Overexpressed Gα13 activates serum response factor through stoichiometric imbalance with Gßγ and mislocalization to the cytoplasm.

Gα13, a heterotrimeric G protein α subunit of the G12/13 subfamily, is an oncogenic driver in multiple cancer types. Unlike other G protein subfamilies that contribute to cancer progression via amino acid substitutions that abolish their deactivating, intrinsic GTPase activity, Gα13 rarely harbors such mutations in tumors and instead appears to stimulate aberrant cell growth via overexpression as a wildtype form. It is not known why this effect is exclusive to the G12/13 subfamily, nor has a mechanism been elucidated for overexpressed Gα13 promoting tumor progression. Using a reporter gene assay for serum response factor (SRF)-mediated transcription in HEK293 cells, we found that transiently expressed, wildtype Gα13 generates a robust SRF signal, approximately half the amplitude observed for GTPase-defective Gα13. When epitope-tagged, wildtype Gα13 was titrated upward in cells, a sharp increase in SRF stimulation was observed coincident with a "spillover" of Gα13 from membrane-associated to a soluble fraction. Overexpressing G protein ß and γ subunits caused both a decrease in this signal and a shift of wildtype Gα13 back to the membranous fraction, suggesting that stoichiometric imbalance in the αßγ heterotrimer results in aberrant subcellular localization and signalling by overexpressed Gα13. We also examined the acylation requirements of wildtype Gα13 for signalling to SRF. Similar to GTPase-defective Gα13, S-palmitoylation of the wildtype α subunit was necessary for SRF activation but could be replaced functionally by an engineered site for N-terminal myristoylation. However, a key difference was observed between wildtype and GTPase-defective Gα13: whereas the latter protein lacking palmitoylation sites was rescued in its SRF signalling by either an engineered polybasic sequence or a C-terminal isoprenylation site, these motifs failed to restore signalling by wildtype, non-palmitoylated Gα13. These findings illuminate several components of the mechanism in which overexpressed, wildtype Gα13 contributes to growth and tumorigenic signalling, and reveal greater stringency in its requirements for post-translational modification in comparison to GTPase-defective Gα13.

Clinical Effectiveness of Intravenous Racemic Ketamine Infusions in a Large Community Sample of Patients With Treatment-Resistant Depression, Suicidal Ideation, and Generalized Anxiety Symptoms: A Retrospective Chart Review.

Introduction: Few studies have been published to date exploring the effectiveness of ketamine for treatment-resistant depression (TRD) in large clinical samples. We report on the clinical outcomes of a large cohort treated with ketamine as part of clinical practice.Methods: Deidentified electronic chart data were obtained from a multisite private ketamine infusion clinic for 424 patients with TRD seen from November 9, 2017, to May 4, 2021. Ketamine infusions were administered at a starting dose of 0.5 mg/kg/40 minutes for 6 infusions within 21 days. Maintenance infusions were offered based on clinical response. Changes in outcome measures (scores on the Patient Health Questionnaire-9 [PHQ-9] and Generalized Anxiety Disorder-7 [GAD-7]) within subjects were analyzed using longitudinal multilevel modeling with Kaplan-Meier estimates. Logistic regression was used to analyze for a priori theorized potential moderators of response.Results: Significant improvements from baseline were observed over time on the main outcomes (all P < .001). Based on PHQ-9 self-report data, within 6 weeks of infusion initiation, a 50% response rate and 20% remission rate for depressive symptoms were observed. Response and remission rates were 72% and 38%, respectively, after 10 infusions, and there was a 50% reduction in self-harm/suicidal ideation (SI) symptom scores within 6 weeks. Half of patients with SI at baseline no longer had it after 6 infusions. A 30% reduction in anxiety symptoms (per the GAD-7) was observed.Conclusions: Ketamine was effective at reducing symptoms of SI, depression, and anxiety. The high rates of response and remission were similar to those for interventional treatments in community samples of TRD. Comparative efficacy trials with other interventions and randomized controlled trials of racemic ketamine infusion as the primary treatment for SI are needed.

Feasibility of bystander-administered naloxone delivered by drone to opioid overdose victims.

BACKGROUND: Currently, ≤5% of bystanders witnessing an opioid overdose (OD) in the US administer antidote to the victim. A possible model to mitigate this crisis would be a system that enables 9-1-1 dispatchers to both rapidly deliver naloxone by drone to bystanders at a suspected opioid OD and direct them to administer it while awaiting EMS arrival. METHODS: A simulated 9-1-1 dispatcher directed thirty subjects via 2-way radio to retrieve naloxone nasal spray from atop a drone located outside the simulation building and then administer it using scripted instructions. The primary outcome measure was time from first contact with the dispatcher to administration of the medication. RESULTS: All subjects administered the medication successfully. The mean time interval from 9 -1-1 contact until antidote administration was 122 [95%CI 109-134] sec. There was a significant reduction in time interval if subjects had prior medical training (p = 0.045) or had prior experience with use of a nasal spray device (p = 0.030). Five subjects had difficulty using the nasal spray and four subjects had minor physical impairments, but these barriers did not result in a significant difference in time to administration (p = 0.467, p = 0.30). A significant number of subjects (29/30 [97%], p = 0.044) indicated that they felt confident they could administer intranasal naloxone to an opioid OD victim after participating in the simulation. CONCLUSIONS: Our results suggest that bystanders can carry out 9-1-1 dispatcher instructions to fetch drone-delivered naloxone and potentially decrease the time interval to intranasal administration which supports further development and testing of a such a system.

Effects of exogenous ghrelin administration and ghrelin receptor blockade, in combination with alcohol, on peripheral inflammatory markers in heavy-drinking individuals: Results from two human laboratory studies.

The ghrelin system has been garnering interest for its role in different neuropsychiatric disorders, including alcohol use disorder (AUD). Accordingly, targeting the ghrelin system is under investigation as a potential novel therapeutic approach. While alcohol provokes the immune system and inflammatory responses, ghrelin has potent immunomodulatory and anti-inflammatory properties. The present study aimed to shed light on the "crosstalk" between ghrelin and inflammation by examining the effects of exogenous ghrelin administration and ghrelin receptor blockade on peripheral inflammatory markers in the context of two human laboratory studies with alcohol administration. Non-treatment-seeking, heavy-drinking individuals with alcohol dependence, the majority of whom were African American males, were enrolled. In the first randomized, crossover, double-blind, placebo-controlled human laboratory study, participants underwent two experimental paradigms - an intravenous alcohol self-administration (IV-ASA) and an intravenous alcohol clamp (IV-AC) - each consisting of two counterbalanced sessions (ghrelin, placebo). A loading dose of intravenous ghrelin (3 mcg/kg) or placebo, followed by a continuous ghrelin (16.9 ng/kg/min) or placebo infusion was administered. In the second dose-escalating, single-blind, placebo-controlled human laboratory phase 1b study, participants were dosed with an oral ghrelin receptor blocker (PF-5190457) and underwent an oral alcohol challenge. Repeated blood samples were collected, and plasma concentrations of the following inflammatory markers were measured: C-reactive protein (CRP), interleukin (IL)-6, IL-10, IL-18, and tumor necrosis factor alpha (TNF-α). During the IV-ASA experiment, significant drug × time interaction effects were observed for IL-6 (F3,36 = 3.345, p = 0.030) and IL-10 (F3,53.2 = 4.638, p = 0.006), indicating that ghrelin, compared to placebo, significantly reduced blood concentrations of the proinflammatory cytokine IL-6, while increasing blood concentrations of the anti-inflammatory cytokine IL-10. No significant drug × time interaction effects were observed during the IV-AC experiment, possibly because of its much shorter duration and/or smaller sample. Treatment with PF-5190457, compared to placebo, had no significant effect on the inflammatory markers investigated. In conclusion, a supraphysiologic pharmacological challenge with exogenous ghrelin in heavy-drinking individuals produced anti-inflammatory effects in the context of intravenous alcohol administration. On the contrary, ghrelin receptor blockade did not lead to any change in the inflammatory markers included in this study. Mechanistic studies are required to better understand the interaction between ghrelin, alcohol, and inflammatory processes.

Effects of oral, smoked, and vaporized cannabis on endocrine pathways related to appetite and metabolism: a randomized, double-blind, placebo-controlled, human laboratory study.

As perspectives on cannabis continue to shift, understanding the physiological and behavioral effects of cannabis use is of paramount importance. Previous data suggest that cannabis use influences food intake, appetite, and metabolism, yet human research in this regard remains scant. The present study investigated the effects of cannabis administration, via different routes, on peripheral concentrations of appetitive and metabolic hormones in a sample of cannabis users. This was a randomized, crossover, double-blind, placebo-controlled study. Twenty participants underwent four experimental sessions during which oral cannabis, smoked cannabis, vaporized cannabis, or placebo was administered. Active compounds contained 6.9 ± 0.95% (~50.6 mg) ∆9-tetrahydrocannabinol (THC). Repeated blood samples were obtained, and the following endocrine markers were measured: total ghrelin, acyl-ghrelin, leptin, glucagon-like peptide-1 (GLP-1), and insulin. Results showed a significant drug main effect (p = 0.001), as well as a significant drug × time-point interaction effect (p = 0.01) on insulin. The spike in blood insulin concentrations observed under the placebo condition (probably due to the intake of brownie) was blunted by cannabis administration. A significant drug main effect (p = 0.001), as well as a trend-level drug × time-point interaction effect (p = 0.08) was also detected for GLP-1, suggesting that GLP-1 concentrations were lower under cannabis, compared to the placebo condition. Finally, a significant drug main effect (p = 0.01) was found for total ghrelin, suggesting that total ghrelin concentrations during the oral cannabis session were higher than the smoked and vaporized cannabis sessions. In conclusion, cannabis administration in this study modulated blood concentrations of some appetitive and metabolic hormones, chiefly insulin, in cannabis users. Understanding the mechanisms underpinning these effects may provide additional information on the cross-talk between cannabinoids and physiological pathways related to appetite and metabolism.

Peripheral proinflammatory markers are upregulated in abstinent alcohol-dependent patients but are not affected by cognitive bias modification: Preliminary findings.

BACKGROUND: Inflammatory pathways are known to be negatively affected in patients with alcohol use disorder (AUD). Cognitive bias modification (CBM), an emerging behavioral treatment that involves the 're-training' of cognitive biases using computerized tasks, has been reported to reduce alcohol craving and relapse rates. The aim of this study was to compare peripheral concentrations of the proinflammatory biomarkers IL-18, IL-6, IL-1ß, TNF-α and CRP in AUD patients versus controls and to identify whether CBM treatment affected these biomarkers in AUD patients. METHODS: This 3-week double-blind randomized controlled study tested 36 male abstinent AUD patients receiving CBM or placebo-training, who were also compared to 18 male healthy controls. The approach avoidance task (AAT) was used to test the AUD patients before and after training. CBM training took place over 6 sessions, using a joystick-based approach-avoidance task. Blood samples were collected after the pre- and post-AAT test sessions for the AUD groups, and during an outpatient appointment with the controls. RESULTS: AUD patients, versus controls, presented with significantly higher plasma levels of TNF- α (P < 0.0001) and CRP (P = 0.0031). No changes in the CBM versus placebo groups were noted in IL-18, TNF-α and CRP concentrations following pre-post change or within group pretest- posttest analysis. IL-6 and IL-1ß levels fell under the lower detection limit, thus were not included in the final analyses. CONCLUSIONS: This study confirms that the inflammatory system is altered in AUD. This was the first study that investigated whether CBM training affected proinflammatory markers in AUD patients.