Astrocyte Ca2+-evoked ATP release regulates myelinated axon excitability and conduction speed

INTRODUCTION: Astrocytes support neuronal function throughout the central nervous system. In the gray matter, they regulate synapse number during development, remove synaptically released neurotransmitters to terminate their action and prevent excitotoxicity, control the extracellular potassium concentration to prevent hyperexcitability, regulate blood flow to ensure an adequate energy supply, provide lactate to neurons for energy, and respond to rises of intracellular calcium concentration ([Ca2+]i) by releasing adenosine triphosphate (ATP) and other gliotransmitters that act on neuronal receptors to modulate information processing. However, their role is unclear in the white matter, which transmits information rapidly between gray matter areas using axons wrapped with capacitance-reducing myelin (although they have been suggested to regulate myelination during development and during normal function). RATIONALE: Recently, it has been suggested that learning and memory may reflect not only changes in synaptic function in the gray matter, but also changes in white matter function. In particular, neural circuit function might be regulated by changes in the conduction speed of myelinated axons that result in an altered arrival time of action potentials at a distant neuron. These speed changes might be brought about by alterations of the properties of the passively conducting myelinated internodes or of the intervening excitable nodes of Ranvier, where the action potential is generated. We applied immunohistochemistry to assess how astrocytes interact with myelinated axons, neuronal stimulation and light-evoked calcium uncaging in astrocytes to evoke Ca2+-dependent release of gliotransmitters, and electrophysiology and pharmacology to characterize how astrocyte-released substances might affect the axon initial segment (AIS) and nodes of Ranvier of myelinated neurons. Measurements of conduction velocity and computer modeling allowed us to interpret the results. RESULTS: Astrocytes closely approach the axons of myelinated neurons in layer V of the cerebral cortex that enter the corpus callosum. Uncaging Ca2+ within astrocytes or stimulating spike trains in neurons evoked a rise of astrocyte [Ca2+]i that triggered the release of ATP-containing vesicles from these cells. This evoked an inward current in the AIS and nodes of Ranvier of the pyramidal neurons. Pharmacology showed that this was mediated by the activation of Gs-linked adenosine A2a receptors (A2aRs), implying that the released ATP was converted to adenosine by extracellular enzymes. The A2aRs raise the intracellular concentration of cyclic AMP, which activates hyperpolarization-activated cyclic nucleotide–gated (HCN) channels mediating the inward hyperpolarization-activated current (Ih) and thus depolarizes the cell. In the AIS, the activation of A2aRs alters excitability and hence action potential generation, whereas in the nodes of Ranvier, it decreases the conduction speed of the action potential along the axon. CONCLUSION: As in the gray matter, astrocyte [Ca2+]i regulates the release of ATP into the extracellular space in the white matter. After conversion to adenosine, this regulates the excitability and conduction speed of myelinated axons. The changes in excitability at the AIS will lead to changes in the relationship between the synaptic input and action potential output of the cell. The altered conduction speed of the myelinated axon may change neural circuit function by changing the action potential arrival time at the cell’s output synapses, thus altering the integration of signals in postsynaptic neurons. Variations in astrocyte-derived adenosine level can occur between wake and sleep states, and the extracellular adenosine concentration rises during energy deprivation conditions. These changes in adenosine level could thus control white matter information flow and neural circuit function

Adiponectin levels in people with Latent Autoimmune Diabetes-a case control study

<p>Abstract</p> <p>Background</p> <p>To examine adiponectin levels in people with Latent Autoimmune Diabetes in Adults using a matched pair case control study.</p> <p>Findings</p> <p>Patients with LADA (n = 64), were matched for sex with type 2 diabetic and non-diabetic controls. A matched paired T-test was used to examine average adiponectin levels in the LADA patients' versus controls. The average adiponectin level for the LADA patients was 9.96 μg/ml compared to 6.4 μg/ml for Type 2 matched controls and 9.6 μg/ml for non-diabetic controls. Mean difference for the LADA-type 2 comparison was calculated after data was log transformed and showed a difference of 1.58 μg/ml (95%CI: 1.28-1.95, p = 0.0001). There was no significant difference between LADA and non-diabetic controls (p = 0.54).</p> <p>Conclusions</p> <p>Adiponectin levels are higher among people with LADA compared to those with type 2 diabetes and are equivalent to levels seen in non-diabetic controls. This suggests that risk of complications in LADA, as with type 1 diabetes may be related more to glycaemic control rather than to factors of the metabolic syndrome.</p

A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening

<p>Abstract</p> <p>Background</p> <p>A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma.</p> <p>Methods</p> <p>Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds.</p> <p>Results</p> <p>Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell <it>in vitro </it>and attenuated active force development of intact tissue <it>ex vivo</it>.</p> <p>Conclusions</p> <p>This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.</p

Family-based factors associated with overweight and obesity among Pakistani primary school children

<p>Abstract</p> <p>Background</p> <p>Childhood obesity epidemic is now penetrating the developing countries including Pakistan, especially in the affluent urban population. There is no data on association of family-based factors with overweight and obesity among school-aged children in Pakistan. The study aimed to explore the family-based factors associated with overweight and obesity among Pakistani primary school children.</p> <p>Methods</p> <p>A population-based cross-sectional study was conducted with a representative multistage cluster sample of 1860 children aged five to twelve years in Lahore, Pakistan. Overweight (> +1SD BMI-for-age z-score) and obesity (> +2SD BMI-for-age z-score) were defined using the World Health Organization reference 2007. Chi-square test was used as the test of trend. Linear regression was used to examine the predictive power of independent variables in relation to BMI. Logistic regression was used to quantify the independent predictors of overweight and adjusted odds ratios (aOR) with 95% confidence intervals (CI) were obtained. All regression analyses were controlled for age and gender and statistical significance was considered at P < 0.05.</p> <p>Results</p> <p>Significant family-based correlates of overweight and obesity included higher parental education (P < 0.001), both parents working (P = 0.002), fewer siblings (P < 0.001), fewer persons in child's living room (P < 0.001) and residence in high-income neighborhoods (P < 0.001). Smoking in living place was not associated with overweight and obesity. Higher parental education (P < 0.001) and living in high-income neighborhoods (P < 0.001) showed a significant independent positive association with BMI while greater number of siblings (P = 0.001) and persons in child's living room (P = 0.022) showed a significant independent inverse association. College-level or higher parental education as compared to high school-level or lower parental education (aOR 2.54, 95% CI 1.76-3.67), living in high-income neighborhoods as compared to low-income neighborhoods (aOR 2.13, 95% CI 1.31-3.46) and three or less siblings as compared to more than three siblings (aOR 1.75, 95% CI 1.26-2.42) were significant independent predictors of overweight.</p> <p>Conclusion</p> <p>Family-based factors were significantly associated with overweight and obesity among school-aged children in Pakistan. Higher parental education, living in high-income neighborhoods and fewer siblings were independent predictors of overweight. These findings support the need to design evidence-based child health policy and implement targeted interventions, considering the impact of family-based factors and involving communities.</p

Applying the ALARA concept to the evaluation of vesicoureteric reflux

The voiding cystourethrogram (VCUG) is a widely used study to define lower urinary tract anatomy and to diagnose vesicoureteric reflux (VUR) in children. We examine the technical advances in the VCUG and other examinations for reflux that have reduced radiation exposure of children, and we give recommendations for the use of imaging studies in four groups of children: (1) children with urinary tract infection, (2) siblings of patients with VUR, (3) infants with antenatal hydronephrosis (ANH), and (4) children with a solitary functioning kidney. By performing examinations with little to no radiation, carefully selecting only the children who need imaging studies and judiciously timing follow-up examinations, we can reduce the radiation exposure of children being studied for reflux

Electron spin resonance in membrane research: protein–lipid interactions from challenging beginnings to state of the art

Conventional electron paramagnetic resonance (EPR) spectra of lipids that are spin-labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane. This allows determination of both the stoichiometry of lipid–protein interaction (i.e., number of lipid sites at the protein perimeter) and the selectivity of the protein for different lipid species (i.e., association constants relative to the background lipid). Spin-label EPR data are summarised for 20 or more different transmembrane peptides and proteins, and 7 distinct species of lipids. Lineshape simulations of the two-component conventional spin-label EPR spectra allow estimation of the rate at which protein-associated lipids exchange with those in the bulk fluid regions of the membrane. For lipids that do not display a selectivity for the protein, the intrinsic off-rates for exchange are in the region of 10 MHz: less than 10× slower than the rates of diffusive exchange in fluid lipid membranes. Lipids with an affinity for the protein, relative to the background lipid, have off-rates for leaving the protein that are correspondingly slower. Non-linear EPR, which depends on saturation of the spectrum at high radiation intensities, is optimally sensitive to dynamics on the timescale of spin-lattice relaxation, i.e., the microsecond regime. Both progressive saturation and saturation transfer EPR experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale. The sensitivity of non-linear EPR to low frequencies of spin exchange also allows the location of spin-labelled membrane protein residues relative to those of spin-labelled lipids, in double-labelling experiments

Synthetic biology to access and expand nature's chemical diversity

Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Accessing these natural products promises to reinvigorate drug discovery pipelines and provide novel routes to synthesize complex chemicals. The pathways leading to the production of these molecules often comprise dozens of genes spanning large areas of the genome and are controlled by complex regulatory networks with some of the most interesting molecules being produced by non-model organisms. In this Review, we discuss how advances in synthetic biology — including novel DNA construction technologies, the use of genetic parts for the precise control of expression and for synthetic regulatory circuits — and multiplexed genome engineering can be used to optimize the design and synthesis of pathways that produce natural products

Myelin Proteomics: Molecular Anatomy of an Insulating Sheath

Fast-transmitting vertebrate axons are electrically insulated with multiple layers of nonconductive plasma membrane of glial cell origin, termed myelin. The myelin membrane is dominated by lipids, and its protein composition has historically been viewed to be of very low complexity. In this review, we discuss an updated reference compendium of 342 proteins associated with central nervous system myelin that represents a valuable resource for analyzing myelin biogenesis and white matter homeostasis. Cataloging the myelin proteome has been made possible by technical advances in the separation and mass spectrometric detection of proteins, also referred to as proteomics. This led to the identification of a large number of novel myelin-associated proteins, many of which represent low abundant components involved in catalytic activities, the cytoskeleton, vesicular trafficking, or cell adhesion. By mass spectrometry-based quantification, proteolipid protein and myelin basic protein constitute 17% and 8% of total myelin protein, respectively, suggesting that their abundance was previously overestimated. As the biochemical profile of myelin-associated proteins is highly reproducible, differential proteome analyses can be applied to material isolated from patients or animal models of myelin-related diseases such as multiple sclerosis and leukodystrophies