RESUMEN
OBJECTIVE: This study assessed predictors of stress, anxiety and depression during the COVID-19 pandemic using a large number of demographic, COVID-19 context and psychological variables. METHODS: Data from 741 adults were drawn from the Boston College daily sleep and well-being survey. Baseline demographics, the long version of the daily surveys and the round one assessment of the survey were utilized for the present study. A Gaussian graphical model (GGM) was estimated as a feature selection technique on a subset of ordinal/continuous variables. An ensemble Random Forest (RF) machine learning algorithm was used for prediction. RESULTS: GGM was found to be an efficient feature selection method and supported the findings derived from the RF machine learning model. Psychological variables were significant predictors of stress, anxiety and depression, while demographic and COVID-19-related factors had minimal predictive value. The outcome variables were mutually predictive of each other, and negative affect and subjective sleep quality were the common predictors of these outcomes of stress, anxiety, and depression. CONCLUSION: The study identifies risk factors for adverse mental health outcomes during the pandemic and informs interventions to mitigate the impact on mental health.
RESUMEN
Ligand-induced activation of G protein-coupled receptors (GPCRs) can initiate signaling through multiple distinct pathways with differing biological and physiological outcomes. There is intense interest in understanding how variation in GPCR ligand structure can be used to promote pathway selective signaling ("biased agonism") with the goal of promoting desirable responses and avoiding deleterious side effects. Here we present an approach in which a conventional peptide ligand for the type 1 parathyroid hormone receptor (PTHR1) is converted from an agonist which induces signaling through all relevant pathways to a compound that is highly selective for a single pathway. This is achieved not through variation in the core structure of the agonist, but rather by linking it to a nanobody tethering agent that binds with high affinity to a separate site on the receptor not involved in signal transduction. The resulting conjugate represents the most biased agonist of PTHR1 reported to date. This approach holds promise for facile generation of pathway selective ligands for other GPCRs.
Asunto(s)
Receptor de Hormona Paratiroídea Tipo 1 , Receptores Acoplados a Proteínas G , Transducción de Señal , Anticuerpos de Dominio Único , Ligandos , Humanos , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/agonistas , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/farmacología , Células HEK293 , Transducción de Señal/efectos de los fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Unión Proteica , Animales , Péptidos/química , Péptidos/farmacología , Péptidos/metabolismoRESUMEN
Ligand-induced activation of G protein-coupled receptors (GPCRs) can initiate signaling through multiple distinct pathways with differing biological and physiological outcomes. There is intense interest in understanding how variation in GPCR ligand structure can be used to promote pathway selective signaling ("biased agonism") with the goal of promoting desirable responses and avoiding deleterious side effects. Here we present a new approach in which a conventional peptide ligand for the type 1 parathyroid hormone receptor (PTHR1) is converted from an agonist which induces signaling through all relevant pathways to a compound that is highly selective for a single pathway. This is achieved not through variation in the core structure of the agonist, but rather by linking it to a nanobody tethering agent that binds with high affinity to a separate site on the receptor not involved in signal transduction. The resulting conjugate represents the most biased agonist of PTHR1 reported to date. This approach holds promise for facile generation of pathway selective ligands for other GPCRs.
RESUMEN
Military organizations often demonstrate contrasting features compared to civilian ones, including indoctrination of military identity and mind-set. Therefore, on returning after retirement, military personnel undergo acculturation to reconnect to the civilian world. Many military retirees face difficulty readjusting in multiple professional and personal life domains, and report decreased life satisfaction due to this transition. The present review conducted a thematic meta-synthesis of 28 studies that had qualitatively assessed military to civilian transition experiences. The aim was to understand the military-civilian culture gap and identify the challenges faced during this transition. The analysis led to six themes - "Military Institutionalization, Military-Civilian Cultural Contrast, The Three S's of Transition Challenges - Stereotypes, Skills, and Support, The Losses of Identity, Reconnecting with Family, Friends, and Civilian Counterparts, and Facilitators in Transition - Covering the Military-Civilian Gap." Based on these findings, the review further presents possible intervention suggestions for retirement adjustment and future research direction.
RESUMEN
OBJECTIVE: The COVID-19 pandemic is a collective trauma causing profound psychological distress and, in some cases, positive psychological changes. The present study ascertained the most influential symptoms and strong connections between posttraumatic stress (PTS) symptoms and posttraumatic growth (PTG) elements. It also examined the symptoms/elements that acted as a bridge between the two, to elucidate how PTS and PTG co-exist. METHOD: The PTSD Checklist (PCL-5) and Posttraumatic Growth Inventory (PTGI) were used to assess PTS symptoms and PTG, respectively, in a sample of 509 adults from Kashmir, India. RESULTS: The network analysis revealed negative trauma-related emotions as the most influential symptom in the PTS network, and the ability to do better things with life as the most influential element in the PTG network. Eight bridge symptoms/elements emerged between the PTS and PTG, namely intrusive thoughts, nightmares, more compassion for others, sleep disturbances, trauma-related amnesia, hypervigilance, willingness to express emotions, and counting on others more. CONCLUSIONS: The study identified the most influential symptom in the PTS network and the bridge symptoms in the combined network of PTS symptoms and PTG elements in the context of the COVID-19 pandemic in the present sample. The bridge symptoms helped in understanding how PTS and PTG co-existed in adults exposed to disasters such as COVID-19. Identification of these symptoms can be highly significant for developing targeted interventions. These implications have been discussed further in the present study. (PsycInfo Database Record (c) 2022 APA, all rights reserved).
RESUMEN
Synthetic molecules that form a covalent bond upon binding to a targeted biomolecule (proximity-induced reactivity) are the subject of intense biomedical interest for the unique pharmacological properties imparted by irreversible binding. However, off-target covalent labeling and the lack of molecules with sufficient specificity limit more widespread applications. We describe the first example of a cross-linking platform that uses a synthetic peptide epitope and a single domain antibody (or nanobody) pair to form a covalent linkage rapidly and specifically. The rate of the cross-linking reaction between peptide and nanobody is faster than most other biocompatible cross-linking reactions, and it can be used to label live cells expressing receptor-nanobody fusions. The rapid kinetics of this system allowed us to probe the consequences on signaling for ligand cross-linking to the A2A-adenosine receptor. Our method may be generally useful to site-specifically link synthetic molecules to receptors on mammalian cell surfaces.
Asunto(s)
Proteínas de la Membrana , Anticuerpos de Dominio Único , Animales , Ligandos , Epítopos , Péptidos/química , MamíferosRESUMEN
Peptide epitope tags offer a valuable means for detection and manipulation of protein targets for which high quality detection reagents are not available. Most commonly used epitope tags are bound by conventional, full-size antibodies (Abs). The complex architecture of Abs complicates their application in protein engineering and intracellular applications. To address these shortcomings, single domain antibodies (nanobodies, Nbs) that recognize short peptide epitopes have become increasingly prized. Here, we characterize the interaction between a Nb (Nb6E) and a 14-mer peptide epitope. We identify residues in the peptide epitope essential for high affinity binding. Using this information in combination with computational modeling we propose a mode of interaction between Nb6E and this epitope. We apply this nanobody-epitope pair to augment the potency of a ligand at an engineered adenosine A2A receptor. This characterization of the nanobody-epitope pair opens the door to diverse applications including mechanistic studies of the G protein-coupled receptor function.
Asunto(s)
Anticuerpos de Dominio Único , Anticuerpos , Epítopos/química , Péptidos/química , Ingeniería de Proteínas , Anticuerpos de Dominio Único/químicaRESUMEN
Proper function of receptors on the cell surface is essential for homeostasis. Compounds that target cell surface receptors to address dysregulation have proven exceptionally successful as therapeutic agents; however, the development of compounds with the desired specificity for receptors, cells, and tissues of choice has proven difficult in some cases. The use of compounds that can engage more than one binding site at the cell surface offers a path toward improving biological specificity or pharmacological properties. In this chapter we summarize historical context for the development of such bivalent compounds. We focus on developments in chemical methods and biological engineering to provide bivalent compounds in which the high affinity and specificity of antibodies are leveraged to create multifunctional conjugates with new and useful properties. The development of methods to meld biological macromolecules with synthetic compounds will facilitate modulation of receptor biology in ways not previously possible.
Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/metabolismo , Biología , Proteínas de la MembranaRESUMEN
Neurons integrate inputs over different time and space scales. Fast excitatory synapses at boutons (ms and µm), and slow modulation over entire dendritic arbors (seconds and mm) are all ultimately combined to produce behavior. Understanding the timing of signaling events mediated by G-protein-coupled receptors is necessary to elucidate the mechanism of action of therapeutics targeting the nervous system. Measuring signaling kinetics in live cells has been transformed by the adoption of fluorescent biosensors and dyes that convert biological signals into optical signals that are conveniently recorded by microscopic imaging or by fluorescence plate readers. Quantifying the timing of signaling has now become routine with the application of equations in familiar curve fitting software to estimate the rates of signaling from the waveform. Here we describe examples of the application of these methods, including (1) Kinetic analysis of opioid signaling dynamics and partial agonism measured using cAMP and arrestin biosensors; (2) Quantifying the signaling activity of illicit synthetic cannabinoid receptor agonists measured using a fluorescent membrane potential dye; (3) Demonstration of multiplicity of arrestin functions from analysis of biosensor waveforms and quantification of the rates of these processes. These examples show how temporal analysis provides additional dimensions to enhance the understanding of GPCR signaling and therapeutic mechanisms in the nervous system.
RESUMEN
Quantitative measurement of receptor signaling by different ligands is important for understanding the mechanism of drug action and screening of drugs. Here, we describe a simple and cost-effective method of measuring adenylyl cyclase inhibition, one of the hallmarks of opioid receptor activation. The assay is based on bioluminescence resonance energy transfer (BRET) that involves transfection of a biosensor in human embryonic kidney (HEK)-293 cells stably transfected with µ-opioid receptor (µ receptor), enabling real-time measurement of cAMP levels.
Asunto(s)
Inhibidores de Adenilato Ciclasa/análisis , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Imagen Molecular/métodos , Toxina de Adenilato Ciclasa , Inhibidores de Adenilato Ciclasa/metabolismo , Adenilil Ciclasas/metabolismo , Analgésicos Opioides , Animales , Colforsina/farmacología , AMP Cíclico , Transferencia de Energía , Células HEK293 , Humanos , Receptores Opioides/química , Receptores Opioides/metabolismo , Receptores Opioides muRESUMEN
Synthetic cannabinoid receptor agonists (SCRAs) are new psychoactive substances associated with acute intoxication and even death. However, the molecular mechanisms through which SCRAs may exert their toxic effects remain unclear-including the potential differential activation of G protein subtypes by cannabinoid receptor type 1 (CB1), a major target of SCRA. We measured CB1-mediated activation of Gαs and Gαi/o proteins by SCRAs by examining stimulation (pertussis toxin, PTX treated) as well as inhibition (non-PTX treated) of forskolin (FSK)-induced cyclic adenosine monophosphate (cAMP) accumulation in human embryonic kidney (HEK) cells stably expressing CB1. Real-time measurements of stimulation and inhibition of cAMP levels were made using a BRET biosensor. We found that the maximum concentration of SCRAs tested (10 µmol L-1 ), increased cAMP levels 12%-45% above that produced by FSK alone, while the phytocannabinoid THC did not significantly alter cAMP levels in PTX-treated HEK-CB1 cells. All SCRAs had greater potency to inhibit FSK-induced cAMP levels than to stimulate cAMP levels. The rank order of potencies for SCRA stimulation of cAMP (Gαs ) was PB-22 > 5F-MDMB-PICA > JWH-018 ≈ AB-FUBINACA > XLR-11. By contrast, the potency of SCRAs for inhibition of cAMP (Gαi/o ) was 5F-MDMB-PICA > AB-FUBINACA > PB-22 > JWH-018 > XLR-11. The different rank order of potency and EMax of the SCRAs to stimulate Gαs -like signaling compared to Gαi/o signaling suggests differences in G protein preference between SCRAs. Understanding the apparent differences among these drugs may contribute to unravelling their complex effects in humans.
Asunto(s)
Agonistas de Receptores de Cannabinoides/farmacología , Proteínas de Unión al GTP/metabolismo , Receptor Cannabinoide CB1/metabolismo , Benzoxazinas/farmacología , Cannabinoides/farmacología , AMP Cíclico/metabolismo , Dronabinol/farmacología , Células HEK293 , Humanos , Indazoles/farmacología , Indoles/farmacología , Morfolinas/farmacología , Naftalenos/farmacología , Quinolinas/farmacología , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/genéticaRESUMEN
BACKGROUND: Synthetic cannabinoids are a commonly used class of recreational drugs that can have significant adverse effects. There have been sporadic reports of co-consumption of illicit drugs with rodenticides such as warfarin and brodifacoum (BFC) over the past 20 years but recently, hundreds of people have been reported to have been poisoned with a mixture of synthetic cannabinoids and BFC. We have sought to establish whether BFC directly affects cannabinoid receptors, or their activation by the synthetic cannabinoid CP55940 or the phytocannabinoid Δ9-tetrahydrocannabinol (Δ9-THC). METHODS: The effects of BFC on the hyperpolarization of wild type AtT20 cells, or AtT20 cells stably expressing human CB1- or CB2- receptors, were studied using a fluorescent assay of membrane potential. The effect of BFC on CB1- and CB2-mediated inhibition of forskolin-stimulated adenylyl cyclase (AC) activation was measured using a BRET assay of cAMP levels in HEK 293 cells stably expressing human CB1 or CB2. RESULTS: BFC did not activate CB1 or CB2 receptors, or affect the hyperpolarization of wild type AtT20 cells produced by somatostatin. BFC (1 µM) did not affect the hyperpolarization of AtT20-CB1 or AtT20-CB2 cells produced by CP55940 or Δ9-THC. BFC (1 µM) did not affect the inhibition of forskolin-stimulated AC activity by CP55940 in HEK 293 cells expressing CB1 or CB2. BFC (1 µM) also failed to affect the desensitization of CB1 and CB2 signaling produced by prolonged (30 min) application of CP55940 or Δ9-THC to AtT20 cells. DISCUSSION: BFC is not a cannabinoid receptor agonist, and appeared not to affect cannabinoid receptor activation. Our data suggests there is no pharmacodynamic rationale for mixing BFC with synthetic cannabinoids; however, it does not speak to whether BFC may affect synthetic cannabinoid metabolism or biodistribution. The reasons underlying the mixing of BFC with synthetic cannabinoids are unknown, and it remains to be established whether the "contamination" was deliberate or accidental. However, the consequences for people who ingested the mixture were often serious, and sometimes fatal, but this seems unlikely to be due to BFC action at cannabinoid receptors.
RESUMEN
Introduction: Compounds present in Cannabis sativa such as phytocannabinoids and terpenoids may act in concert to elicit therapeutic effects. Cannabinoids such as Δ9-tetrahydrocannabinol (Δ9-THC) directly activate cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2); however, it is not known if terpenoids present in Cannabis also affect cannabinoid receptor signaling. Therefore, we examined six common terpenoids alone, and in combination with cannabinoid receptor agonists, on CB1 and CB2 signaling in vitro. Materials and Methods: Potassium channel activity in AtT20 FlpIn cells transfected with human CB1 or CB2 receptors was measured in real time using FLIPR® membrane potential dye in a FlexStation 3 plate reader. Terpenoids were tested individually and in combination for periods up to 30 min. Endogenous somatostatin receptors served as a control for direct effects of drugs on potassium channels. Results: α-Pinene, ß-pinene, ß-caryophyllene, linalool, limonene, and ß-myrcene (up to 30-100 µM) did not change membrane potential in AtT20 cells expressing CB1 or CB2, or affect the response to a maximally effective concentration of the synthetic cannabinoid CP55,940. The presence of individual or a combination of terpenoids did not affect the hyperpolarization produced by Δ9-THC (10 µM): (CB1: control, 59%±7%; with terpenoids (10 µM each) 55%±4%; CB2: Δ9-THC 16%±5%, with terpenoids (10 µM each) 17%±4%). To investigate possible effect on desensitization of CB1 responses, all six terpenoids were added together with Δ9-THC and signaling measured continuously over 30 min. Terpenoids did not affect desensitization, after 30 min the control hyperpolarization recovered by 63%±6% in the presence of the terpenoids recovery was 61%±5%. Discussion: None of the six of the most common terpenoids in Cannabis directly activated CB1 or CB2, or modulated the signaling of the phytocannabinoid agonist Δ9-THC. These results suggest that if a phytocannabinoid-terpenoid entourage effect exists, it is not at the CB1 or CB2 receptor level. It remains possible that terpenoids activate CB1 and CB2 signaling pathways that do not involve potassium channels; however, it seems more likely that they may act at different molecular target(s) in the neuronal circuits important for the behavioral effect of Cannabis.
RESUMEN
BACKGROUND AND PURPOSE: The morbidity and mortality associated with recreational use of synthetic cannabinoid receptor agonists (SCRAs) may reflect strong activation of CB1 receptors and is a major health concern. The properties of SCRA at CB1 receptors are not well defined. Here we have developed an assay to determine acute CB1 receptor efficacy using receptor depletion with the irreversible CB1 receptor antagonist AM6544, with application of the Black and Leff operational model to calculate efficacy. EXPERIMENTAL APPROACH: Receptor depletion in mouse AtT-20 pituitary adenoma cells stably expressing human CB1 receptors was achieved by pretreatment of cells with AM6544 (10 µM, 60 min). The CB1 receptor-mediated hyperpolarisation of AtT-20 cells was measured using fluorescence-based membrane potential dye. From data fit to the operational model, the efficacy (τ) and affinity (KA ) parameters were obtained for each drug. KEY RESULTS: AM6544 did not affect the potency or maximal effect of native somatostatin receptor-induced hyperpolarization. The τ value of ∆9 -THC was 80-fold less than the reference CB receptor agonist CP55940 and 260-fold less than the highest efficacy SCRA, 5F-MDMB-PICA. The operational efficacy of SCRAs ranged from 233 (5F-MDMB-PICA) to 28 (AB-PINACA), with CP55940 in the middle of the efficacy rank order. There was no correlation between the τ and KA values. CONCLUSIONS AND IMPLICATIONS: All SCRAs tested showed substantially higher efficacy at CB1 receptors than ∆9 -THC, which may contribute to the adverse effects seen with these drugs but not ∆9 -THC.
Asunto(s)
Agonistas de Receptores de Cannabinoides/farmacología , Antagonistas de Receptores de Cannabinoides/farmacología , Ciclohexanoles/farmacología , Drogas Ilícitas/farmacología , Receptor Cannabinoide CB1/agonistas , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Receptor Cannabinoide CB1/antagonistas & inhibidoresRESUMEN
Indole and indazole synthetic cannabinoids (SCs) featuring l-valinate or l-tert-leucinate pendant group have recently emerged as prevalent recreational drugs, and their use has been associated with serious adverse health effects. Due to the limited pharmacological data available for these compounds, 5F-AMBICA, 5F-AMB, 5F-ADB, AMB-FUBINACA, MDMB-FUBINACA, MDMB-CHMICA, and their analogues were synthesized and assessed for cannabimimetic activity in vitro and in vivo. All SCs acted as potent, highly efficacious agonists at CB1 (EC50 = 0.45-36 nM) and CB2 (EC50 = 4.6-128 nM) receptors in a fluorometric assay of membrane potential, with a general preference for CB1 activation. The cannabimimetic properties of two prevalent compounds with confirmed toxicity in humans, 5F-AMB and MDMB-FUBINACA, were demonstrated in vivo using biotelemetry in rats. Bradycardia and hypothermia were induced by 5F-AMB and MDMB-FUBINACA doses of 0.1-1 mg/kg (and 3 mg/kg for 5F-AMB), with MDMB-FUBINACA showing the most dramatic hypothermic response recorded in our laboratory for any SC (>3 °C at 0.3 mg/kg). Reversal of hypothermia by pretreatment with a CB1, but not CB2, antagonist was demonstrated for 5F-AMB and MDMB-FUBINACA, consistent with CB1-mediated effects in vivo. The in vitro and in vivo data indicate that these SCs act as highly efficacious CB receptor agonists with greater potency than Δ(9)-THC and earlier generations of SCs.