Mass Spectrometry and Genetic Analysis of Biological Barrier Lipids

Malnutrition during embryonic development can have significant health ramifications for the adult organism. Such developmental programming effects have been studied in a variety of different organs but no research to date has looked at the largest organ in the body, the skin. One important and evolutionarily conserved role of the skin is to provide a lipid barrier against stresses in the external environment. Here, I utilise the short lifespan and powerful genetics of Drosophila melanogaster to investigate how cuticular barrier lipids are regulated by developmental diet and environmental stresses in the adult. The Drosophila cuticle is significantly different from the mammalian skin, yet both are coated with a waterproof lipid blend synthesised by specialised subepidermal cells - sebocytes in mammals and oenocytes in Drosophila. Research on the composition and regulation of lipid barriers has been hampered, at least in part, by the lack of methods for resolving surface lipids from intracellular lipids. I therefore optimise several techniques for analysing surface lipids, including a new cryogenic platform for mass spectrometry imaging. I demonstrate that developmental diet programmes the blend of adult cuticular lipids in both Drosophila and mice by increasing the adult skin wax esters. In Drosophila, I also show how adult environmental stresses impact upon the cuticle lipid blend. Combining mass spectrometry imaging with genetic analysis in Drosophila, I then identify a physiological feedback circuit that regulates the blend of adult cuticular hydrocarbons in response to increased housing density. This circuit involves the detection of housing density by an as yet unknown mechanism, information relay to oenocytes, and export of hydrocarbons to the cuticle surface via Obp99b/Obp99d odorant-binding proteins. Finally, I discuss the wider implications of this study and how mammals may regulate their skin lipids in line with a changing environment

Self-employment among the Armed Forces Community

The Institute for Employment Research at the University of Warwick, QinetiQ and X-Forces Enterprise were commissioned by Forces in Mind Trust to understand what more could be done to support the Armed Forces Community in pursuing self-employment and thereby help to maximise their chances of a successful and sustainable transition. This research seeks to fill the current gap in knowledge and contribute to policy-making and service delivery

Impact of contextual factors on patient outcomes following conservative low back pain treatment: systematic review

Background and objectiveChronic low back pain is pervasive, societally impactful, and current treatments only provide moderate relief. Exploring whether therapeutic elements, either unrecognised or perceived as implicit within clinical encounters, are acknowledged and deliberately targeted may improve treatment efficacy. Contextual factors (specifically, patient’s and practitioner’s beliefs/characteristics; patient-practitioner relationships; the therapeutic setting/environment; and treatment characteristics) could be important, but there is limited evidence regarding their influence. This research aims to review the impact of interventions modifying contextual factors during conservative care on patient’s pain and physical functioning.Databases and data treatmentFour electronic databases (Medline, CINAHL, PsycINFO and AMED) were searched from 2009 until 15th February 2022, using tailored search strategies, and resulted in 3476 unique citations. After initial screening, 170 full-text records were potentially eligible and assessed against the inclusion–exclusion criteria. Thereafter, studies were assessed for methodological quality using a modified Downs and Black scale, data extracted, and synthesised using a narrative approach.ResultsTwenty-one primary studies (N = 3075 participants), were included in this review. Eight studies reported significant improvements in pain intensity, and seven in physical functioning, in favour of the contextual factor intervention(s). Notable contextual factors included: addressing maladaptive illness beliefs; verbal suggestions to influence symptom change expectations; visual or physical cues to suggest pain-relieving treatment properties; and positive communication such as empathy to enhance the therapeutic alliance.ConclusionThis review identified influential contextual factors which may augment conservative chronic low back pain care. The heterogeneity of interventions suggests modifying more than one contextual factor may be more impactful on patients’ clinical outcomes, although these findings require judicious interpretation

Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP

The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudomonas fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high affinity binding was also observed for the type III secretion system homolog HrcN and the type VI ATPase ClpB2. The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins

Protein CoAlation: a redox-regulated protein modification by coenzyme A in mammalian cells.

Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry, we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation). We show that protein CoAlation is a reversible post-translational modification that is induced in mammalian cells and tissues by oxidising agents and metabolic stress. Many key cellular enzymes were found to be CoAlated in vitro and in vivo in ways that modified their activities. Our study reveals that protein CoAlation is a widespread post-translational modification which may play an important role in redox regulation under physiological and pathophysiological conditions

Metformin Alters Human Host Responses to Mycobacterium tuberculosis in Healthy Subjects.

BACKGROUND: Metformin, the most widely administered diabetes drug, has been proposed as a candidate adjunctive host-directed therapy for tuberculosis, but little is known about its effects on human host responses to Mycobacterium tuberculosis. METHODS: We investigated in vitro and in vivo effects of metformin in humans. RESULTS: Metformin added to peripheral blood mononuclear cells from healthy volunteers enhanced in vitro cellular metabolism while inhibiting the mammalian target of rapamycin targets p70S6K and 4EBP1, with decreased cytokine production and cellular proliferation and increased phagocytosis activity. Metformin administered to healthy human volunteers led to significant downregulation of genes involved in oxidative phosphorylation, mammalian target of rapamycin signaling, and type I interferon response pathways, particularly following stimulation with M. tuberculosis, and upregulation of genes involved in phagocytosis and reactive oxygen species production was increased. These in vivo effects were accompanied by a metformin-induced shift in myeloid cells from classical to nonclassical monocytes. At a functional level, metformin lowered ex vivo production of tumor necrosis factor α, interferon γ, and interleukin 1β but increased phagocytosis activity and reactive oxygen species production. CONCLUSION: Metformin has a range of potentially beneficial effects on cellular metabolism, immune function, and gene transcription involved in innate host responses to M. tuberculosis

The Importance of Brain Banks for Molecular Neuropathological Research: The New South Wales Tissue Resource Centre Experience

New developments in molecular neuropathology have evoked increased demands for postmortem human brain tissue. The New South Wales Tissue Resource Centre (TRC) at The University of Sydney has grown from a small tissue collection into one of the leading international brain banking facilities, which operates with best practice and quality control protocols. The focus of this tissue collection is on schizophrenia and allied disorders, alcohol use disorders and controls. This review highlights changes in TRC operational procedures dictated by modern neuroscience, and provides examples of applications of modern molecular techniques to study the neuropathogenesis of many different brain disorders

The correlation of ULF waves and auroral intensity before, during and after substorm expansion phase onset

We present case studies of the evolution of magnetic wave amplitudes and auroral intensity through the late growth phase and the expansion phase of the substorm cycle. We present strong evidence that substorm-related auroral enhancements are clearly and demonstrably linked to ULF wave amplitudes observed at the same location. In most cases, we find that the highest correlations are observed when the magnetometer time series is advanced in time, indicating that the ULF wave amplitudes start to grow before measured auroral intensities, though interestingly this is not always the case. Further we discuss the four possible reasons that may be able to explain both the timing and the high correlations between these two phenomena, including: a simple coincidence, an artifact of instrumental effects, the response of the ionosphere to magnetic waves and auroral particle precipitation, and finally that ULF waves and auroral particle precipitation are physically linked. We discount coincidence and instrumental effects since in the studies presented here they are unlikely or in general will contribute negligible effects, and we find that the ionospheric response to waves and precipitation can explain some, but not all of the results contained within this paper. Specifically, ionospheric response to substorm waves and auroral precipitation cannot explain that the result that previous studies have shown, that onset of ULF wave activity and the onset of auroral particle precipitation occur at the same time and in the same location. This leaves the possibility that ULF waves and auroral particles are physically linked