelation of metabolism, capsule and virulence.

A study into the effects of amorphous nano-SiO2 particles on A549 lung epithelial cells was undertaken using proteomics to understand the interactions that occur and the biological consequences of exposure of lung to nanoparticles. Suitable conditions for treatment, where A549 cells remained viable for the exposure period, were established by following changes in cell morphology, flow cytometry, and MTT reduction. Label-free proteomics was used to estimate the relative level of proteins from their component tryptic peptides detected by mass spectrometry. It was found that A549 cells tolerated treatment with 100 mu g/ml nano-SiO2 in the presence of 1.25% serum for at least 4 h. After this time detrimental changes in cell morphology, flow cytometry, and MTT reduction were evident. Proteomics performed after 4 h indicated changes in the expression of 47 proteins. Most of the proteins affected fell into four functional groups, indicating that the most prominent cellular changes were those that affected apoptosis regulation (e.g. UCP2 and calpain-12), structural reorganisation and regulation of actin cytoskeleton (e.g. PHACTR1), the unfolded protein response (e.g. HSP 90), and proteins involved in protein synthesis (e.g. ribosomal proteins). Treatment with just 10 mu g/ml nano-SiO2 particles in serum-free medium resulted in a rapid deterioration of the cells and in medium containing 10% serum the cells were resistant to up to 1000 mu g/ml nano-SiO2 particles, suggesting interaction of serum components with the nanoparticles. A variety of serum proteins were found which bound to nano-SiO2 particles, the most prominent of which were albumin, apolipoprotein A-I, hemoglobin, vitronectin and fibronectin. The use of a proteomics platform, with appropriately designed experimental conditions, enabled the early biological perturbations induced by nano-SiO2 in a model target cell system to be identified. The approach facilitates the design of more focused test systems for use in tiered evaluations of nanomaterials.