T394A Mutation at the µ Opioid Receptor Blocks Opioid Tolerance and Increases Vulnerability to Heroin Self-Administration in Mice.

The etiology and pathophysiology underlying opioid tolerance and dependence are still unknown. Because mu opioid receptor (MOR) plays an essential role in opioid action, many vulnerability-related studies have focused on single nucleotide polymorphisms of MOR, particularly on A118G. In this study, we found that a single-point mutation at the MOR T394 phosphorylation site could be another important susceptive factor in the development of opioid tolerance and dependence in mice. T394A mutation, in which a threonine at 394 was replaced by an alanine, did not alter agonist binding to MOR and opioid analgesia, but resulted in loss of etorphine-induced MOR internalization in spinal dorsal horn neurons and opioid analgesic tolerance induced by either morphine or etorphine. In addition, this mutation also caused an increase in intravenous heroin self-administration and in nucleus accumbens dopamine response to heroin. These findings suggest that T394 phosphorylation following MOR activation causes MOR internalization and desensitization, which subsequently contributes to the development of tolerance in both opioid analgesia and opioid reward. Accordingly, T394A mutation blocks opioid tolerance and leads to an increase in brain dopamine response to opioids and in opioid-taking behavior. Thus, the T394 may serve as a new drug target for modulating opioid tolerance and the development of opioid abuse and addiction. SIGNIFICANCE STATEMENT: The mechanisms underlying opioid tolerance and susceptibility to opioid addiction remain unclear. The present studies demonstrate that a single-point mutation at the T394 phosphorylation site in the C-terminal of mu opioid receptor (MOR) results in loss of opioid tolerance and enhanced vulnerability to heroin self-administration. These findings suggest that modulation of the MOR-T394 phosphorylation or dephosphorylation status may have therapeutic potential in management of pain, opioid tolerance, and opioid abuse and addiction. Accordingly, MOR-T394 mutation or polymorphisms could be a risk factor in developing opioid abuse and addiction and therefore be used as a new biomarker in prediction and prevention of opioid abuse and addiction.

Agonist-Dependent and -Independent κ Opioid Receptor Phosphorylation: Distinct Phosphorylation Patterns and Different Cellular Outcomes.

We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse κ opioid receptor (KOPR) at residues S356, T357, T363, and S369. Here, we found that agonist (U50,488H)-dependent KOPR phosphorylation at all the residues was mediated by Gi/o α proteins and multiple protein kinases [GRK2, GRK3, GRK5, GRK6 and protein kinase C (PKC)]. In addition, PKC activation by phorbol ester induced agonist-independent KOPR phosphorylation. Compared with U50,488H, PKC activation promoted much higher S356/T357 phosphorylation, much lower T363 phosphorylation, and similar levels of S369 phosphorylation. After U50,488H treatment, GRKs, but not PKC, were involved in agonist-induced KOPR internalization. In contrast, PKC activation caused a lower level of agonist-independent KOPR internalization, compared with U50,488H. U50,488H-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was G protein-, but not ß-arrestin-, dependent. After U50,488H treatment, GRK-mediated, but not PKC-mediated, KOPR phosphorylation followed by ß-arrestin recruitment desensitized U50,488H-induced ERK1/2 response. Therefore, agonist-dependent (GRK- and PKC-mediated) and agonist-independent (PKC-promoted) KOPR phosphorylations show distinct phosphorylation patterns, leading to diverse cellular outcomes.

Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated following U50,488H and etorphine respectively. Thus KOPR internalization requires receptor phosphorylation above a certain threshold, and higher-order KOPR phosphorylation may be disproportionally important.

An alternative method to quantify 2-MIB producing cyanobacteria in drinking water reservoirs: Method development and field applications.

2-Methylisoborneol (2-MIB) is a commonly detected cyanobacterial odorant in drinking water sources in many countries. To provide safe and high-quality water, development of a monitoring method for the detection of 2-MIB-synthesis (mibC) genes is very important. In this study, new primers MIBS02F/R intended specifically for the mibC gene were developed and tested. Experimental results show that the MIBS02F/R primer set was able to capture 13 2-MIB producing cyanobacterial strains grown in the laboratory, and to effectively amplify the targeted DNA region from 17 2-MIB-producing cyanobacterial strains listed in the literature. The primers were further coupled with a TaqMan probe to detect 2-MIB producers in 29 drinking water reservoirs (DWRs). The results showed statistically significant correlations between mibC genes and 2-MIB concentrations for the data from each reservoir (R2=0.413-0.998; p<0.05), from all reservoirs in each of the three islands (R2=0.302-0.796; p<0.01), and from all data of the three islands (R2=0.473-0.479; p<0.01). The results demonstrate that the real-time PCR can be an alternative method to provide information to managers of reservoirs and water utilities facing 2-MIB-related incidents.

Proteomics analysis of EV71-infected cells reveals the involvement of host protein NEDD4L in EV71 replication.

Enterovirus 71 (EV71) is a human enterovirus that has seriously affected the Asia-Pacific area for the past two decades. EV71 infection can result in mild hand-foot-and-mouth disease and herpangina and may occasionally lead to severe neurological complications in children. However, the specific biological processes that become altered during EV71 infection remain unclear. To further explore host responses upon EV71 infection, we identified proteins differentially expressed in EV71-infected human glioblastoma SF268 cells using isobaric mass tag (iTRAQ) labeling coupled with multidimensional liquid chromatography-mass spectrometry (LC-MS/MS). Network analysis of proteins altered in cells infected with EV71 revealed that the changed biological processes are related to protein and ion transport, regulation of protein degradation, and homeostatic processes. We confirmed that the levels of NEDD4L and PSMF1 were increased and reduced, respectively, in EV71-infected cells compared to mock-infected control cells. To determine the physiological relevance of our findings, we investigated the consequences of EV71 infection in cells with NEDD4L or PSMF1 depletion. We found that the depletion of NEDD4L significantly reduced the replication of EV71, whereas PSMF1 knockdown enhanced EV71 replication. Collectively, our findings provide the first evidence of proteome-wide dysregulation by EV71 infection and suggest a novel role for the host protein NEDD4L in the replication of this virus.

Illuminating the understudied GPCR-ome.

G-protein-coupled receptors (GPCRs) are the target of >30% of approved drugs. Despite their popularity, many of the >800 human GPCRs remain understudied. The Illuminating the Druggable Genome (IDG) project has generated many tools leading to important insights into the function and druggability of these so-called 'dark' receptors. These tools include assays, such as PRESTO-TANGO and TRUPATH, billions of small molecules made available via the ZINC virtual library, solved orphan GPCR structures, GPCR knock-in mice, and more. Together, these tools are illuminating the remaining 'dark' GPCRs.

Neuropsychological features of somatic symptom disorder and depression/anxiety in Taiwan: An analysis based on the comorbidity status.

Individuals with somatic symptom disorder (SSD) often have comorbid depression or anxiety, but whether SSD is associated with specific neuropsychological functions has yet to be fully examined. We analyzed which neuropsychological features are more closely associated with SSD, anxiety, and depression. In this case-control study, we recruited 140 individuals with SSD, 104 individuals with affective disorders without SSD, and 159 healthy controls in Taiwan. We collected DSM-5 diagnoses, questionnaire scores, and performance on eight tasks from the Cambridge Neuropsychological Test Automated Battery (CANTAB) for each participant. Several CANTAB tasks involving attention, executive function, and social cognition showed significant group differences. In the adjusted analysis, the tasks significantly associated with SSD were the Match to Sample Visual Search (MTS) and the Emotion Recognition Task (ERT). Among the questionnaires, the Cognitions about Body and Health Questionnaire showed the most significant associations with the tasks, specifically with Rapid Visual Information Processing, MTS, Paired Associates Learning, Spatial Working Memory, Intra-Extra Dimensional Set Shift, and ERT. We conclude that the MTS and ERT tasks show significant relationships with both SSD diagnosis and related questionnaires. These tasks primarily involve selective attention and negative emotion regulation.

Impact of viral filters on accuracy of cardiopulmonary testing and spirometry.

The present study found that using viral filters at the proximal end of a spirometry and CPET test circuit did not significantly alter the test results, with the exception of a marginal decrease noted in peak work rate https://bit.ly/3Vkew95.

Association of categorical diagnoses and psychopathologies with quality of life in patients with depression, anxiety, and somatic symptoms: A cross-sectional study.

OBJECTIVE: Major depressive disorder (MDD), anxiety disorders, and somatic symptom disorder (SSD) are associated with quality of life (QoL) reduction. This cross-sectional study investigated the relationship between these conditions as categorical diagnoses and related psychopathologies with QoL, recognizing their frequent overlap. METHODS: We recruited a total of 403 clinical patients and healthy individuals, administering diagnostic interviews based on the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition. QoL and psychopathologies were assessed using the WHO Quality of Life-BREF (WHOQOL-BREF) and several self-administered questionnaires, respectively. Multiple linear regression analyses examined the associations between psychiatric diagnoses, psychopathologies, and QoL. RESULTS: SSD and MDD were independently associated with impaired global (ß = -0.318 and - 0.287) and all QoL domains (ß = -0.307, -0.150, -0.125, and - 0.133, in physical, psychological, social, and environmental domains respectively for SSD; ß = -0.278, -0.344, -0.275, and - 0.268 for MDD). The Beck Depression Inventory-II score showed pervasive associations with QoL (ß = -0.390, -0.408, -0.685, -0.463, and - 0.420, in global, physical, psychological, social, and environmental domains). The Patient Health Questionnaire-15 and Health Anxiety Questionnaire scores were associated with global (ß = -0.168 and - 0.181) and physical (ß = -0.293 and - 0.121) QoL domain, while the Cognitions About Body and Health Questionnaire score was only associated with environmental QoL domain (ß = -0.157). CONCLUSION: SSD and MDD were independently associated with QoL impairment. Depressive symptoms were associated with all QoL domains, whereas somatic symptom burden and health anxiety primarily affected the physical QoL domain. Clinicians should consider concomitant psychopathologies when managing patients with depression, anxiety, or somatic symptoms.

Performances of full-scale biological nitrogen pre-treatment process for drinking water source: Seasonal variations and microbial community.

This study investigated the performance of the full-scale unit over a two-year period to enhance nitrification efficiency and provide operational strategies. Results indicated that raw water quality from Donggan River was notably influenced by seasonal variations, particularly during dry and wet seasons, impacting the nitrification efficiency of the biological pretreatment process. Factors such as influent concentrations of ammonia and total Kjeldahl nitrogen were found to have significant effects on nitrification, with temperature and conductivity also showing correlations. The specific rate of ammonia removal was calculated to be approximately 0.1 kg-N/m3/d under the existing operational setup. Moreover, elevating dissolved oxygen levels above 4 mg/L was proposed to potentially boost ammonia oxidation based on findings from experiments conducted in lab-scale bioreactors. In times of increased influent ammonia levels, the elimination of about 1-3 mg-N/L of total nitrogen signified the activation of denitrification processes. This observation was corroborated by results from next-generation sequencing techniques, verifying the existence of denitrifying microorganisms. The real-time PCR analysis results indicated that the abundance of comammox amoA gene was comparable with the abundance of the AOB amoA gene, indicating the presence of comammox Nitrospira and their potential role on nitrification in the system.

Clarifying the link between psychopathologies and heart rate variability, and the sex differences: Can neuropsychological features serve as mediators?

BACKGROUND: Patients with common mental disorders (depression, anxiety, and somatic symptoms) often exhibit lower heart rate variability (HRV) than healthy individuals. Under the hypothesis that disorder status affects cognitive function, we examined whether neuropsychological features can be mediators between psychopathologies and HRV and possible sex differences. METHODS: We recruited 359 individuals (aged 42.47 ± 12.23) with and without common mental disorders. Questionnaires were used to measure their psychopathologies. Eight tests from the Cambridge Neuropsychological Test Automated Battery were selected to measure neuropsychological functions. Resting-state HRV measurements were obtained for 5 min. The associations among these constructs were analyzed using multiple linear regression analysis and structural equation modelling. RESULTS: Among women, Reaction Time (RTI, a task of psychomotor speed) indicator mediated the relationship between depression and low-frequency power (LF); Emotion Recognition Task (ERT, a task of emotional regulation) indicator mediated the relationship between health anxiety and high-frequency power (HF). Among men, Intra-Extra Dimensional Set Shift (IED, a task of shifting) indicator mediated the relationship between depression and LF; Match to Sample Visual Search (MTS, a task of selective attention) indicator mediated the relationship between health cognition and HF. The depression-RTI-LF pathway in women tended to lower HRV; whereas health anxiety-ERT-HF in women, depression-IED-LF and health cognition-MTS-HF in men tended to increase HRV. LIMITATIONS: Possible medication effects; not directly measuring brain activity; only gathering resting-state HRV. CONCLUSION: Our findings support the notion that neuropsychological features mediate the relationship between psychopathologies and HRV, and that sex differences exist.

A suite of engineered mice for interrogating psychedelic drug actions.

Psychedelic drugs like lysergic acid diethylamide (LSD) and psilocybin have emerged as potentially transformative therapeutics for many neuropsychiatric diseases, including depression, anxiety, post-traumatic stress disorder, migraine, and cluster headaches. LSD and psilocybin exert their psychedelic effects via activation of the 5-hydroxytryptamine 2A receptor (HTR2A). Here we provide a suite of engineered mice useful for clarifying the role of HTR2A and HTR2A-expressing neurons in psychedelic drug actions. We first generated Htr2a-EGFP-CT-IRES-CreERT2 mice (CT:C-terminus) to independently identify both HTR2A-EGFP-CT receptors and HTR2A-containing cells thereby providing a detailed anatomical map of HTR2A and identifying cell types that express HTR2A. We also generated a humanized Htr2a mouse line and an additional constitutive Htr2A-Cre mouse line. Psychedelics induced a variety of known behavioral changes in our mice validating their utility for behavioral studies. Finally, electrophysiology studies revealed that extracellular 5-HT elicited a HTR2A-mediated robust increase in firing of genetically-identified pyramidal neurons--consistent with a plasma membrane localization and mode of action. These mouse lines represent invaluable tools for elucidating the molecular, cellular, pharmacological, physiological, behavioral, and other actions of psychedelic drugs in vivo.

Quantitative PCR based detection system for cyanobacterial geosmin/2-methylisoborneol (2-MIB) events in drinking water sources: Current status and challenges.

Taste and odor (T&O) are an important issue in drinking water, aquaculture, recreation and a few other associated industries, and cyanobacteria-relevant geosmin and 2-methylisoborneol (2-MIB) are the two most commonly detected T&O compounds worldwide. A rise in the cyanobacterial blooms and associated geosmin/2-MIB episodes due to anthropogenic activities as well as climate change has led to global concerns for drinking water quality. The increasing awareness for the safe drinking, aquaculture or recreational water systems has boost the demand for rapid, robust, on-site early detection and monitoring system for cyanobacterial geosmin/2-MIB events. In past years, research has indicated quantitative PCR (qPCR) as one of the promising tools for detection of geosmin/2-MIB episodes. It offers advantages of detecting the source organism even at very low concentrations, distinction of odor-producing cyanobacterial strains from non-producers and evaluation of odor producing potential of the cyanobacteria at much faster rates compared to conventional techniques.The present review aims at examining the current status of developed qPCR primers and probes in identifying and detecting the cyanobacterial blooms along with geosmin/2-MIB events. Among the more than 100 articles about cyanobacteria associated geosmin/2-MIB in drinking water systems published after 1990, limited reports (approx. 10 each for geosmin and 2-MIB) focused on qPCR detection and its application in the field. Based on the review of literature, a comprehensive open access global cyanobacterial geosmin/2-MIB events database (CyanoGM Explorer) is curated. It acts as a single platform to access updated information related to origin and geographical distribution of geosmin/2-MIB events, cyanobacterial producers, frequency, and techniques associated with the monitoring of the events. Although a total of 132 cyanobacterial strains from 21 genera and 72 cyanobacterial strains from 13 genera have been reported for geosmin and 2-MIB production, respectively, only 58 geosmin and 28 2-MIB synthesis regions have been assembled in the NCBI database. Based on the identity, geosmin sequences were found to be more diverse in the geosmin synthase conserved/primer design region, compared to 2-MIB synthesis region, hindering the design of universal primers/probes. Emerging technologies such as the bioelectronic nose, Surface Enhanced Raman Scattering (SERS), and nanopore sequencing are discussed for future applications in early on-site detection of geosmin/2-MIB and producers. In the end, the paper also highlights various challenges in applying qPCR as a universal system of monitoring and development of response system for geosmin/2-MIB episodes.

LSD-stimulated behaviors in mice require ß-arrestin 2 but not ß-arrestin 1.

Recent evidence suggests that psychedelic drugs can exert beneficial effects on anxiety, depression, and ethanol and nicotine abuse in humans. However, their hallucinogenic side-effects often preclude their clinical use. Lysergic acid diethylamide (LSD) is a prototypical hallucinogen and its psychedelic actions are exerted through the 5-HT2A serotonin receptor (5-HT2AR). 5-HT2AR activation stimulates Gq- and ß-arrestin- (ßArr) mediated signaling. To separate these signaling modalities, we have used ßArr1 and ßArr2 mice. We find that LSD stimulates motor activities to similar extents in WT and ßArr1-KO mice, without effects in ßArr2-KOs. LSD robustly stimulates many surrogates of psychedelic drug actions including head twitches, grooming, retrograde walking, and nose-poking in WT and ßArr1-KO animals. By contrast, in ßArr2-KO mice head twitch responses are low with LSD and this psychedelic is without effects on other surrogates. The 5-HT2AR antagonist MDL100907 (MDL) blocks the LSD effects. LSD also disrupts prepulse inhibition (PPI) in WT and ßArr1-KOs, but not in ßArr2-KOs. MDL restores LSD-mediated disruption of PPI in WT mice; haloperidol is required for normalization of PPI in ßArr1-KOs. Collectively, these results reveal that LSD's psychedelic drug-like actions appear to require ßArr2.

Pharmacological and phosphoproteomic approaches to roles of protein kinase C in kappa opioid receptor-mediated effects in mice.

Kappa opioid receptor (KOR) agonists possess adverse dysphoric and psychotomimetic effects, thus limiting their applications as non-addictive anti-pruritic and analgesic agents. Here, we showed that protein kinase C (PKC) inhibition preserved the beneficial antinociceptive and antipruritic effects of KOR agonists, but attenuated the adverse condition placed aversion (CPA), sedation, and motor incoordination in mice. Using a large-scale mass spectrometry-based phosphoproteomics of KOR-mediated signaling in the mouse brain, we observed PKC-dependent modulation of G protein-coupled receptor kinases and Wnt pathways at 5 min; stress signaling, cytoskeleton, mTOR signaling and receptor phosphorylation, including cannabinoid receptor CB1 at 30 min. We further demonstrated that inhibition of CB1 attenuated KOR-mediated CPA. Our results demonstrated the feasibility of in vivo biochemical dissection of signaling pathways that lead to side effects.

Comparison of Pharmacological Properties between the Kappa Opioid Receptor Agonist Nalfurafine and 42B, Its 3-Dehydroxy Analogue: Disconnect between in Vitro Agonist Bias and in Vivo Pharmacological Effects.

Nalfurafine, a moderately selective kappa opioid receptor (KOR) agonist, is used in Japan for treatment of itch without causing dysphoria or psychotomimesis. Here we characterized the pharmacology of compound 42B, a 3-dehydroxy analogue of nalfurafine and compared with that of nalfurafine. Nalfurafine and 42B acted as full KOR agonists and partial µ opioid receptor (MOR) agonists, but 42B showed much lower potency for both receptors and lower KOR/MOR selectivity, different from previous reports. Molecular modeling revealed that water-mediated hydrogen-bond formation between 3-OH of nalfurafine and KOR accounted for its higher KOR potency than 42B. The higher potency of both at KOR over MOR may be due to hydrogen-bond formation between nonconserved Y7.35 of KOR and their carbonyl groups. Both showed modest G protein signaling biases. In mice, like nalfurafine, 42B produced antinociceptive and antiscratch effects and did not cause conditioned place aversion (CPA) in the effective dose ranges. Unlike nalfurafine, 42B caused motor incoordination and hypolocomotion. As both agonists showed G protein biases, yet produced different effects on locomotor activity and motor incoordination, the findings and those in the literature suggest caution in correlating in vitro biochemical data with in vivo behavior effects. The factors contributing to the disconnect, including pharmacodynamic and pharmacokinetic issues, are discussed. In addition, our results suggest that among the KOR-induced adverse behaviors, CPA can be separated from motor incoordination and hypolocomotion.

A molecular-based method to estimate the risk associated with cyanotoxins and odor compounds in drinking water sources.

A biomolecular-based monitoring approach for the assessment of water quality hazards and risks associated with cyanobacteria was developed and validated in drinking reservoirs in Taiwan and the Philippines. The approach was based upon the measurement of gene abundances of toxigenic Microcystis and Cylindrospermopsis; for cyanotoxins; and for aesthetically offensive earthy-musty odor compounds. This was compared to conventional monitoring approaches, which included cell enumeration by microscopy, and toxin and odor compound analysis by instrumental analytical methods and immunoassays as appropriate for the metabolites. The validation involved samples from ten major reservoirs on Taiwan's main island, nineteen reservoirs on the offshore islands, and Laguna de Bay in the Philippines. The gene-based approach was successfully validated statistically and compared to conventional widely utilized risk assessment schemes which have employed 'Alert Levels' for toxic cyanobacteria. In this case a new integrated scheme of 'Response Levels' is proposed which incorporates odor metabolite hazards in addition to cyanotoxins and is based upon gene copy numbers to derive quantitative triggers. The comprehensive scheme evaluated from these locations is considered to be more precise and efficient for both monitoring and as a risk assessment diagnostic tool, given that it offers the capacity for analysis of the abundance of genes for cyanobacterial metabolites in large numbers of natural water samples in a significantly reduced period of time compared to the approaches of cell enumeration by microscopy or metabolite analytical techniques. This approach is the first time both the hazard and risk for both odors and cyanotoxins from cyanobacteria have been considered together in a monitoring scheme and offers an improved means for determining the Response Levels in the risk assessment process for cyanobacteria and their metabolites in drinking water sources.

Phosphoproteomic approach for agonist-specific signaling in mouse brains: mTOR pathway is involved in κ opioid aversion.

Kappa opioid receptor (KOR) agonists produce analgesic and anti-pruritic effects, but their clinical application was limited by dysphoria and hallucinations. Nalfurafine, a clinically used KOR agonist, does not cause dysphoria or hallucinations at therapeutic doses in humans. We found that in CD-1 mice nalfurafine produced analgesic and anti-scratch effects dose-dependently, like the prototypic KOR agonist U50,488H. In contrast, unlike U50,488H, nalfurafine caused no aversion, anhedonia, or sedation or and a low level of motor incoordination at the effective analgesia and anti-scratch doses. Thus, we established a mouse model that recapitulated important aspects of the clinical observations. We then employed a phosphoproteomics approach to investigate mechanisms underlying differential KOR-mediated effects. A large-scale mass spectrometry (MS)-based analysis on brains revealed that nalfurafine perturbed phosphoproteomes differently from U50,488H in a brain-region specific manner after 30-min treatment. In particular, U50,488H and nalfurafine imparted phosphorylation changes to proteins found in different cellular components or signaling pathways in different brain regions. Notably, we observed that U50,488H, but not nalfurafine, activated the mammalian target of rapamycin (mTOR) pathway in the striatum and cortex. Inhibition of the mTOR pathway by rapamycin abolished U50,488H-induced aversion, without affecting analgesic, anti-scratch, and sedative effects and motor incoordination. The results indicate that the mTOR pathway is involved in KOR agonist-induced aversion. This is the first demonstration that phosphoproteomics can be applied to agonist-specific signaling of G protein-coupled receptors (GPCRs) in mouse brains to unravel pharmacologically important pathways. Furthermore, this is one of the first two reports that the mTOR pathway mediates aversion caused by KOR activation.

Regulation of type V adenylate cyclase by Ric8a, a guanine nucleotide exchange factor.

In the present study, we demonstrate that AC5 (type V adenylate cyclase) interacts with Ric8a through directly interacting at its N-terminus. Ric8a was shown to be a GEF (guanine nucleotide exchange factor) for several alpha subunits of heterotrimeric GTP binding proteins (Galpha proteins) in vitro. Selective Galpha targets of Ric8a have not yet been revealed in vivo. An interaction between AC5 and Ric8a was verified by pull-down assays, co-immunoprecipitation analyses, and co-localization in the brain. Expression of Ric8a selectively suppressed AC5 activity. Treating cells with pertussis toxin or expressing a dominant negative Galphai mutant abolished the suppressive effect of Ric8a, suggesting that interaction between the N-terminus of AC5 and a GEF (Ric8a) provides a novel pathway to fine-tune AC5 activity via a Galphai-mediated pathway.

A Simple Alternative Method for Preservation of 2-Methylisoborneol in Water Samples.

2-Methylisoborneol (2-MIB) is one of the most commonly observed taste and odor (T&O) compounds present in drinking water sources. As it is biodegradable, a preservation agent, typically mercury chloride, is needed if the water is not analyzed right after sampling. Since mercury is a toxic metal, an alternative chemical that is cheaper and less toxic is desirable. In this study, two chemicals commonly used in water treatment processes, chlorine (as sodium hypochlorite) and KMnO4 (potassium permanganate), are studied to determine their feasibility as preservation agents for 2-MIB in water. Preservation experiments were first conducted in deionized water spiked with 2-MIB and with chlorine or permanganate at 4 and 25 °C. The results indicate that 2-MIB concentrations in the water samples spiked with both chemicals remained almost constant within 14 days for all the tested conditions, suggesting that oxidation and volatilization did not cause the loss of 2-MIB in the system. The experiments were further conducted for three different reservoir water samples with 30⁻60 ng/L of indulgent 2-MIB. The experimental results demonstrated that preservation with permanganate may have underestimated the 2-MIB concentration in the samples as a result of the formation of manganese dioxide particles in natural water and adsorption of 2-MIB onto the particles. Chlorine was demonstrated to be a good preservation agent for all three tested natural waters since oxidation of 2-MIB was negligible and biodegradation was inhibited. When the residual chlorine concentrations were controlled to be higher than 0.5 mg/L on the final day (day 14) of the experiments, the concentration reduction of 2-MIB became lower than 13% at both of the tested temperatures. The results demonstrated that sodium hypochlorite can be used as an alternative preservation agent for 2-MIB in water before analysis.