An investigation into protein and lipid binding by the phosphatidylinositol transfer protein RdgBβ

The phosphatidylinositol transfer proteins (PITPs) are a family of lipid carrier proteins that bind and transfer phosphatidylinositol (PI) and phosphatidylcholine (PC) between membranes. PITPs are commonly involved in phosphoinositide-requiring processes, including phospholipase C and PI 3-kinase signalling, and membrane trafficking. In this study I focus on the uncharacterised soluble PITP, RdgBβ (PITPNC1). The lipid binding and transfer properties of RdgBβ have scarcely been characterised, and the function of RdgBβ is completely unknown. I uncover that RdgBβ interacts with 14-3-3 through its long, disordered C-terminus. RdgBβ is ubiquitinated and subject to rapid degradation in cells, and binding of 14-3-3 via two phosphorylated residues may serve to protect the protein from protease digestion. Whereas RdgBβ binds 14-3-3 under basal conditions, I deduce that, upon stimulation of cells with phorbol ester, RdgBβ binds the Angiotensin II receptor (AT1R)-associated protein, ATRAP, via its N-terminal PITP domain. Others have shown that ATRAP suppresses Angiotensin II signalling by uncoupling AT1R from G proteins and promoting AT1R internalisation. I find that the RdgBβ-ATRAP interaction is blocked by inhibition of protein kinase C or protein synthesis, and may function to re-localise RdgBβ to the membrane in stimulated cells. Unexpectedly, I find that RdgBβ binds PI and phosphatic acid (PA), rather than PI and PC, and that binding of PA is increased by stimulation of cells with GTPγS. Mass spectrometry is used to analyse the molecular species of PI and PA bound by RdgBβ, and reveals that whereas RdgBβ is non-selective in its binding of PI, it selects short-chain monounsaturated or saturated PA species, likely derived from the hydrolysis of PC by phospholipase D

Internal lee wave closures: Parameter sensitivity and comparison to observations

This is the final version. Available from AGU via the DOI in this recordThe SOFine and DIMES data analyzed in this paper can be obtained through the British Oceanographic Data Centre (BODC) by navigating the following links, respectively: http://archive.noc.ac.uk/SOFINE/and http://dimes.ucsd.edu/en/data/This paper examines two internal lee wave closures that have been used together with ocean models to predict the time‐averaged global energy conversion rate into lee waves and dissipation rate associated with lee waves and topographic blocking: the Garner (2005) scheme and the Bell (1975) theory. The closure predictions in two Southern Ocean regions where geostrophic flows dominate over tides are examined and compared to microstructure profiler observations of the turbulent kinetic energy dissipation rate, where the latter are assumed to reflect the dissipation associated with topographic blocking and generated lee wave energy. It is shown that when applied to these Southern Ocean regions, the two closures differ most in their treatment of topographic blocking. For several reasons, pointwise validation of the closures is not possible using existing observations, but horizontally averaged comparisons between closure predictions and observations are made. When anisotropy of the underlying topography is accounted for, the two horizontally averaged closure predictions near the seafloor are approximately equal. The dissipation associated with topographic blocking is predicted by the Garner (2005) scheme to account for the majority of the depth‐integrated dissipation over the bottom 1000 m of the water column, where the horizontally averaged predictions lie well within the spatial variability of the horizontally averaged observations. Simplifications made by the Garner (2005) scheme that are inappropriate for the oceanic context, together with imperfect observational information, can partially account for the prediction‐observation disagreement, particularly in the upper water column.D. S. Trossman and B. K. Arbic gratefully acknowledge support from National Science Foundation (NSF) grant OCE‐0960820 and Office of Naval Research (ONR) grant N00014‐11‐1‐0487. S. Waterman gratefully acknowledges support from the Australian Research Council (grants DE120102927 and CE110001028) and the National Science and Engineering Research Council of Canada (grant 22R23085)

Mathematical modeling of gonadotropin-releasing hormone signaling.

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.Gonadotropin-releasing hormone (GnRH) acts via G-protein coupled receptors on pituitary gonadotropes to control of reproduction. These are Gq-coupled receptors that mediate acute effects of GnRH on the exocytotic secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as the chronic regulation of their synthesis. GnRH is secreted in short pulses and GnRH effects on its target cells are dependent upon the dynamics of these pulses. Here we overview GnRH receptors and their signaling network, placing emphasis on pulsatile signaling, and how mechanistic mathematical models and an information theoretic approach have helped further this field.This work was funded Project Grants from MRC (93447) and the BBSRC (J014699). KTA and MV gratefully acknowledge the financial support of the EPSRC via grant EP/N014391/1 and an MRC Biomedical Informatics Fellowship (MR/K021826/1), respectively

Gonadotropin-releasing hormone signaling: An information theoretic approach

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.Gonadotropin-releasing hormone (GnRH) is a peptide hormone that mediates central control of reproduction, acting via G-protein coupled receptors that are primarily Gq coupled and mediate GnRH effects on the synthesis and secretion of luteinizing hormone and follicle-stimulating hormone. A great deal is known about the GnRH receptor signaling network but GnRH is secreted in short pulses and much less is known about how gonadotropes decode this pulsatile signal. Similarly, single cell measures reveal considerable cell-cell heterogeneity in responses to GnRH but the impact of this variability on signaling is largely unknown. Ordinary differential equation-based mathematical models have been used to explore the decoding of pulse dynamics and information theory-derived statistical measures are increasingly used to address the influence of cell-cell variability on the amount of information transferred by signaling pathways. Here, we describe both approaches for GnRH signaling, with emphasis on novel insights gained from the information theoretic approach and on the fundamental question of why GnRH is secreted in pulses.This work was funded Project Grants from MRC (93447) and the BBSRC (J014699). KTA and MV gratefully acknowledge the financial support of the EPSRC via grant EP/N014391/1 and an MRC Biomedical Informatics Fellowship (MR/K021826/1), respectively

Development of a Highly Differentiated Human Primary Proximal Tubule MPS Model (aProximate MPS Flow)

\ua9 2023 by the authors.The kidney proximal tubule (PT) mediates renal drug elimination in vivo and is a major site of drug-induced toxicity. To reliably assess drug efficacy, it is crucial to construct a model in which PT functions are replicated. Current animal studies have proven poorly predictive of human outcome. To address this, we developed a physiologically relevant micro-physiological system (MPS) model of the human PT, the aProximate MPS Flow platform (Patent No: G001336.GB). In this model, primary human PT cells (hPTCs) are subjected to fluidic media flow and a shear stress of 0.01–0.2 Pa. We observe that these cells replicate the polarity of hPTCs and exhibit a higher expression of all the key transporters of SLC22A6 (OAT1), SLC22A8 (OAT3), SLC22A2 (OCT2), SLC47A1 (MATE1), SLC22A12 (URAT1), SLC2A9 (GLUT9), ABCB1 (MDR1), ABCC2 (MRP2), LRP2 (megalin), CUBN (cubilin), compared with cells grown under static conditions. Immunofluorescence microscopy confirmed an increase in OAT1, OAT3, and cilia protein expression. Increased sensitivity to nephrotoxic protein cisplatin was observed; creatinine and FITC-albumin uptake was significantly increased under fluidic shear stress conditions. Taken together, these data suggest that growing human PT cells under media flow significantly improves the phenotype and function of hPTC monolayers and has benefits to the utility and near-physiology of the model

Information Transfer via Gonadotropin-Releasing Hormone Receptors to ERK and NFAT: Sensing GnRH and Sensing Dynamics

This is the final version of the article. Available from Oxford University Press via the DOI in this record.Information theoretic approaches can be used to quantify information transfer via cell signaling networks. In this study, we do so for gonadotropin-releasing hormone (GnRH) activation of extracellular signal-regulated kinase (ERK) and nuclear factor of activated T cells (NFAT) in large numbers of individual fixed LβT2 and HeLa cells. Information transfer, measured by mutual information between GnRH and ERK or NFAT, was <1 bit (despite 3-bit system inputs). It was increased by sensing both ERK and NFAT, but the increase was <50%. In live cells, information transfer via GnRH receptors to NFAT was also <1 bit and was increased by consideration of response trajectory, but the increase was <10%. GnRH secretion is pulsatile, so we explored information gained by sensing a second pulse, developing a model of GnRH signaling to NFAT with variability introduced by allowing effectors to fluctuate. Simulations revealed that when cell–cell variability reflects rapidly fluctuating effector levels, additional information is gained by sensing two GnRH pulses, but where it is due to slowly fluctuating effectors, responses in one pulse are predictive of those in another, so little information is gained from sensing both. Wet laboratory experiments revealed that the latter scenario holds true for GnRH signaling; within the timescale of our experiments (1 to 2 hours), cell–cell variability in the NFAT pathway remains relatively constant, so trajectories are reproducible from pulse to pulse. Accordingly, joint sensing, sensing of response trajectories, and sensing of repeated pulses can all increase information transfer via GnRH receptors, but in each case the increase is small.This work was supported by Biochemical and Biophysical Science Research Council Grant BBSRC BB/J014699/1 (to C.A.M. and K.T.-A.). M.V. acknowledges the support of the Medical Research Council (a strategic skills development fellowship in biomedical informatics) and the Engineering and Physical Sciences Research Council via Grant EP/N014391/1

Unhealthy weight control behaviours in adolescent girls: a process model based on self-determination theory

This study used self-determination theory (Deci, E.L., & Ryan, R.M. (2000). The 'what' and 'why' of goal pursuits: Human needs and the self-determination of behavior. Psychological Inquiry, 11, 227-268.) to examine predictors of body image concerns and unhealthy weight control behaviours in a sample of 350 Greek adolescent girls. A process model was tested which proposed that perceptions of parental autonomy support and two life goals (health and image) would predict adolescents' degree of satisfaction of their basic psychological needs. In turn, psychological need satisfaction was hypothesised to negatively predict body image concerns (i.e. drive for thinness and body dissatisfaction) and, indirectly, unhealthy weight control behaviours. The predictions of the model were largely supported indicating that parental autonomy support and adaptive life goals can indirectly impact upon the extent to which female adolescents engage in unhealthy weight control behaviours via facilitating the latter's psychological need satisfaction

Information Transfer in Gonadotropin-releasing Hormone (GnRH) Signaling: extracellular signal-regulated kinase (ERK)-mediated feedback loops control hormone sensing

The computation model used in the study of GnRH signalling which was used to generate the data appearing in this paper is in ORE at http://hdl.handle.net/10871/27844Cell signaling pathways are noisy communication channels, and statistical measures derived from information theory can be used to quantify the information they transfer. Here we use single cell signaling measures to calculate mutual information as a measure of information transfer via gonadotropin-releasing hormone (GnRH) receptors (GnRHR) to extracellular signal-regulated kinase (ERK) or nuclear factor of activated T-cells (NFAT). This revealed mutual information values <1 bit, implying that individual GnRH-responsive cells cannot unambiguously differentiate even two equally probable input concentrations. Addressing possible mechanisms for mitigation of information loss, we focused on the ERK pathway and developed a stochastic activation model incorporating negative feedback and constitutive activity. Model simulations revealed interplay between fast (min) and slow (min-h) negative feedback loops with maximal information transfer at intermediate feedback levels. Consistent with this, experiments revealed that reducing negative feedback (by expressing catalytically inactive ERK2) and increasing negative feedback (by Egr1-driven expression of dual-specificity phosphatase 5 (DUSP5)) both reduced information transfer from GnRHR to ERK. It was also reduced by blocking protein synthesis (to prevent GnRH from increasing DUSP expression) but did not differ for different GnRHRs that do or do not undergo rapid homologous desensitization. Thus, the first statistical measures of information transfer via these receptors reveals that individual cells are unreliable sensors of GnRH concentration and that this reliability is maximal at intermediate levels of ERK-mediated negative feedback but is not influenced by receptor desensitization.This work was supported by a Biochemical and Biophysical Science Research Council award (BBSRC BB/J014699/1; to C. A. M. and K. T.-A.)

An information theoretic approach to insulin sensing by human kidney podocytes

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this recordPodocytes are key components of the glomerular filtration barrier (GFB). They are insulin-responsive but can become insulin-resistant, causing features of the leading global cause of kidney failure, diabetic nephropathy. Insulin acts via insulin receptors to control activities fundamental to GFB integrity, but the amount of information transferred is unknown. Here we measure this in human podocytes, using information theory-derived statistics that take into account cell-cell variability. High content imaging was used to measure insulin effects on Akt, FOXO and ERK. Mutual Information (MI) and Channel Capacity (CC) were calculated as measures of information transfer. We find that insulin acts via noisy communication channels with more information flow to Akt than to ERK. Information flow estimates were increased by consideration of joint sensing (ERK and Akt) and response trajectory (live cell imaging of FOXO1-clover translocation). Nevertheless, MI values were always <1Bit as most information was lost through signaling. Constitutive PI3K activity is a predominant feature of the system that restricts the proportion of CC engaged by insulin. Negative feedback from Akt supressed this activity and thereby improved insulin sensing, whereas sensing was robust to manipulation of feedforward signaling by inhibiting PI3K, PTEN or PTP1B. The decisions made by individual podocytes dictate GFB integrity, so we suggest that understanding the information on which the decisions are based will improve understanding of diabetic kidney disease and its treatment.Kidney Research UK Gran

Quality of private and public ambulatory health care in low and middle income countries: systematic review of comparative studies

Paul Garner and colleagues conducted a systematic review of 80 studies to compare the quality of private versus public ambulatory health care in low- and middle-income countries