Protein kinase C regulates tonic GABAA receptor-mediated inhibition in the hippocampus and thalamus.

Tonic inhibition mediated by extrasynaptic GABAA receptors (GABAA Rs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABAA Rs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABAA R-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABAA R activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABAA Rs, which represent a key extrasynaptic GABAA R isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABAA Rs. The inhibitory effects of PKC activation on α4β2δ GABAA R activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABAA R-mediated inhibition

Probing GABAA receptors with inhibitory neurosteroids

γ-aminobutyric acid type A receptors (GABAARs) are important components of the central nervous system and they are functionally tasked with controlling neuronal excitability. These receptors are subject to post-translational modification and also to modulation by endogenous regulators, such as the neurosteroids. These modulators can either potentiate or inhibit GABAAR function. Whilst the former class of neurosteroids are considered to bind to and act from the transmembrane domain of the receptor, the domains that are important for the inhibitory neurosteroids remain less clear. In this study, we systematically compare a panel of recombinant synaptic-type and extrasynaptic-type GABAARs expressed in heterologous cell systems for their sensitivity to inhibition by the classic inhibitory neurosteroid, pregnenolone sulphate. Generally, peak GABA current responses were inhibited less compared to steady-state currents, implicating the desensitised state in inhibition. Moreover, pregnenolone sulphate inhibition increased with GABA concentration, but showed minimal voltage dependence. There was no strong dependence of inhibition on receptor subunit composition, the exception being the ρ1 receptor, which is markedly less sensitive. By using competition experiments with pregnenolone sulphate and the GABA channel blocker picrotoxinin, discrete binding sites are proposed. Furthermore, by assessing inhibition using site-directed mutagenesis and receptor chimeras comprising α, β or γ subunits with ρ1 subunits, the receptor transmembrane domains are strongly implicated in mediating inhibition and most likely the binding location for pregnenolone sulphate in GABAARs

Intracellular Chloride Ions Regulate the Time Course of GABA-Mediated Inhibitory Synaptic Transmission

The time-dependent integration of excitatory and inhibitory synaptic currents is an important process for shaping the input - output profiles of individual excitable cells, and therefore the activity of neuronal networks. Here, we show that the decay time course of GABAergic inhibitory synaptic currents is considerably faster when recorded with physiological internal Cl- concentrations than with symmetrical Cl- solutions. This effect of intracellular Cl- is due to a direct modulation of the GABA(A) receptor that is independent of the net direction of current flow through the ion channel. As a consequence, the time window during which GABAergic inhibition can counteract coincident excitatory inputs is much shorter, under physiological conditions, than that previously measured using high internal Cl-. This is expected to have implications for neuronal network excitability and neurodevelopment, and for our understanding of pathological conditions, such as epilepsy and chronic pain, where intracellular Cl- concentrations can be altered

Structural determinants and regulation of spontaneous activity in GABAA receptors

GABA_{A} receptors are vital for controlling neuronal excitability and can display significant levels of constitutive activity that contributes to tonic inhibition. However, the mechanisms underlying spontaneity are poorly understood. Here we demonstrate a strict requirement for β3 subunit incorporation into receptors for spontaneous gating, facilitated by α4, α6 and δ subunits. The crucial molecular determinant involves four amino acids (GKER) in the β3 subunit's extracellular domain, which interacts with adjacent receptor subunits to promote transition to activated, open channel conformations. Spontaneous activity is further regulated by β3 subunit phosphorylation and by allosteric modulators including neurosteroids and benzodiazepines. Promoting spontaneous activity reduced neuronal excitability, indicating that spontaneous currents will alter neural network activity. This study demonstrates how regional diversity in GABA_{A} receptor isoform, protein kinase activity, and neurosteroid levels, can impact on tonic inhibition through the modulation of spontaneous GABA_{A} receptor gating

Risk of subsequent primary neoplasms in survivors of adolescent and young adult cancer (Teenage and Young Adult Cancer Survivor Study): a population-based, cohort study.

Background Few studies have investigated the risks of subsequent primary neoplasms after adolescent and young adult (AYA) cancer. We investigated the risks of specific subsequent primary neoplasms after each of 16 types of AYA cancer. Methods The Teenage and Young Adult Cancer Survivor Study is a population-based cohort of 200 945 survivors of cancer diagnosed when aged 15–39 years in England and Wales from Jan 1, 1971, to Dec 31, 2006. The cohort was established using cancer registrations from the Office for National Statistics and the Welsh Cancer registry. Follow-up was from 5-year survival until the first occurrence of death, emigration, or study end date (Dec 31, 2012). In this analysis, we focus on the risk of specific subsequent primary neoplasms after 16 types of AYA cancer: breast; cervical; testicular; Hodgkin lymphoma (female); Hodgkin lymphoma (male); melanoma; CNS (intracranial); colorectal; non-Hodgkin lymphoma; thyroid; soft-tissue sarcoma; ovarian; bladder; other female genital; leukaemia; and head and neck cancer. We report absolute excess risks (AERs; per 10 000 person-years) and cumulative incidence of specific types of subsequent primary neoplasm after each type of AYA cancer. Findings During the 2 631 326 person-years of follow-up (median follow-up 16·8 years, IQR 10·5–25·2), 12 321 subsequent primary neoplasms were diagnosed in 11 565 survivors, most frequently among survivors of breast cancer, cervical cancer, testicular cancer, and Hodgkin lymphoma. AERs of any subsequent primary neoplasms were 19·5 per 10 000 person-years (95% CI 17·4–21·5) in survivors of breast cancer, 10·2 (8·0–12·4) in survivors of cervical cancer, 18·9 (16·6–21·1) in survivors of testicular cancer, 55·7 (50·4–61·1) in female survivors of Hodgkin lymphoma, and 29·9 (26·3–33·6) in male survivors of Hodgkin lymphoma. The cumulative incidence of all subsequent primary neoplasms 35 years after diagnosis was 11·9% (95% CI 11·3–12·6) in survivors of breast cancer, 15·8% (14·8–16·7) in survivors of cervical cancer, 20·2% (18·9–21·5) in survivors of testicular cancer, 26·6% (24·7–28·6) in female survivors of Hodgkin lymphoma, and 16·5% (15·2–18·0) in male survivors of Hodgkin lymphoma. In patients who had survived at least 30 years from diagnosis of cervical cancer, testicular cancer, Hodgkin lymphoma in women, breast cancer, and Hodgkin lymphoma in men, we identified a small number of specific subsequent primary neoplasms that account for 82%, 61%, 58%, 45%, and 41% of the total excess number of neoplasms, respectively. Lung cancer accounted for a notable proportion of the excess number of neoplasms across all AYA groups investigated. Interpretation Our finding that a small number of specific subsequent primary neoplasms account for a large percentage of the total excess number of neoplasms in long-term survivors of cervical, breast, and testicular cancer, and Hodgkin lymphoma provides an evidence base to inform priorities for clinical long-term follow-up. The prominence of lung cancer after each of these AYA cancers indicates the need for further work aimed at preventing and reducing the burden of this cancer in future survivors of AYA cancer. Funding Cancer Research UK, National Institute for Health Research, Academy of Medical Sciences, and Children with Cancer UK

Measurement of D-s(+) and D-s(*+) production in B meson decays and from continuum e(+)e(-) annihilation at √s=10.6 GeV

This is the pre-print version of the Article. The official published version can be accessed from the links below. Copyright @ 2002 APSNew measurements of Ds+ and Ds*+ meson production rates from B decays and from qq̅ continuum events near the Υ(4S) resonance are presented. Using 20.8 fb-1 of data on the Υ(4S) resonance and 2.6 fb-1 off-resonance, we find the inclusive branching fractions B(B⃗Ds+X)=(10.93±0.19±0.58±2.73)% and B(B⃗Ds*+X)=(7.9±0.8±0.7±2.0)%, where the first error is statistical, the second is systematic, and the third is due to the Ds+→φπ+ branching fraction uncertainty. The production cross sections σ(e+e-→Ds+X)×B(Ds+→φπ+)=7.55±0.20±0.34pb and σ(e+e-→Ds*±X)×B(Ds+→φπ+)=5.8±0.7±0.5pb are measured at center-of-mass energies about 40 MeV below the Υ(4S) mass. The branching fractions ΣB(B⃗Ds(*)+D(*))=(5.07±0.14±0.30±1.27)% and ΣB(B⃗Ds*+D(*))=(4.1±0.2±0.4±1.0)% are determined from the Ds(*)+ momentum spectra. The mass difference m(Ds+)-m(D+)=98.4±0.1±0.3MeV/c2 is also measured.This work was supported by DOE and NSF (USA), NSERC (Canada), IHEP (China), CEA and CNRS-IN2P3 (France), BMBF (Germany), INFN (Italy), NFR (Norway), MIST (Russia), and PPARC (United Kingdom). Individuals have received support from the Swiss NSF, A. P. Sloan Foundation, Research Corporation, and Alexander von Humboldt Foundation

Results of a randomized, double blind, placebo controlled, crossover trial of melatonin for treatment of Nocturia in adults with multiple sclerosis (MeNiMS)

© 2018 The Author(s). Background: Nocturia is a common urinary symptom of multiple sclerosis (MS) which can affect quality of life (QoL) adversely. Melatonin is a hormone known to regulate circadian rhythm and reduce smooth muscle activity such as in the bladder. There is limited evidence supporting use of melatonin to alleviate urinary frequency at night in the treatment of nocturia. The aim of this study was to evaluate the effect of melatonin on the mean number of nocturia episodes per night in patients with MS. Methods: A randomized, double blind, placebo controlled crossover trial was conducted. 34 patients with nocturia secondary to multiple sclerosis underwent a 4-day pre-treatment monitoring phase. The patients were randomized to receive either 2 mg per night (taken at bedtime) of capsulated sustained-release melatonin (Circadin®) or 1 placebo capsule for 6 weeks followed by a crossover to the other regimen for an additional 6 weeks after a 1-month washout period. Results: From the 26 patients who completed the study, there was no significant difference observed in the signs or symptoms of nocturia when taking 2 mg melatonin compared to placebo. The primary outcome measure, mean number of nocturia episodes on bladder diaries, was 1.8/night at baseline, and 1.4/night on melatonin, compared with 1.6 for placebo (Medians 1.70, 1.50, and 1.30 respectively, p = 0.85). There was also no significant difference seen in LUTS, QoL and sleep quality when taking melatonin. No significant safety concerns arose. Conclusions: This small study suggests that a low dose of melatonin taken at bedtime may be ineffective therapy for nocturia in MS. Trial registration: (EudraCT reference) 2012-00418321 registered: 25/01/13. ISRCTN Registry: ISRCTN38687869

Measurement of the inclusive and dijet cross-sections of b-jets in pp collisions at sqrt(s) = 7 TeV with the ATLAS detector

The inclusive and dijet production cross-sections have been measured for jets containing b-hadrons (b-jets) in proton-proton collisions at a centre-of-mass energy of sqrt(s) = 7 TeV, using the ATLAS detector at the LHC. The measurements use data corresponding to an integrated luminosity of 34 pb^-1. The b-jets are identified using either a lifetime-based method, where secondary decay vertices of b-hadrons in jets are reconstructed using information from the tracking detectors, or a muon-based method where the presence of a muon is used to identify semileptonic decays of b-hadrons inside jets. The inclusive b-jet cross-section is measured as a function of transverse momentum in the range 20 < pT < 400 GeV and rapidity in the range |y| < 2.1. The bbbar-dijet cross-section is measured as a function of the dijet invariant mass in the range 110 < m_jj < 760 GeV, the azimuthal angle difference between the two jets and the angular variable chi in two dijet mass regions. The results are compared with next-to-leading-order QCD predictions. Good agreement is observed between the measured cross-sections and the predictions obtained using POWHEG + Pythia. MC@NLO + Herwig shows good agreement with the measured bbbar-dijet cross-section. However, it does not reproduce the measured inclusive cross-section well, particularly for central b-jets with large transverse momenta.Comment: 10 pages plus author list (21 pages total), 8 figures, 1 table, final version published in European Physical Journal

A propofol binding site on mammalian GABAA receptors identified by photolabeling

Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABA(A) receptors, but where it binds to this receptor is not known and has been a matter of some controversy. We have synthesized a novel propofol analogue photolabeling reagent that has a biological activity very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin which have been identified using X-ray crystallography. Using a combination of the protiated label and a deuterated version, and mammalian receptors labeled in intact membranes, we have identified a novel binding site for propofol in GABA(A) receptors consisting of both β(3) homopentamers and α(1)β(3) heteropentamers. The binding site is located within the β subunit, at the interface between the transmembrane domains and the extracellular domain, and lies close to known determinants of anesthetic sensitivity in transmembrane segments TM1 and TM2

Replacement of α-galactosidase A in Fabry disease: effect on fibroblast cultures compared with biopsied tissues of treated patients

The function and intracellular delivery of enzyme therapeutics for Fabry disease were studied in cultured fibroblasts and in the biopsied tissues of two male patients to show diversity of affected cells in response to treatment. In the mutant fibroblasts cultures, the final cellular level of endocytosed recombinant α-galactosidases A (agalsidases, FabrazymeTM, and ReplagalTM) exceeded, by several fold, the amount in control fibroblasts and led to efficient direct intra-lysosomal hydrolysis of (3H)Gb3Cer. In contrast, in the samples from the heart and some other tissues biopsied after several months of enzyme replacement therapy (ERT) with FabrazymeTM, only the endothelial cells were free of storage. Persistent Gb3Cer storage was found in cardiocytes (accompanied by increase of lipopigment), smooth muscle cells, fibroblasts, sweat glands, and skeletal muscle. Immunohistochemistry of cardiocytes demonstrated, for the first time, the presence of a considerable amount of the active enzyme in intimate contact with the storage compartment. Factors responsible for the limited ERT effectiveness are discussed, namely post-mitotic status of storage cells preventing their replacement by enzyme supplied precursors, modification of the lysosomal system by longstanding storage, and possible relative lack of Sap B. These observations support the strategy of early treatment for prevention of lysosomal storage