Optimization of ACE-tRNAs function in translation for suppression of nonsense mutations.

Nonsense suppressor transfer RNAs (tRNAs) or AntiCodon-Edited tRNAs (ACE-tRNAs) have long been envisioned as a therapeutic approach to overcome genetic diseases resulting from the introduction of premature termination codons (PTCs). The ACE-tRNA approach for the rescue of PTCs has been hampered by ineffective delivery through available modalities for gene therapy. Here we have screened a series of ACE-tRNA expression cassette sequence libraries containing >1800 members in an effort to optimize ACE-tRNA function and provide a roadmap for optimization in the future.

Gz Enhanced Signal Transduction assaY (GESTY) for GPCR deorphanization.

G protein-coupled receptors (GPCRs) are key pharmacological targets, yet many remain underutilized due to unknown activation mechanisms and ligands. Orphan GPCRs, lacking identified natural ligands, are a high priority for research, as identifying their ligands will aid in understanding their functions and potential as drug targets. Most GPCRs, including orphans, couple to G family members, however current assays to detect their activation are limited, hindering ligand identification efforts.

Effects of (2R,6R)-hydroxynorketamine in assays of acute pain-stimulated and pain-depressed behaviors in mice.

Ketamine has been shown to produce analgesia in various acute and chronic pain states; however, abuse liability concerns have limited its utility. The ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) has been shown to produce antidepressant-like effects similar to ketamine without abuse liability concerns. (2R,6R)-HNK produces sustained analgesia in models of chronic pain, but has yet to be evaluated in models of acute pain.

Examination of the mechanisms underlying the discriminative stimulus properties of the atypical antipsychotic amisulpride.

Amisulpride is an atypical benzamide antipsychotic/antidepressant, whose mechanism of action is thought to depend mainly on dopamine D2/3 receptor activity, but also with some serotonin 5-HT2B/7 effects. The present study examined the role of D2/3 receptors and 5-HT2B/7 receptors in amisulpride's discriminative stimulus. Selective agonists and antagonists of the above receptors were tested in adult, male C57BL/6 mice trained to discriminate 10 mg/kg amisulpride from vehicle in a two-lever drug discrimination assay.

Efficient suppression of endogenous CFTR nonsense mutations using anticodon-engineered transfer RNAs.

Nonsense mutations or premature termination codons (PTCs) comprise ∼11% of all genetic lesions, which result in over 7,000 distinct genetic diseases. Due to their outsized impact on human health, considerable effort has been made to find therapies for nonsense-associated diseases. Suppressor tRNAs have long been identified as a possible therapeutic for nonsense-associated diseases; however, their ability to inhibit nonsense-mediated mRNA decay (NMD) and support significant protein translation from endogenous transcripts has not been determined in mammalian cells.

How to account for hallucinations in the interpretation of the antidepressant effects of psychedelics: a translational framework.

Rationale: Recent trials with psychedelics in major depressive disorder and treatment-resistant depression showed remarkable improvements in depressive symptoms that can last for up to several months after even a single administration. The lack of an appropriate placebo control group-as patients are often able to discriminate the subjective effects of the drug-and an incomplete understanding of the role of the hallucinogenic and mystical experience, hampers the interpretation of these therapeutic effects.

Objectives: To control for these factors, we developed a translational framework based on establishing pharmacokinetic/pharmacodynamic (PK/PD) relationships in rodents and humans for hallucinogenic (i.

Effects of subanesthetic ketamine and (2R,6R) hydroxynorketamine on working memory and synaptic transmission in the nucleus reuniens in mice.

Rationale: Acute cognitive impairment and abuse potential of ketamine incentivizes the search for alternatives to ketamine for clinical management of treatment-resistant depression. Recently, (2R,6R) hydroxynorketamine ((2R,6R)-HNK), a metabolite of ketamine, has shown promise due to its reported lack of ketamine-like reinforcing properties. Nonetheless, the effect of (2R,6R)-HNK on cognition has not been reported.

Preliminary assessment of the subjective effects of electronic-cigarettes in young-adult low-dose electronic-cigarette users: Effects of nicotine dose and e-liquid flavor.

Objective: The present study evaluated the effects of nicotine concentration (0-10 mg/ml) and flavor (gummy bear vs unflavored) on the subjective experiences of vaporized nicotine in young adult low-dose nicotine (3 mg/ml) ECIG users.

Participants: Eight young adult ECIG users were recruited.

Methods: A single blinded crossover study was used.

Opioid receptor system contributes to the acute and sustained antidepressant-like effects, but not the hyperactivity motor effects of ketamine in mice.

In 2000, a subanesthetic dose (0.5 mg/kg i.v.

Therapeutic promise of engineered nonsense suppressor tRNAs.

Nonsense mutations change an amino acid codon to a premature termination codon (PTC) generally through a single-nucleotide substitution. The generation of a PTC results in a defective truncated protein and often in severe forms of disease. Because of the exceedingly high prevalence of nonsense-associated diseases and a unifying mechanism, there has been a concerted effort to identify PTC therapeutics.

Dissecting Optical Response and Molecular Structure of Fluorescent Proteins With Non-canonical Chromophores.

Tracking the structural dynamics of fluorescent protein chromophores holds the key to unlocking the fluorescence mechanisms in real time and enabling rational design principles of these powerful and versatile bioimaging probes. By combining recent chemical biology and ultrafast spectroscopy advances, we prepared the superfolder green fluorescent protein (sfGFP) and its non-canonical amino acid (ncAA) derivatives with a single chlorine, bromine, and nitro substituent at the site to the phenolate oxygen of the embedded chromophore, and characterized them using an integrated toolset of femtosecond transient absorption and tunable femtosecond stimulated Raman spectroscopy (FSRS), aided by quantum calculations of the vibrational normal modes. A dominant vibrational cooling time constant of ~4 and 11 ps is revealed in Cl-GFP and Br-GFP, respectively, facilitating a ~30 and 12% increase of the fluorescent quantum yield vs.

Efficient Site-Specific Prokaryotic and Eukaryotic Incorporation of Halotyrosine Amino Acids into Proteins.

Post-translational modifications (PTMs) of protein tyrosine (Tyr) residues can serve as a molecular fingerprint of exposure to distinct oxidative pathways and are observed in abnormally high abundance in the majority of human inflammatory pathologies. Reactive oxidants generated during inflammation include hypohalous acids and nitric oxide-derived oxidants, which oxidatively modify protein Tyr residues via halogenation and nitration, respectively, forming 3-chloroTyr, 3-bromoTyr, and 3-nitroTyr. Traditional methods for generating oxidized or halogenated proteins involve nonspecific chemical reactions that result in complex protein mixtures, making it difficult to ascribe observed functional changes to a site-specific PTM or to generate antibodies sensitive to site-specific oxidative PTMs.

Tyrosine nitration on calmodulin enhances calcium-dependent association and activation of nitric-oxide synthase.

Production of reactive oxygen species caused by dysregulated endothelial nitric-oxide synthase (eNOS) activity is linked to vascular dysfunction. eNOS is a major target protein of the primary calcium-sensing protein calmodulin. Calmodulin is often modified by the main biomarker of nitroxidative stress, 3-nitrotyrosine (nitroTyr).

Genetically Encoded Protein Tyrosine Nitration in Mammalian Cells.

Tyrosine nitration has served as a major biomarker for oxidative stress and is present in high abundance in over 50 disease pathologies in humans. While data mounts on specific disease pathways from specific sites of tyrosine nitration, the role of these modifications is still largely unclear. Strategies for installing site-specific tyrosine nitration in target proteins in eukaryotic cells, through routes not dependent on oxidative stress, would provide a powerful method to address the consequences of tyrosine nitration.

Discriminative stimulus properties of the typical antipsychotic haloperidol compared to other antipsychotic drugs in C57BL/6 mice.

Haloperidol (HAL) was developed in 1958 for the treatment of schizophrenia and is classified as a typical antipsychotic drug (APD). Effective in treating positive symptoms of schizophrenia, it does not treat negative symptoms and produces extrapyramidal motor side-effects. Atypical APDs like clozapine treat both positive and negative symptoms of schizophrenia, have reduced extrapyramidal motor side-effects and possess other clinical advantages.

Vortioxetine Differentially Modulates MK-801-Induced Changes in Visual Signal Detection Task Performance and Locomotor Activity.

Attention impairment is a common feature of Major Depressive Disorder (MDD), and MDD-associated cognitive dysfunction may play an important role in determining functional status among this patient population. Vortioxetine is a multimodal antidepressant that may improve some aspects of cognitive function in MDD patients, and may indirectly increase glutamate neurotransmission in brain regions classically associated with attention function. Previous non-clinical research suggests that vortioxetine has limited effects on attention.

Genetic Code Expansion: A Powerful Tool for Understanding the Physiological Consequences of Oxidative Stress Protein Modifications.

Posttranslational modifications resulting from oxidation of proteins (Ox-PTMs) are present intracellularly under conditions of oxidative stress as well as basal conditions. In the past, these modifications were thought to be generic protein damage, but it has become increasingly clear that Ox-PTMs can have specific physiological effects. It is an arduous task to distinguish between the two cases, as multiple Ox-PTMs occur simultaneously on the same protein, convoluting analysis.

Drug Discrimination: Historical Origins, Important Concepts, and Principles.

Research on the stimulus properties of drugs began with studies on state dependent learning during the first half of the twentieth century. From that research, an entirely new approach evolved called drug discrimination. Animals (including humans) could discriminate the presence or absence of a drug; once learned, the drug could serve as a discriminative stimulus, signaling the availability or nonavailability of reinforcement.

The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice.

Clozapine-N-oxide (CNO) has long been the ligand of choice for selectively activating Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). However, recent studies have challenged the long-held assertion that CNO is otherwise pharmacologically inert. The present study aimed to 1) determine whether CNO is reverse-metabolized to its parent compound clozapine in mice (as has recently been reported in rats), and 2) determine whether CNO exerts clozapine-like interoceptive stimulus effects in rats and/or mice.

Structural insights into a thermostable variant of human carbonic anhydrase II.

Carbonic anhydrase is an enzyme of interest for many biotechnological developments including carbon sequestration. These applications often require harsh conditions, so there is a need for the development of thermostable variants. One of the most thermostable human carbonic anhydrase II (HCAIIts) variants was patented in 2006.

Discriminative stimulus properties of the atypical antipsychotic amisulpride: comparison to its isomers and to other benzamide derivatives, antipsychotic, antidepressant, and antianxiety drugs in C57BL/6 mice.

Rationale: Racemic (RS)-amisulpride (Solian) is an atypical antipsychotic drug used to treat schizophrenia and dysthymia. Blockade of dopamine D/D and/or serotonin 5-HT receptors is implicated in its pharmacological effects. While the (S)-amisulpride isomer possesses a robust discriminative cue, discriminative stimulus properties of (RS)-amisulpride have not been evaluated.