Nanoflow-nanospray mass spectrometry metabolomics reveals disruption of the urinary metabolite profiles of HIV-positive patients on combination antiretroviral therapy

Background: The use of combination antiretroviral therapy (cART) has substantially improved the outlook for patients with HIV infection. However, lifelong exposure to cART is also associated with adverse metabolic changes and an enhanced risk of renal, hepatic and cardiovascular dysfunction. This study investigated disruptions of the urinary metabolome of cART-exposed patients, thereby furthering our understanding of some of the side effects of pharmaceutical intervention. Methods: HIV-positive patients were recruited from an HIV clinic and divided into cART-naive and cART-exposed groups. HIV-negative patients were recruited from a sexual health clinic. All 89 subjects were white males. Targeted biochemistry analyses were performed on plasma samples. Urine samples were collected following an overnight fast and analysed with a highly sensitive untargeted metabolomic method using nanoflow/nanospray liquid chromatography-time of flight mass spectrometry. Datasets were analysed using projection modelling to detect metabolite markers of cART exposure. Results: Metabolites or parent compounds of all cART drugs were detected in urine extracts of all but one of the cART-exposed patients confirming adherence to the pharmaceutical regimen. Analysis of urine samples from patients on cART revealed significant reductions in selected bile acids, lipid, nucleoside and androgen metabolites. However, plasma concentrations of free or conjugated testosterone were unchanged indicating possible disruption of androgen transport or excretion in urine of patients on cART. Conclusions: Discovery-based metabolomics reveals the potential to identify novel markers of cART intervention and metabolite disruption in HIV-positive patients, which may enable the efficacy, compliance and side effects of these pharmaceutical mixtures to be investigated

Damage localisation in a stiffened composite panel

This work was conducted as part of the Aircraft Reliability Through Intelligent Materials Application (ARTIMA) European Union project. It presents a case study of damage detection in a curved carbon-fibre reinforced panel with two omega stiffeners which was investigated using ultrasonic Lamb waves. The statistical technique of outlier analysis was used here as a way of pre-processing experimental data prior to damage classification. Multilayer perceptron neural networks were used here for both classification and regression problems of damage detection. It was then investigated whether using wavelet analysis to perform prior wavelet decompositions of experimental data could facilitate damage classification

Dock2 generates characteristic spatiotemporal patterns of Rac activity to regulate neutrophil polarisation, migration and phagocytosis

IntroductionRac-GTPases and their Rac-GEF activators play important roles in neutrophil-mediated host defence. These proteins control the adhesion molecules and cytoskeletal dynamics required for neutrophil recruitment to inflamed and infected organs, and the neutrophil effector responses that kill pathogens.MethodsHere, we used live cell TIRF-FRET imaging in neutrophils from Rac-FRET reporter mice with deficiencies in the Rac-GEFs Dock2, Tiam1 or Prex1/Vav1 to evaluate if these proteins activate spatiotemporally distinct pools of Rac, and to correlate patterns of Rac activity with the neutrophil responses they control.ResultsAll the GEFs were required for neutrophil adhesion, and Prex1/Vav1 were important during spreading and for the velocity of migration during chemotaxis. However, Dock2 emerged as the prominent regulator of neutrophil responses, as this GEF was required for neutrophil polarisation and random migration, for migration velocity during chemokinesis, for the likelihood to migrate and for the speed of migration and of turning during chemotaxis, as well as for rapid particle engulfment during phagocytosis. We identified characteristic spatiotemporal patterns of Rac activity generated by Dock2 which correlate with the importance of the Rac-GEF in these neutrophil responses. We also demonstrate a requirement for Dock2 in neutrophil recruitment during aseptic peritonitis.DiscussionCollectively, our data provide a first direct comparison of the pools of Rac activity generated by different types of Rac-GEFs, and identify Dock2 as a key regulator of polarisation, migration and phagocytosis in primary neutrophils

The Rac-GEF Tiam1 controls integrin-dependent neutrophil responses

Rac GTPases are required for neutrophil adhesion and migration, and for the neutrophil effector responses that kill pathogens. These Rac-dependent functions are impaired when neutrophils lack the activators of Rac, Rac-GEFs from the Prex, Vav, and Dock families. In this study, we demonstrate that Tiam1 is also expressed in neutrophils, governing focal complexes, actin cytoskeletal dynamics, polarisation, and migration, in a manner depending on the integrin ligand to which the cells adhere. Tiam1 is dispensable for the generation of reactive oxygen species but mediates degranulation and NETs release in adherent neutrophils, as well as the killing of bacteria. In vivo, Tiam1 is required for neutrophil recruitment during aseptic peritonitis and for the clearance of Streptococcus pneumoniae during pulmonary infection. However, Tiam1 functions differently to other Rac-GEFs. Instead of promoting neutrophil adhesion to ICAM1 and stimulating β2 integrin activity as could be expected, Tiam1 restricts these processes. In accordance with these paradoxical inhibitory roles, Tiam1 limits the fMLP-stimulated activation of Rac1 and Rac2 in adherent neutrophils, rather than activating Rac as expected. Tiam1 promotes the expression of several regulators of small GTPases and cytoskeletal dynamics, including αPix, Psd4, Rasa3, and Tiam2. It also controls the association of Rasa3, and potentially αPix, Git2, Psd4, and 14-3-3ζ/δ, with Rac. We propose these latter roles of Tiam1 underlie its effects on Rac and β2 integrin activity and on cell responses. Hence, Tiam1 is a novel regulator of Rac-dependent neutrophil responses that functions differently to other known neutrophil Rac-GEFs

Investigating the immunosuppressive functions of the GPCR adapter protein Norbin

This project investigated the roles of Norbin, an adaptor protein which regulates trafficking and signalling of G protein-coupled receptors (GPCRs). Norbin was originally expected to be exclusively neuronal. However, previous PhD students in our laboratory found that Norbin is also expressed in myeloid cells and made mice with myeloid Norbin deficiency (NcdnΔmye) to investigate its role in these cells. They showed that NcdnΔmye mice have increased immunity during pulmonary *S. pneumoniae* infection, and that neutrophils from these mice kill bacteria better, in a reactive oxygen species (ROS)-dependent manner, and produce more ROS in response to a wide range of stimuli. I followed up on their work using immune cell depletion to demonstrate that the elevated immunity of NcdnΔmye mice is neutrophil-derived rather than depending on macrophages. I showed that neutrophils arrive more rapidly in the airways of NcdnΔmye mice during infection with *S. pneumoniae*, and they degranulate in the lungs to a greater extent, although the systemic levels of inflammatory cytokines are normal. I also found ten-fold elevated immunity of NcdnΔmye mice during septic peritonitis. I showed that NcdnΔmye neutrophils degranulate more and produce more neutrophils extracellular traps (NETs) than control neutrophils, and their elevated killing of *S. aureus* is dependent on NETs, whereas their production of inflammatory cytokines is normal. I showed that Norbin controls the Rac-GEFs Vav, which is constitutively active in NcdnΔmye neutrophils, and Tiam1 which is upregulated to cell surface upon C5a-stimulation. I also showed that the elevated ROS production in these cells can be reversed by titrating inhibitors of Rac and Erk. Arguably my most important contribution was to investigate the role of Norbin in neutrophil GPCR trafficking. Norbin is known to interact directly with numerous GPCRs, influencing their trafficking and signalling. However, apart from Norbin needing to interact directly with a GPCR, little is known about the mechanisms. I identified novel direct interactions between Norbin and the C-terminal tails of the GPCRs CXCR4 and C5aR1. I showed that the cell surface levels of CXCR4 and C5aR1 are elevated in NcdnΔmye neutrophils whereas the total cellular levels of these receptors are normal. I used various pulse-chase experiments to show that constitutive trafficking of these GPCRs is unaffected, but their agonist-induced internalisation is altered in NcdnΔmye neutrophils, and their recycling back to the plasma membrane faster. Altered binding kinetics of β-arrestin with stimulated C5aR1 may underlie the mechanisms behind these trafficking differences, and the resulting elevated GPCR cell surface levels can explain some of the increased responsiveness of NcdnΔmye neutrophils.MR

Use of a pre-analysis osmolality normalisation method to correct for variable urine concentrations and for improved metabolomic analyses

Metabolomics analyses of urine have the potential to provide new information on the detection and progression of many disease processes. However, urine samples can vary significantly in total solute concentration and this presents a challenge to achieve high quality metabolomic datasets and the detection of biomarkers of disease or environmental exposures. This study investigated the efficacy of pre- and post-analysis normalisation methods to analyse metabolomic datasets obtained from neat and diluted urine samples from five individuals. Urine samples were extracted by solid phase extraction (SPE) prior to metabolomic analyses using a sensitive nanoflow/nanospray LC–MS technique and the data analysed by principal component analyses (PCA). Post-analysis normalisation ofthe datasets to either creatinine or osmolality concentration, or to mass spectrum total signal (MSTS), revealed that sample discrimination was driven by the dilution factor of urine rather than the individual providing the sample. Normalisation of urine samples to equal osmolality concentration prior to LC–MS analysis resulted in clustering of the PCAscores plot according to sample source and significantimprovements inthenumber of peaks common to samples of all three dilutions from each individual. In addition, the ability to identify discriminating markers, using orthogonal partial least squared-discriminant analysis (OPLS-DA), was greatly improved when pre-analysis normalisation to osmolality was compared with post-analysis normalisation to osmolality and non-normalised datasets. Further improvements for peak area repeatability were observed in some samples when the pre-analysis normalisation to osmolality was combined with a post-analysis mass spectrum total useful signal (MSTUS) or MSTS normalisation. Future adoption of such normalisation methods may reduce the variability in metabolomics analyses due to differing urine concentrations and improve the discovery of discriminating metabolites associated with sample source

Design and forward kinematics of the compliant micro-manipulator with lever mechanisms

This paper presents the forward kinematics of a five-bar compliant micro-manipulator. To overcome the limited displacement of such a flexure-based mechanism driven by piezoelectric actuators, lever mechanisms are utilized to enlarge the working range. The mechanical design of the micro-manipulator is firstly described. Mathematical formulations for the five-bar mechanism are described and the solutions are developed to decide the end-effector position in Cartesian space. The amplification factor of the lever mechanism is also derived based on the analytical solution of the four-bar linkages. The velocity of the end-effector is obtained by differentiating the forward position kinematic equation, and the local mobility index of the five-bar compliant mechanism is determined and analysed. Based on linearization of trigonometric functions and constant Jacobian matrix, numerical simulations are carried out to investigate the performance of the five-bar compliant manipulator and to determine the optimal geometric parameters for the configuration. The comparisons between the exact solution and simplified methodologies are conducted. Experiments are carried out to validate the established model and the performance of the developed micro-manipulator

The GPCR adaptor protein Norbin regulates S1PR1 trafficking and the morphology, cell cycle and survival of PC12 cells

Abstract Norbin is an adaptor protein that binds numerous G protein-coupled receptors (GPCRs), is highly expressed in neurons, and is essential for a functioning nervous system in rodent models. Yet, beyond its control of neurite outgrowth and synaptic plasticity, few cellular roles of Norbin have been investigated to date. Furthermore, while Norbin is known to regulate the steady-state cell surface levels of several GPCRs, only in one case has the protein been shown to control the agonist-induced receptor internalisation which serves to attenuate GPCR signalling. Here, we generated a Norbin-deficient PC12 cell line which enabled us to study both the cellular functions of Norbin and its roles in GPCR trafficking and signalling. We show that Norbin limits cell size and spreading, and is required for the growth, viability and cell cycle progression of PC12 cells. We also found that Norbin regulates both the steady-state surface level and agonist-induced internalisation of the GPCR sphingosine-1-phosphate receptor 1 (S1PR1) in these cells, suggesting that its role in agonist-dependent GPCR trafficking is more widespread than previously appreciated. Finally, we show that Norbin limits the S1P-stimulated activation of Akt and p38 Mapk, and is required for the activation of Erk in PC12 cells. Together, our findings provide a better understanding of the cellular functions of Norbin and its control of GPCR trafficking