The Study of the Effect of Drugs of Abuse on Protein Kinase A Activity in Mouse Brain and Spinal Cord

Morphine and Δ9-THC are drugs that produce analgesia and rewarding effects. However, chronic treatment with morphine and Δ9-THC produces problematic side-effects including tolerance and physical dependence. The cellular mechanisms underlying opioid and cannabinoid antinociceptive tolerance have been studied for years. Research has demonstrated that the expression of morphine and Δ9-THC antinociceptive tolerance may be mediated through intracellular signaling pathways, such as the adenylyl cyclase /Protein Kinase A (PKA) cascade. The present study investigated the role of PKA in the expression of morphine and Δ9-THC antinociceptive tolerance. Male Swiss Webster mice were treated chronically with morphine or Δ9-THC and the warm-water tail-flick test was used to assess antinociception. These studies revealed that the level and the duration of morphine antinociceptive tolerance both influenced whether PKA activity was increased in mouse brain and spinal cord. Cytosolic PKA activity was increased in the thalamus of 3-day morphine-tolerant mice expressing a 45-fold level of tolerance, but not in mice that expressed a 10-fold level of tolerance. In addition, cytosolic PKA activity was increased in the lumbar spinal cord (LSC) of 15-day morphine-tolerant mice. However, chronic treatment with A9-THC had no effect on neuronal PKA activity even in mice that expressed a high level of antinociceptive tolerance. The absence of an effect of chronic treatment with A9-THC on neuronal PKA activity was supported by the development of a positive control in which the PKA activator Sp-8-Br-cAMPS was administered intracerebroventricularly (i.c.v.) and intrathecally (i.t.) in drug-naYve mice and increases in PKA activity were observed in several brain regions and LSC. Finally, the i.c.v. injection of two peptide fragments of native Protein Kinase A inhibitor (PKI) peptide, PKI-(6-22)-amide and PKI-(Myr- 14-22)- amide, significantly reversed antinociceptive tolerance in mice treated chronically with morphine. PKI-(6-22)-amide (i.c.v.) also inhibited PKA activity in brain regions (thalamus, periaqueductal gray (PAG), and medulla) and LSC, which studies have shown play a role in morphine-induced analgesia. Moreover, PKI-(6-22)-amide reduced the increase in PKA activity in thalamus and LSC observed with chronic morphine treatment. Overall, these studies provide evidence that PKA plays a role in morphine tolerance, but not Δ9-THC tolerance at the doses and times tested

CB1 cannabinoid receptors promote maximal FAK catalytic activity by stimulating cooperative signaling between receptor tyrosine kinases and integrins in neuronal cells

Tyrosine phosphorylation (Tyr-P) of focal adhesion kinase (FAK) regulates FAK activation. Phosphorylated FAK Tyr 397 binds Src family kinases (Src), which in turn directly phosphorylate FAK Tyr 576/577 to produce maximal FAK enzymatic activity. CB1 cannabinoid receptors (CB1) are abundantly expressed in the nervous system and influence FAK activation by presently unknown mechanisms. The current investigation determined CB1-stimulated maximal FAK catalytic activity is mediated by Gi/o proteins in N18TG2 neuronal cells, and that G12/13 regulation of Rac1 and RhoA occurs concomitantly. Immunoblotting analyses using antibodies against FAK phospho-Tyr 397 and phospho-Tyr 576/577 demonstrated the time-course of CB1-stimulated FAK 576/577 Tyr-P occurred in three phases: Phase I (0-2 min) maximal Tyr-P, Phase II (5-20 min) rapid decline in Tyr-P, and Phase III (>20 min) plateau in Tyr-P at submaximal levels. In contrast, FAK 397 Tyr-P was monophasic and significantly lower in magnitude. FAK 397 Tyr-P and Phase I FAK 576/577 Tyr-P involved protein tyrosine phosphatase (PTP1B, Shp1/Shp2)-mediated Src activation, Protein Kinase A (PKA) inhibition, and integrin activation. Phase I maximal FAK 576/577 Tyr-P also required cooperative signaling between receptor tyrosine kinases (RTKs) and integrins. The integrin antagonist RGDS peptide, Flk-1 vascular endothelial growth factor receptor (VEGFR) antagonist SU5416, and epidermal growth factor receptor (EGFR) antagonist AG 1478 blocked Phase I FAK 576/577 Tyr-P. CB1 agonists failed to stimulate FAK Tyr-P in the absence of integrin activation upon suspension in serum-free culture media. In contrast, cells grown on the integrin ligands fibronectin and laminin displayed increased FAK 576/577 Tyr-P that was augmented by CB1 agonists and blocked by the Src inhibitor PP2 and Flk-1 VEGFR antagonist SU5416. Taken together, these studies have identified a complex integrative pathway utilized by CB1 to stimulate maximal FAK 576/577 Tyr-P in neuronal cells

Student perspectives on creating a positive classroom dynamic: science education in prison

Detailed student perspectives on their involvement in prison education are limited in published literature, yet such contributions are invaluable to education practitioners wanting to create inclusive learning environments. This article focuses on the student experience of taking part in a science outreach programme teaching science in prison in England, which was designed to build confidence in students who face challenges in accessing education pathways. Here, former students share their experiences of the programme, as well as other education courses in prison, and offer guidance on best practices for those engaging in outreach or research with the prison population. In particular, their reflections highlight that by creating and maintaining an environment that is accessible, inclusive and relatable, students from all backgrounds are able to engage in course content, and overcome hidden barriers to accessing education. Furthermore, based on their lived experience, the students offer practical advice with regard to improving future access to education in prison. The aim of this article is to give a voice to students in prison about their education experience, highlighting which aspects of this outreach programme (and other education courses) were impactful for them

Use of Sperm In Vitro Capacitation and Flow Cytometry to Estimate Bull Fertility

Study Description: Frozen-thawed semen from five bulls previously identified as high (48.1% and 47.7%, bulls A and B, respectively), intermediary (45.5%, bull C) or low (43.1% and 40.7%, bulls D and E, respectively) fertility, based on pregnancy per AI, were evaluated with several laboratory measures. Measures included total motility, sperm plasma membrane integrity (viability), acrosome integrity, reactive oxygen species (ROS), mitochondrial membrane energy potential (mito-potential), zinc signatures (signatures 1 to 4), and CD9 protein populations at pre-wash, post-wash, h 0 (diluted with non-capacitation media), and at 0, 3, 6, and 24 h after dilution with capacitation media and incubation at 37 ºC. Data were analyzed using the GLIMMIX procedure of SAS for repeated measures with bull, time, and the interaction as fixed effects. Bull by time interaction was significant (P ≤ 0.01) for total motility and viability. There tended (P = 0.06) to be a bull by time interaction for zinc signatures 1 + 2 combined. There was a significant effect of bull (P ≤ 0.03) for viability, viable sperm with disrupted acrosome, zinc signatures 1, 2, and 1 + 2, viable CD9- (CD9 negative), and dead CD9+ (CD9 positive). High and intermediary field fertility bulls had greater (P ≤ 0.04) percentages of viable sperm, zinc signature 2, and zinc signature 1 + 2 compared to low fertility bulls. High and intermediary fertility bulls had decreased (P ≤ 0.05) percentage of dead CD9+ compared to low fertility bulls. There was or tended to be a positive correlation between pregnancy per AI and viability (P = 0.10; r = 0.81), zinc signature 2 (P = 0.04; r = 0.89), and zinc signature 1 + 2 (P = 0.10; r = 0.80)

Work-Unit Absenteeism: Effects of Satisfaction, Commitment, Labor Market Conditions, and Time

Prior research is limited in explaining absenteeism at the unit level and over time. We developed and tested a model of unit-level absenteeism using five waves of data collected over six years from 115 work units in a large state agency. Unit-level job satisfaction, organizational commitment, and local unemployment were modeled as time-varying predictors of absenteeism. Shared satisfaction and commitment interacted in predicting absenteeism but were not related to the rate of change in absenteeism over time. Unit-level satisfaction and commitment were more strongly related to absenteeism when units were located in areas with plentiful job alternatives

Evaluation of stroke services in Anglia Stroke Clinical Network to examine the variation in acute services and stroke outcomes.

BACKGROUND: Stroke is the third leading cause of death in developed countries and the leading cause of long-term disability worldwide. A series of national stroke audits in the UK highlighted the differences in stroke care between hospitals. The study aims to describe variation in outcomes following stroke and to identify the characteristics of services that are associated with better outcomes, after accounting for case mix differences and individual prognostic factors. METHODS/DESIGN: We will conduct a cohort study in eight acute NHS trusts within East of England, with at least one year of follow-up after stroke. The study population will be a systematically selected representative sample of patients admitted with stroke during the study period, recruited within each hospital. We will collect individual patient data on prognostic characteristics, health care received, outcomes and costs of care and we will also record relevant characteristics of each provider organisation. The determinants of one year outcome including patient reported outcome will be assessed statistically with proportional hazards regression models. Self (or proxy) completed EuroQol (EQ-5D) questionnaires will measure quality of life at baseline and follow-up for cost utility analyses. DISCUSSION: This study will provide observational data about health service factors associated with variations in patient outcomes and health care costs following hospital admission for acute stroke. This will form the basis for future RCTs by identifying promising health service interventions, assessing the feasibility of recruiting and following up trial patients, and provide evidence about frequency and variances in outcomes, and intra-cluster correlation of outcomes, for sample size calculations. The results will inform clinicians, public, service providers, commissioners and policy makers to drive further improvement in health services which will bring direct benefit to the patients.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Teleost Growth Factor Independence (Gfi) Genes Differentially Regulate Successive Waves of Hematopoiesis

Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.Stem Cell and Regenerative Biolog

Neuroactive steroids in depression and anxiety disorders: Clinical studies

Certain neuroactive steroids modulate ligand-gated ion channels via non-genomic mechanisms. Especially 3 alpha-reduced pregnane steroids are potent positive allosteric modulators of the gamma-aminobutyric acid type A (GABA(A)) receptor. During major depression, there is a disequilibrium of 3 alpha-reduced neuroactive steroids, which is corrected by clinically effective pharmacological treatment. To investigate whether these alterations are a general principle of successful antidepressant treatment, we studied the impact of nonpharmacological treatment options on neuroactive steroid concentrations during major depression. Neither partial sleep deprivation, transcranial magnetic stimulation, nor electroconvulsive therapy affected neuroactive steroid levels irrespectively of the response to these treatments. These studies suggest that the changes in neuroactive steroid concentrations observed after antidepressant pharmacotherapy more likely reflect distinct pharmacological properties of antidepressants rather than the clinical response. In patients with panic disorder, changes in neuroactive steroid composition have been observed opposite to those seen in depression. However, during experimentally induced panic induction either with cholecystokinine-tetrapeptide or sodium lactate, there was a pronounced decline in the concentrations of 3 alpha-reduced neuroactive steroids in patients with panic disorder, which might result in a decreased GABAergic tone. In contrast, no changes in neuroactive steroid concentrations could be observed in healthy controls with the exception of 3 alpha,5 alpha-tetrahydrodeoxycorticosterone. The modulation of GABA(A) receptors by neuroactive steroids might contribute to the pathophysiology of depression and anxiety disorders and might offer new targets for the development of novel anxiolytic compounds. Copyright (c) 2006 S. Karger AG, Basel

Atypical disengagement from faces and its modulation by the control of eye fixation in children with Autism Spectrum Disorder

By using the gap overlap task, we investigated disengagement from faces and objects in children (9–17 years old) with and without autism spectrum disorder (ASD) and its neurophysiological correlates. In typically developing (TD) children, faces elicited larger gap effect, an index of attentional engagement, and larger saccade-related event-related potentials (ERPs), compared to objects. In children with ASD, by contrast, neither gap effect nor ERPs differ between faces and objects. Follow-up experiments demonstrated that instructed fixation on the eyes induces larger gap effect for faces in children with ASD, whereas instructed fixation on the mouth can disrupt larger gap effect in TD children. These results suggest a critical role of eye fixation on attentional engagement to faces in both groups

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following SARS-CoV-2 infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor-binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an N-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multi-donor class of “public” antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that “public” NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape