Identification of secretory granule phosphatidylinositol 4,5-bisphosphateinteracting proteins using an affinity pulldown strategy

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2) synthesis is required for calcium-dependent exocytosis in neurosecretory cells. We developed a PtdIns(4,5)P-2 bead pulldown strategy combined with subcellular fractionation to identify endogenous chromaffin granule proteins that interact with PtdIns(4,5)P-2. We identified two synaptotagmin isoforms, synaptotagmins 1 and 7; spectrin; alpha-adaptin; and synaptotagmin-like protein 4 (granuphilin) by mass spectrometry and Western blotting. The interaction between synaptotagmin 7 and PtdIns(4,5)P-2 and its functional relevance was investigated. The 45-kDa isoform of synaptotagmin 7 was found to be highly expressed in adrenal chromaffin cells compared with PC12 cells and to mainly localize to secretory granules by subcellular fractionation, immunoisolation, and immunocytochemistry. We demonstrated that synaptotagmin 7 binds PtdIns(4,5)P-2 via the C2B domain in the absence of calcium and via both the C2A and C2B domains in the presence of calcium. We mutated the polylysine stretch in synaptotagmin 7 C2B and demonstrated that this mutant domain lacks the calcium-independent PtdIns(4,5)P-2 binding. Synaptotagmin 7 C2B domain inhibited catecholamine release from digitonin-permeabilized chromaffin cells, and this inhibition was abrogated with the C2B polylysine mutant. These data indicate that synaptotagmin 7 C2B-effector interactions, which occur via the polylysine stretch, including calcium-independent PtdIns(4,5)P-2 binding, are important for chromaffin granule exocytosis

Phosphatidylinositol(4,5)bisphosphate coordinates actin-mediated mobilization and translocation of secretory vesicles to the plasma membrane of chromaffin cells

ORP5 and ORP8, members of the oxysterol-binding protein (OSBP)-related proteins (ORP) family, are endoplasmic reticulum membrane proteins implicated in lipid trafficking. ORP5 and ORP8 are reported to localize to endoplasmic reticulum-plasma membrane junctions via binding to phosphatidylinositol-4-phosphate (PtdIns(4)P), and act as a PtdIns(4)P/phosphatidylserine counter exchanger between the endoplasmic reticulum and plasma membrane. Here we provide evidence that the pleckstrin homology domain of ORP5/8 via PtdIns(4,5)P 2, and not PtdIns(4)P binding mediates the recruitment of ORP5/8 to endoplasmic reticulum-plasma membrane contact sites. The OSBP-related domain of ORP8 can extract and transport multiple phosphoinositides in vitro, and knocking down both ORP5 and ORP8 in cells increases the plasma membrane level of PtdIns(4,5)P 2 with little effect on PtdIns(4)P. Overall, our data show, for the first time, that phosphoinositides other than PtdIns(4)P can also serve as co-exchangers for the transport of cargo lipids by ORPs.ORP5/8 are endoplasmic reticulum (ER) membrane proteins implicated in lipid trafficking that localize to ER-plasma membrane (PM) contacts and maintain membrane homeostasis. Here the authors show that PtdIns(4,5)P 2 plays a critical role in the targeting and function of ORP5/8 at the PM

The combined diabetes and renal control trial (C-DIRECT) - a feasibility randomised controlled trial to evaluate outcomes in multi-morbid patients with diabetes and on dialysis using a mixed methods approach

Background: This cluster randomised controlled trial set out to investigate the feasibility and acceptability of the “Combined Diabetes and Renal Control Trial” (C-DIRECT) intervention, a nurse-led intervention based on motivational interviewing and self-management in patients with coexisting end stage renal diseases and diabetes mellitus (DM ESRD). Its efficacy to improve glycaemic control, as well as psychosocial and self-care outcomes were also evaluated as secondary outcomes. Methods: An assessor-blinded, clustered randomised-controlled trial was conducted with 44 haemodialysis patients with DM ESRD and ≥ 8% glycated haemoglobin (HbA1c), in dialysis centres across Singapore. Patients were randomised according to dialysis shifts. 20 patients were assigned to intervention and 24 were in usual care. The C-DIRECT intervention consisted of three weekly chair-side sessions delivered by diabetes specialist nurses. Data on recruitment, randomisation, and retention, and secondary outcomes such as clinical endpoints, emotional distress, adherence, and self-management skills measures were obtained at baseline and at 12 weeks follow-up. A qualitative evaluation using interviews was conducted at the end of the trial. Results: Of the 44 recruited at baseline, 42 patients were evaluated at follow-up. One patient died, and one discontinued the study due to deteriorating health. Recruitment, retention, and acceptability rates of C-DIRECT were generally satisfactory HbA1c levels decreased in both groups, but C-DIRECT had more participants with HbA1c < 8% at follow up compared to usual care. Significant improvements in role limitations due to physical health were noted for C-DIRECT whereas levels remained stable in usual care. No statistically significant differences between groups were observed for other clinical markers and other patient-reported outcomes. There were no adverse effects. Conclusions: The trial demonstrated satisfactory feasibility. A brief intervention delivered on bedside as part of routine dialysis care showed some benefits in glycaemic control and on QOL domain compared with usual care, although no effect was observed in other secondary outcomes. Further research is needed to design and assess interventions to promote diabetes self-management in socially vulnerable patients

Lactobacillus rhamnosus GG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants.

Dietary intervention with extensively hydrolyzed casein formula supplemented with Lactobacillus rhamnosus GG (EHCF+LGG) accelerates tolerance acquisition in infants with cow's milk allergy (CMA). We examined whether this effect is attributable, at least in part, to an influence on the gut microbiota. Fecal samples from healthy controls (n=20) and from CMA infants (n=19) before and after treatment with EHCF with (n=12) and without (n=7) supplementation with LGG were compared by 16S rRNA-based operational taxonomic unit clustering and oligotyping. Differential feature selection and generalized linear model fitting revealed that the CMA infants have a diverse gut microbial community structure dominated by Lachnospiraceae (20.5±9.7%) and Ruminococcaceae (16.2±9.1%). Blautia, Roseburia and Coprococcus were significantly enriched following treatment with EHCF and LGG, but only one genus, Oscillospira, was significantly different between infants that became tolerant and those that remained allergic. However, most tolerant infants showed a significant increase in fecal butyrate levels, and those taxa that were significantly enriched in these samples, Blautia and Roseburia, exhibited specific strain-level demarcations between tolerant and allergic infants. Our data suggest that EHCF+LGG promotes tolerance in infants with CMA, in part, by influencing the strain-level bacterial community structure of the infant gut

Infectious Disease Ontology

Technological developments have resulted in tremendous increases in the volume and diversity of the data and information that must be processed in the course of biomedical and clinical research and practice. Researchers are at the same time under ever greater pressure to share data and to take steps to ensure that data resources are interoperable. The use of ontologies to annotate data has proven successful in supporting these goals and in providing new possibilities for the automated processing of data and information. In this chapter, we describe different types of vocabulary resources and emphasize those features of formal ontologies that make them most useful for computational applications. We describe current uses of ontologies and discuss future goals for ontology-based computing, focusing on its use in the field of infectious diseases. We review the largest and most widely used vocabulary resources relevant to the study of infectious diseases and conclude with a description of the Infectious Disease Ontology (IDO) suite of interoperable ontology modules that together cover the entire infectious disease domain

The role of multiple marks in epigenetic silencing and the emergence of a stable bivalent chromatin state

We introduce and analyze a minimal model of epigenetic silencing in budding yeast, built upon known biomolecular interactions in the system. Doing so, we identify the epigenetic marks essential for the bistability of epigenetic states. The model explicitly incorporates two key chromatin marks, namely H4K16 acetylation and H3K79 methylation, and explores whether the presence of multiple marks lead to a qualitatively different systems behavior. We find that having both modifications is important for the robustness of epigenetic silencing. Besides the silenced and transcriptionally active fate of chromatin, our model leads to a novel state with bivalent (i.e., both active and silencing) marks under certain perturbations (knock-out mutations, inhibition or enhancement of enzymatic activity). The bivalent state appears under several perturbations and is shown to result in patchy silencing. We also show that the titration effect, owing to a limited supply of silencing proteins, can result in counter-intuitive responses. The design principles of the silencing system is systematically investigated and disparate experimental observations are assessed within a single theoretical framework. Specifically, we discuss the behavior of Sir protein recruitment, spreading and stability of silenced regions in commonly-studied mutants (e.g., sas2, dot1) illuminating the controversial role of Dot1 in the systems biology of yeast silencing.Comment: Supplementary Material, 14 page

Representation of Time-Varying Stimuli by a Network Exhibiting Oscillations on a Faster Time Scale

Sensory processing is associated with gamma frequency oscillations (30–80 Hz) in sensory cortices. This raises the question whether gamma oscillations can be directly involved in the representation of time-varying stimuli, including stimuli whose time scale is longer than a gamma cycle. We are interested in the ability of the system to reliably distinguish different stimuli while being robust to stimulus variations such as uniform time-warp. We address this issue with a dynamical model of spiking neurons and study the response to an asymmetric sawtooth input current over a range of shape parameters. These parameters describe how fast the input current rises and falls in time. Our network consists of inhibitory and excitatory populations that are sufficient for generating oscillations in the gamma range. The oscillations period is about one-third of the stimulus duration. Embedded in this network is a subpopulation of excitatory cells that respond to the sawtooth stimulus and a subpopulation of cells that respond to an onset cue. The intrinsic gamma oscillations generate a temporally sparse code for the external stimuli. In this code, an excitatory cell may fire a single spike during a gamma cycle, depending on its tuning properties and on the temporal structure of the specific input; the identity of the stimulus is coded by the list of excitatory cells that fire during each cycle. We quantify the properties of this representation in a series of simulations and show that the sparseness of the code makes it robust to uniform warping of the time scale. We find that resetting of the oscillation phase at stimulus onset is important for a reliable representation of the stimulus and that there is a tradeoff between the resolution of the neural representation of the stimulus and robustness to time-warp. Author Summary Sensory processing of time-varying stimuli, such as speech, is associated with high-frequency oscillatory cortical activity, the functional significance of which is still unknown. One possibility is that the oscillations are part of a stimulus-encoding mechanism. Here, we investigate a computational model of such a mechanism, a spiking neuronal network whose intrinsic oscillations interact with external input (waveforms simulating short speech segments in a single acoustic frequency band) to encode stimuli that extend over a time interval longer than the oscillation's period. The network implements a temporally sparse encoding, whose robustness to time warping and neuronal noise we quantify. To our knowledge, this study is the first to demonstrate that a biophysically plausible model of oscillations occurring in the processing of auditory input may generate a representation of signals that span multiple oscillation cycles.National Science Foundation (DMS-0211505); Burroughs Wellcome Fund; U.S. Air Force Office of Scientific Researc

Why is Behavioral Game a Game for Economists? : The concept of beliefs in equilibrium

The interdisciplinary exchange between economists and psychologists has so far been more active and fruitful in the modifications of Expected Utility Theory than in those of Game Theory. We argue that this asymmetry may be explained by economists' specific way of doing equilibrium analysis of aggregate-level outcomes in their practice, and by psychologists' reluctance to fully engage with such practice. We focus on the notion of belief that is embedded in economists' practice of equilibrium analysis, more specifically Nash equilibrium, and argue that its difference from the psychological counterpart is one of the factors that makes interdisciplinary exchange in behavioral game theory more difficult.Peer reviewe