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Abstract We compared liquid chromatography tandem mass spectrometry (LC–MS/MS) against Binding Site immunonephelometry (BSIN) with regards to these methods’ abilities to diagnose IgG4-related disease (IgG4-RD). IgG subclasses were gathered from laboratory from December 2011 to December 2020. The IgG4-RD positive and negative patients were diagnosed according to the ACR/EULAR classification criteria by extensive chart review. Both methods’ results were compared in terms of test characteristics. For BSIN, there were 43 IgG4-RD positive cases and 174 disease negative cases, while for LC–MS/MS, there were 102 IgG4-RD positive cases and 562 disease negative cases. The majority of IgG4-RD patients by BSIN and LC–MS/MS had an elevated IgG4 level, 81% and 86%, respectively. For BSIN, the ROC curve, cut-off value of 1.25 g/L, had a sensitivity of 81% and a specificity of 84%. For LC–MS/MS, the ROC curve, cut-off value of 1.25 g/L, had a sensitivity of 86% and a specificity of 84%. The responder index score to IgG4 level r-correlation value for BSIN and LC–MS/MS was 0.5 and 0.6, respectively. In our center, LC–MS/MS and BSIN are equivalent test methods in IgG4-RD diagnosis. IgG4 level does correlate with disease activity by the responder index. LC–MS/MS is a valid and equally reliable alternative to BSIN in the diagnosis of IgG4-related disease.

Abstract Background Advanced spark ignition engines require high performance fuels with improved resistance to autoignition. Biologically derived olefinic alcohols have arisen as promising blendstock candidates due to favorable octane numbers and synergistic blending characteristics. However, production and downstream separation of these alcohols are limited by their intrinsic toxicity and high aqueous solubility, respectively. Bioproduction of carboxylate esters of alcohols can improve partitioning and reduce toxicity, but in practice has been limited to saturated esters with characteristically low octane sensitivity. If olefinic esters retain the synergistic blending characteristics of their alcohol counterparts, they could improve the bioblendstock combustion performance while also retaining the production advantages of the ester moiety. Results Optimization of Escherichia coli isoprenoid pathways has led to high titers of isoprenol and prenol, which are not only excellent standalone biofuel and blend candidates, but also novel targets for esterification. Here, a selection of olefinic esters enhanced blendstock performance according to their degree of unsaturation and branching. E. coli strains harboring optimized mevalonate pathways, thioester pathways, and heterologous alcohol acyltransferases (ATF1, ATF2, and SAAT) were engineered for the bioproduction of four novel olefinic esters. Although prenyl and isoprenyl lactate titers were limited to 1.48 ± 0.41 mg/L and 5.57 ± 1.36 mg/L, strains engineered for prenyl and isoprenyl acetate attained titers of 176.3 ± 16.0 mg/L and 3.08 ± 0.27 g/L, respectively. Furthermore, prenyl acetate (20% bRON = 125.8) and isoprenyl acetate (20% bRON = 108.4) exhibited blend properties comparable to ethanol and significantly better than any saturated ester. By further scaling cultures to a 2-L bioreactor under fed-batch conditions, 15.0 ± 0.9 g/L isoprenyl acetate was achieved on minimal medium. Metabolic engineering of acetate pathway flux further improved titer to attain an unprecedented 28.0 ± 1.0 g/L isoprenyl acetate, accounting for 75.7% theoretical yield from glucose. Conclusion Our study demonstrated novel bioproduction of four isoprenoid oxygenates for fuel blending. Our optimized E. coli production strain generated an unprecedented titer of isoprenyl acetate and when paired with its favorable blend properties, may enable rapid scale-up of olefinic alcohol esters for use as a fuel blend additive or as a precursor for longer-chain biofuels and biochemicals.

Advances in synthetic biology have radically changed our ability to rewire microorganisms and significantly improved the scalable production of a vast array of drop-in biopolymers and biofuels. The success of a drop-in bioproduct is contingent on market competition with petrochemical analogues and weighted upon relative economic and environmental metrics. While the quantification of comparative trade-offs is critical for accurate process-level decision making, the translation of industrial ecology to synthetic biology is often ambiguous and assessment accuracy has proven challenging. In this review, we explore strategies for evaluating industrial biotechnology through life cycle and techno-economic assessment, then contextualize how recent developments in synthetic biology have improved process viability by expanding feedstock availability and the productivity of microbes. By juxtaposing biological and industrial constraints, we highlight major obstacles between the disparate disciplines that hinder accurate process evaluation. The convergence of these disciplines is crucial in shifting towards carbon neutrality and a circular bioeconomy.

Isoprenoid compounds are biologically ubiquitous, and their characteristic modularity has afforded products ranging from pharmaceuticals to biofuels. Isoprenoid production has been largely successful in Escherichia coli and Saccharomyces cerevisiae with metabolic engineering of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways coupled with the expression of heterologous terpene synthases. Yet conventional microbial chassis pose several major obstacles to successful commercialization including the affordability of sugar substrates at scale, precursor flux limitations, and intermediate feedback-inhibition. Now, recent studies have challenged typical isoprenoid paradigms by expanding the boundaries of terpene biosynthesis and using non-model organisms including those capable of metabolizing atypical C1 substrates. Conversely, investigations of non-model organisms have historically informed optimization in conventional microbes by tuning heterologous gene expression. Here, we review advances in isoprenoid biosynthesis with specific focus on the synergy between model and non-model organisms that may elevate the commercial viability of isoprenoid platforms by addressing the dichotomy between high titer production and inexpensive substrates.

IgG4-related disease is a fibro-inflammatory condition that can affect nearly any organ system. Common presentations include major salivary and lacrimal gland enlargement, orbital disease, autoimmune pancreatitis, retroperitoneal fibrosis and tubulointerstitial nephritis. This review focuses on the hematologic manifestations of IgG4-related disease, including lymphadenopathy, eosinophilia, and polyclonal hypergammaglobulinemia. The disease can easily be missed by unsuspecting hematologists, as patients may present with clinical problems that mimic disorders such as multicentric Castleman disease, lymphoma, plasma cell neoplasms and hypereosinophilic syndromes. When IgG4-related disease is suspected, serum protein electrophoresis and IgG subclasses are helpful as initial tests but a firm histological diagnosis is essential both to confirm the diagnosis and to rule out mimickers. The central histopathological features are a dense, polyclonal, lymphoplasmacytic infiltrate enriched with IgG4-positive plasma cells (with an IgG4/IgG ratio >40%), storiform fibrosis, and obliterative phlebitis. Importantly for hematologists, the latter two features are seen in all tissues except bone marrow and lymph nodes, making these two sites suboptimal for histological confirmation. Many patients follow an indolent course and respond well to treatment, but a significant proportion may have highly morbid or fatal complications such as periaortitis, severe retroperitoneal fibrosis or pachymeningitis. Corticosteroids are effective but cause new or worsening diabetes in about 40% of patients. Initial response rates to rituximab are high but durable remissions are rare. More intensive lymphoma chemotherapy regimens may be required in rare cases of severe, refractory disease, and targeted therapy against plasmablasts, IgE and other disease biomarkers warrant further exploration.

IgG4-related disease (IgG4-RD) is a multiorgan inflammatory disease in which diverse organ manifestations are linked by common histopathological and immunohistochemical features. Prospective studies of IgG4-RD patients are required to clarify the natural history, long-term prognosis, and treatment approaches in this recently recognized condition. Patients with IgG4-RD have different organ manifestations and are followed by multiple specialties. Divergent approaches to the assessment of patients can complicate the interpretation of studies, emphasizing the critical need for validated outcome measures, particularly assessments of disease activity and response to treatment. We developed a prototype IgG4-RD Responder Index (IgG4-RD RI) based on the approach used in the development of the Birmingham Vasculitis Activity Score for Wegener’s granulomatosis (BVAS/WG). The IgG4-RD RI was refined by members of the International IgG4-RD Symposium Organizing Committee in a paper case exercise. The revised instrument was applied retrospectively to fifteen IgG4-RD patients at our institution. Those scores were compared to physician’s global assessment scale for the same visits. This paper describes the philosophy and goals of the IgG4-RD RI, the steps in the development of this instrument to date, and future plans for validation of this instrument as an outcome measure.

In recent years, methamphetamine METH misuse in the US has been rapidly increasing and there is no FDA-approved pharmacotherapy for METH use disorder (MUD). In addition to being dependent on the drug, people with MUD develop a variety of neurological problems related to the toxicity of this drug. A variety of molecular mechanisms underlying METH neurotoxicity has been identified, including dysfunction of the neuroprotective protein parkin. However, it is not known whether parkin loss of function within striatal dopaminergic (DAergic) terminals translates into a decrease in DA storage capacity. This study examined the relationship between parkin, its substrate cell division cycle related-1 (CDCrel-1), and vesicular monoamine transporter-2 (VMAT2) in METH neurotoxicity in male Sprague Dawley rats. To also assess individual differences in response to METH's neurotoxic effects, a large group of rats was treated with binge METH or saline and sacrificed 1h or 24h later. This study is the first to show that binge METH alters the levels and subcellular localization of CDCrel-1 and that CDCrel-1 interacts with VMAT2 and increases its levels at the plasma membrane. Furthermore, we found wide individual differences in the responses of measured indices to METH. Proteomic analysis of VMAT-2-associated proteins revealed upregulation of several proteins involved in the exocytosis/endocytosis cycle. The results suggest that at 1h after METH binge, DAergic neurons are engaged in counteracting METH-induced toxic effects, including oxidative stress- and hyperthermia-induced inhibition of synaptic vesicle cycling, with the responses varying between individual rats. Studying CDCrel-1, VMAT2, and other proteins in large groups of outbred rats can help define individual genetic and molecular differences in responses to METH neurotoxicity which, in turn, will aid treating humans suffering from METH use disorder and its neurological consequences.

The inhibition of emopamil binding protein (EBP), a sterol isomerase within the cholesterol biosynthesis pathway, promotes oligodendrocyte formation, which has been proposed as a potential therapeutic approach for treating multiple sclerosis. Herein, we describe the discovery and optimization of brain-penetrant, orally bioavailable inhibitors of EBP. A structure-based drug design approach from literature compound 1 led to the discovery of a hydantoin-based scaffold, which provided balanced physicochemical properties and potency and an improved in vitro safety profile. The long half-lives of early hydantoin-based EBP inhibitors in rodents prompted an unconventional optimization strategy, focused on increasing metabolic turnover while maintaining potency and a brain-penetrant profile. The resulting EBP inhibitor 11 demonstrated strong in vivo target engagement in the brain, as illustrated by the accumulation of EBP substrate zymostenol after repeated dosing. Furthermore, compound 11 enhanced the formation of oligodendrocytes in human cortical organoids, providing additional support for our therapeutic hypothesis.

Neuroblastoma is a highly metastatic cancer, and thus is one of the leading causes of cancer-related mortalities in pediatric patients. More than 50% of NB cases exhibit 17q21-ter partial chromosomal gain, which is independently associated with poor survival, suggesting the clinical importance of genes at this locus in NB. IGF2BP1 is one such proto-oncogene located at 17q locus, and was found to be upregulated in patients with metastatic NBs. Here, utilizing multiple immunocompetent mouse models, along with our newly developed highly metastatic NB cell line, we demonstrate the role of IGF2BP1 in promoting NB metastasis. Importantly, we show the significance of small extracellular vesicles (EVs) in NB progression, and determine the pro-metastatic function of IGF2BP1 by regulating the NB-EV-protein cargo. Through unbiased proteomic analysis of EVs, we discovered two novel targets (SEMA3A and SHMT2) of IGF2BP1, and reveal the mechanism of IGF2BP1 in NB metastasis. We demonstrate that IGF2BP1 directly binds and governs the expression of SEMA3A/SHMT2 in NB cells, thereby modulating their protein levels in NB-EVs. IGF2BP1-affected levels of SEMA3A and SHMT2 in the EVs, regulate the formation of pro-metastatic microenvironment at potential metastatic organs. Finally, higher levels of SEMA3A/SHMT2 proteins in the EVs derived from NB-PDX models indicate the clinical significance of the two proteins and IGF2BP1-SEMA3A/SHMT2 axis in NB metastasis., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Retinal diseases like diabetic retinopathy and age-related macular degeneration affect millions of individuals worldwide and often lead to vision loss. Vitreous fluid abuts the retina, is accessible for sampling, and contains many proteins related to retinal disease. Therefore, analysis of vitreous is an important tool for studying retinal disease. Because it is rich in proteins and extracellular vesicles, mass spectrometry-based proteomics is an excellent method for vitreous analysis. Here, we discuss important variables to consider when performing vitreous proteomics via mass spectrometry., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Transient receptor potential melastatin type 2 (TRPM2) is a cation channel activated by free intracellular ADP-ribose and reactive oxygen species. TRPM2 signaling has been linked to the pathophysiology of CNS disorders such as neuropathic pain, bipolar disorder and Alzheimer's disease. In this manuscript, we describe the discovery of JNJ-28583113, a potent brain penetrant TRPM2 antagonist. Ca 2+ flux assays in cells overexpressing TRPM2 and electrophysiological recordings were used to test the pharmacology of JNJ-28583113. JNJ-28583113 was assayed in vitro on GSK-3 phosphorylation levels, cell death, cytokine release in microglia and unbiased morphological phenotypic analysis. Finally, we dosed animals to evaluate its pharmacokinetic properties. Our results showed that JNJ-28583113 is a potent (126 ± 0.5 nM) TRPM2 antagonist. Blocking TRPM2 caused phosphorylation of GSK3α and β subunits. JNJ-28583113 also protected cells from oxidative stress induced cell death as well as morphological changes induced by non-cytotoxic concentrations of H 2 O 2 . In addition, inhibiting TRPM2 blunted cytokine release in response to pro-inflammatory stimuli in microglia. Lastly, we showed that JNJ-28583113 was brain penetrant but not suitable for systemic dosing as it was rapidly metabolized in vivo. While the in-vitro pharmacology of JNJ-28583113 is the best in class, its in-vivo properties would need optimization to assist in further probing key roles of TRPM2 in CNS pathophysiology., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: Sleeve gastrectomy is now the commonest bariatric procedure worldwide, making it important to determine 5-year weight loss and micronutrient deficiency outcomes following primary, stand-alone sleeve gastrectomy (SG) with adequate follow-up. The purpose of this study was to evaluate weight loss, co-morbidity resolution, and haematological outcomes at 5 years with primary, stand-alone SG in our unit., Materials and Methods: Baseline data were obtained from our prospectively maintained database and patients were invited for a 5-year follow-up. For those who could not attend, further information was obtained by telephoning them and contacting their general practitioners., Results: A total of 92 patients (66 females) underwent primary SG at our centre in 2011. The median age was 46.0 years. The median weight and the body mass index (BMI) were 133.0 kg and 47.0 kg/m 2 respectively. After 5 years, 7 (7.6%) patients had died due to unrelated causes and 11 (12.0%) had been converted to Roux-en-Y gastric bypass (RYGB). Of the remaining 74, we were able to obtain follow-up data on 64 (86.5%). Despite all efforts, 10 (13.5%) patients were lost to follow-up. The median total weight loss was 21.9% and median excess weight loss (EWL) was 48.0%. Patients developed a range of micronutrient deficiencies over the follow-up period., Conclusion: This study shows acceptable results with SG at 5 years in our unit, but a large number of patients experienced GORD and some patients needed additional micronutrient supplementation over and above our unit recommendation of a single multivitamin/mineral A-Z tablet daily.

Emerging data continues to point towards a relationship between neuroinflammation and neuropsychiatric disorders. ATP-induced activation of P2X7 results in IL-1β release causing neuroinflammation and microglial activation. This study describes the in-vitro and in-vivo neuropharmacology of a novel brain-penetrant P2X7 antagonist, JNJ-55308942, currently in clinical development. JNJ-55308942 is a high-affinity, selective, brain-penetrant (brain/plasma of 1) P2X7 functional antagonist. In human blood and in mouse blood and microglia, JNJ-55308942 attenuated IL-1β release in a potent and concentration-dependent manner. After oral dosing, the compound exhibited both dose and concentration-dependent occupancy of rat brain P2X7 with an ED 50 of 0.07 mg/kg. The P2X7 antagonist (3 mg/kg, oral) blocked Bz-ATP-induced brain IL-1β release in conscious rats, demonstrating functional effects of target engagement in the brain. JNJ-55308942 (30 mg/kg, oral) attenuated LPS-induced microglial activation in mice, assessed at day 2 after a single systemic LPS injection (0.8 mg/kg, i.p.), suggesting a role for P2X7 in microglial activation. In a model of BCG-induced depression, JNJ-55308942 dosed orally (30 mg/kg), reversed the BCG-induced deficits of sucrose preference and social interaction, indicating for the first time a role of P2X7 in the BCG model of depression, probably due to the neuroinflammatory component induced by BCG inoculation. Finally, in a rat model of chronic stress induced sucrose intake deficit, JNJ-55308942 reversed the deficit with concurrent high P2X7 brain occupancy as measured by autoradiography. This body of data demonstrates that JNJ-55308942 is a potent P2X7 antagonist, engages the target in brain, modulates IL-1β release and microglial activation leading to efficacy in two models of anhedonia in rodents.

Background: Epidemiological evidence and animal models suggest that exposure to low and non-neurotoxic concentrations of mercury may contribute to idiosyncratic autoimmune disease. Since defects in function and signaling in B cells are often associated with autoimmunity, we investigated whether mercury exposure might alter B cell responsiveness to self-antigens by interfering with B cell receptor (BCR) signal transduction. In this study we determined the effects of mercury on the protein tyrosine kinase SYK, a critical protein involved in regulation of the BCR signaling pathway., Methods: Phosphorylation sites of murine SYK were mapped before and after treatment of WEHI cell cultures with mercury, or with anti-IgM antibody (positive control) or pervanadate (a potent phosphatase inhibitor). Phosphopeptides were enriched by either titanium dioxide chromatography or anti-phosphotyrosine immunoaffinity, and analyzed by liquid chromatography-mass spectrometry. Select SYK phosphosite cluster regions were profiled for responsiveness to treatments using multiple reaction monitoring (MRM) methodology., Results: A total of 23 phosphosites were identified with high probability in endogenous SYK, including 19 tyrosine and 4 serine residues. For 10 of these sites phosphorylation levels were increased following BCR activation. Using MRM to profile changes in phosphorylation status we found that 4 cluster regions, encompassing 8 phosphosites, were activated by mercury and differentially responsive to all 3 treatments. Phosphorylation of tyrosine-342 and -346 residues were most sensitive to mercury exposure. This cluster is known to propagate normal BCR signal transduction by recruiting adaptor proteins such as PLC-γ and Vav-1 to SYK during formation of the BCR signalosome., Conclusions: Our data shows that mercury alters the phosphorylation status of SYK on tyrosine sites known to have a role in promoting BCR signals. Considering the importance of SYK in the BCR signaling pathway, these data suggest that mercury can alter BCR signaling in B cells, which might affect B cell responsiveness to self-antigen and have implications with respect to autoimmunity and autoimmune disease.

Orexin neurons originating in the perifornical and lateral hypothalamic area are highly reactive to anxiogenic stimuli and have strong projections to anxiety and panic-associated circuitry. Recent studies support a role for the orexin system and in particular the orexin 1 receptor (OX1R) in coordinating an integrative stress response. However, no selective OX1R antagonist has been systematically tested in two preclinical models of using panicogenic stimuli that induce panic attack in the majority of people with panic disorder, namely an acute hypercapnia-panic provocation model and a model involving chronic inhibition of GABA synthesis in the perifornical hypothalamic area followed by intravenous sodium lactate infusion. Here we report on a novel brain penetrant, selective and high affinity OX1R antagonist JNJ-54717793 (1S,2R,4R)-7-([(3-fluoro-2-pyrimidin-2-ylphenyl)carbonyl]- N -[5-(trifluoromethyl)pyrazin-2-yl]-7-azabicyclo[2.2.1]heptan-2-amine). JNJ-54717793 is a high affinity/potent OX1R antagonist and has an excellent selectivity profile including 50 fold versus the OX2R. Ex vivo receptor binding studies demonstrated that after oral administration JNJ-54717793 crossed the blood brain barrier and occupied OX1Rs in the rat brain. While JNJ-54717793 had minimal effect on spontaneous sleep in rats and in wild-type mice, its administration in OX2R knockout mice, selectively promoted rapid eye movement sleep, demonstrating target engagement and specific OX1R blockade. JNJ-54717793 attenuated CO 2 and sodium lactate induced panic-like behaviors and cardiovascular responses without altering baseline locomotor or autonomic activity. These data confirm that selective OX1R antagonism may represent a novel approach of treating anxiety disorders, with no apparent sedative effects.

Background and Objective: Stable isotope labeling by amino acids in cell culture (SILAC) is a practical and powerful approach for quantitative proteomic analysis. A key advantage of SILAC is the ability to simultaneously detect the isotopically labeled peptides in a single instrument run and so guarantee relative quantitation for a large number of peptides without introducing any variation caused by separate experiment. However, there are a few approaches available to assessing protein ratios and none of the existing algorithms pays considerable attention to the proteins having only one peptide hit., Methods: We introduce new quantitative approaches to dealing with SILAC protein-level summary using classification-based methodologies, such as Gaussian mixture models with EM algorithms and its Bayesian approach as well as K-means clustering. In addition, a new approach is developed using Gaussian mixture model and a stochastic, metaheuristic global optimization algorithm, particle swarm optimization (PSO), to avoid either a premature convergence or being stuck in a local optimum., Results: Our simulation studies show that the newly developed PSO-based method performs the best among others in terms of F1 score and the proposed methods further demonstrate the ability of detecting potential markers through real SILAC experimental data., Conclusions: No matter how many peptide hits the protein has, the developed approach can be applicable, rescuing many proteins doomed to removal. Furthermore, no additional correction for multiple comparisons is necessary for the developed methods, enabling direct interpretation of the analysis outcomes., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

GPR139 is an orphan G-protein-coupled receptor expressed in the central nervous system. To identify its physiologic ligand, we measured GPR139 receptor activity from recombinant cells after treatment with amino acids, orphan ligands, serum, and tissue extracts. GPR139 activity was measured using guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding, calcium mobilization, and extracellular signal-regulated kinases phosphorylation assays. Amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) activated GPR139, with EC50 values in the 30- to 300-μM range, consistent with the physiologic concentrations of L-Trp and L-Phe in tissues. Chromatography of rat brain, rat serum, and human serum extracts revealed two peaks of GPR139 activity, which corresponded to the elution peaks of L-Trp and L-Phe. With the purpose of identifying novel tools to study GPR139 function, a high-throughput screening campaign led to the identification of a selective small-molecule agonist [JNJ-63533054, (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl) benzamide]. The tritium-labeled JNJ-63533054 bound to cell membranes expressing GPR139 and could be specifically displaced by L-Trp and L-Phe. Sequence alignment revealed that GPR139 is highly conserved across species, and RNA sequencing studies of rat and human tissues indicated its exclusive expression in the brain and pituitary gland. Immunohistochemical analysis showed specific expression of the receptor in circumventricular regions of the habenula and septum in mice. Together, these findings suggest that L-Trp and L-Phe are candidate physiologic ligands for GPR139, and we hypothesize that this receptor may act as a sensor to detect dynamic changes of L-Trp and L-Phe in the brain., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Orexins (OXs) are peptides produced by perifornical (PeF) and lateral hypothalamic neurons that exert a prominent role in arousal-related processes, including stress. A critical role for the orexin-1 receptor (OX1R) in complex emotional behavior is emerging, such as overactivation of the OX1R pathway being associated with panic or anxiety states. Here we characterize a brain-penetrant, selective, and high-affinity OX1R antagonist, compound 56 [N-({3-[(3-ethoxy-6-methylpyridin-2-yl)carbonyl]-3-azabicyclo[4.1.0]hept-4-yl}methyl)-5-(trifluoromethyl)pyrimidin-2-amine]. Ex vivo receptor binding studies demonstrated that, after subcutaneous administration, compound 56 crossed the blood-brain barrier and occupied OX1Rs in the rat brain at lower doses than standard OX1R antagonists GSK-1059865 [5-bromo-N-({1-[(3-fluoro-2-methoxyphenyl)carbonyl]-5-methylpiperidin-2-yl}methyl)pyridin-2-amine], SB-334867 [1-(2-methyl-1,3-benzoxazol-6-yl)-3-(1,5-naphthyridin-4-yl)urea], and SB-408124 [1-(6,8-difluoro-2-methylquinolin-4-yl)-3-[4-(dimethylamino)phenyl]urea]. Although compound 56 did not alter spontaneous sleep in rats and in wild-type mice, its administration in orexin-2 receptor knockout mice selectively promoted rapid eye movement sleep, demonstrating target engagement and specific OX1R blockade. In a rat model of psychological stress induced by cage exchange, the OX1R antagonist prevented the prolongation of sleep onset without affecting sleep duration. In a rat model of panic vulnerability (involving disinhibition of the PeF OX region) to threatening internal state changes (i.e., intravenous sodium lactate infusion), compound 56 attenuated sodium lactate-induced panic-like behaviors and cardiovascular responses without altering baseline locomotor or autonomic activity. In conclusion, OX1R antagonism represents a novel therapeutic strategy for the treatment of various psychiatric disorders associated with stress or hyperarousal states., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

In the central nervous system, the ATP-gated Purinergic receptor P2X ligand-gated ion channel 7 (P2X7) is expressed in glial cells and modulates neurophysiology via release of gliotransmitters, including the proinflammatory cytokine interleukin (IL)-1β. In this study, we characterized JNJ-42253432 [2-methyl-N-([1-(4-phenylpiperazin-1-yl)cyclohexyl]methyl)-1,2,3,4-tetrahydroisoquinoline-5-carboxamide] as a centrally permeable (brain-to-plasma ratio of 1), high-affinity P2X7 antagonist with desirable pharmacokinetic and pharmacodynamic properties for in vivo testing in rodents. JNJ-42253432 is a high-affinity antagonist for the rat (pKi 9.1 ± 0.07) and human (pKi 7.9 ± 0.08) P2X7 channel. The compound blocked the ATP-induced current and Bz-ATP [2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate tri(triethylammonium)]-induced release of IL-1β in a concentration-dependent manner. When dosed in rats, JNJ-42253432 occupied the brain P2X7 channel with an ED50 of 0.3 mg/kg, corresponding to a mean plasma concentration of 42 ng/ml. The compound blocked the release of IL-1β induced by Bz-ATP in freely moving rat brain. At higher doses/exposure, JNJ-42253432 also increased serotonin levels in the rat brain, which is due to antagonism of the serotonin transporter (SERT) resulting in an ED50 of 10 mg/kg for SERT occupancy. JNJ-42253432 reduced electroencephalography spectral power in the α-1 band in a dose-dependent manner; the compound also attenuated amphetamine-induced hyperactivity. JNJ-42253432 significantly increased both overall social interaction and social preference, an effect that was independent of stress induced by foot-shock. Surprisingly, there was no effect of the compound on either neuropathic pain or inflammatory pain behaviors. In summary, in this study, we characterize JNJ-42253432 as a novel brain-penetrant P2X7 antagonist with high affinity and selectivity for the P2X7 channel., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Unlabelled: Neuropeptide Y2 (NPY2) receptors are implicated in diverse brain disorders, but no suitable PET radiotracers are currently available for studying NPY2 receptors in the living brain. We developed a novel positron-emitting radioligand based on the NPY2 receptor antagonist JNJ-31020028 (N-(4-(4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl)-3-fluorophenyl)-2-pyridin-3-ylbenzamide) and used the radiotracer for PET brain imaging in pigs., Methods: In vitro receptor autoradiography studies were performed to establish the anatomic distribution of NPY2 receptors in the pig brain. In vivo, baseline 90-min PET recordings of N-(11)C-methyl-JNJ-31020028 were conducted in anesthetized Yorkshire x Landrace pigs, concurrent with arterial blood sampling. Postchallenge scans were conducted after injection of unlabeled JNJ-31020028 as a pharmacologic intervention. Cyclosporine A was used to enhance levels of the PET radiotracer in the brain. The PET images were manually coregistered to a MR imaging atlas of the pig brain. Maps of the N-(11)C-methyl-JNJ-31020028 volume of distribution in the brain were prepared, and regional binding potentials of NPY2 receptors toward the radioligand were calculated using the simplified reference tissue method., Results: In autoradiography studies, N-(11)C-methyl-JNJ-31020028 receptor binding sites were observed primarily in the hippocampus and were inhibited by unlabeled JNJ-31020028. In PET studies, N-(11)C-methyl-JNJ-31020028 was metabolized slowly in the bloodstream, with 25% of the (11)C-labeled parent compound remaining 30 min after injection. PET imaging showed baseline binding potentials of 0.64 ± 0.07 in the thalamus, 0.55 ± 0.02 in the caudate, and 0.49 ± 0.03 in the hippocampus. Graphical reference region analyses demonstrated that N-(11)C-methyl-JNJ-31020028 binding was reversible; infusion of unlabeled JNJ-31020028 markedly displaced the PET radioligand from binding sites in the hippocampus, thalamus, caudate nucleus, and cerebellum but not in the corpus callosum, which served as reference region for nonspecific binding., Conclusion: N-(11)C-methyl-JNJ-31020028 has several suitable properties for PET neuroimaging of NPY2 receptors. First, it is metabolized slowly in the bloodstream of pigs. Second, using cyclosporine, the target-to-background ratio of N-(11)C-methyl-JNJ-31020028 is sufficient for estimating pharmacokinetic parameters. Third, N-(11)C-methyl-JNJ-31020028 binds reversibly and competitively to cerebral sites known to contain relatively high numbers of NPY2 receptors, such as the hippocampus, thalamus, caudate nucleus, and cerebellum. Fourth, white matter such as corpus callosum, known to contain negligible numbers of NPY2 receptors, can serve as a reference region for estimating binding potentials in brain regions. To our knowledge, there is no other radioligand with these favorable properties and with this specificity for NPY2 receptors, which makes N-(11)C-methyl-JNJ-31020028 the first candidate radioligand for PET investigations of NPY2 receptors in the living brain.

In rodents 5-hydroxytryptamine type 7 (5-HT(7)) receptor blockade has been shown to be effective in models of depression and to increase the latency to rapid eye movement (REM) sleep and decrease REM duration. In the clinic, the REM sleep reduction observed with many antidepressants may serve as a biomarker. We report here the preclinical and clinical evaluation of a 5-HT(7) receptor antagonist, (3-(4-chlorophenyl)-1,4,5,6,7,8-hexahydro-1-(phenylmethyl)pyrazolo[3,4-d]azepine 2-hydroxy-1,2,3-propanetricarboxylate) (JNJ-18038683). In rodents, JNJ-18038683 increased the latency to REM sleep and decreased REM duration, and this effect was maintained after repeated administration for 7 days. The compound was effective in the mouse tail suspension test. JNJ-18038683 enhanced serotonin transmission, antidepressant-like behavior, and REM sleep suppression induced by citalopram in rodents. In healthy human volunteers JNJ-18038683 prolonged REM latency and reduced REM sleep duration, demonstrating that the effect of 5-HT(7) blockade on REM sleep translated from rodents to humans. Like in rats, JNJ-18038683 enhanced REM sleep suppression induced by citalopram in humans, although a drug-drug interaction could not be ruled out. In a double-blind, active, and placebo-controlled clinical trial in 225 patients suffering from major depressive disorder, neither treatment with pharmacologically active doses of JNJ-18038683 or escitalopram separated from placebo, indicating a failed study lacking assay sensitivity. Post hoc analyses using an enrichment window strategy, where all the efficacy data from sites with an implausible high placebo response [placebo group Montgomery-Åsberg Depression Rating Scale (MADRS) < = 12] and from sites with no placebo response (MADRS > = 28) are removed, there was a clinically meaningful difference between JNJ-18038683 and placebo. Further clinical studies are required to characterize the potential antidepressant efficacy of JNJ-18038683.

Niacin raises high-density lipoprotein and lowers low-density lipoprotein through the activation of the β-hydroxybutyrate receptor hydroxycarboxylic acid 2 (HCA2) (aka GPR109a) but with an unwanted side effect of cutaneous flushing caused by vascular dilation because of the stimulation of HCA2 receptors in Langerhans cells in skin. HCA1 (aka GPR81), predominantly expressed in adipocytes, was recently identified as a receptor for lactate. Activation of HCA1 in adipocytes by lactate results in the inhibition of lipolysis, suggesting that agonists for HCA1 may be useful for the treatment of dyslipidemia. Lactate is a metabolite of glucose, suggesting that HCA1 may also be involved in the regulation of glucose metabolism. The low potency of lactate to activate HCA1, coupled with its fast turnover rate in vivo, render it an inadequate tool for studying the biological role of lactate/HCA1 in vivo. In this article, we demonstrate the identification of 3-hydroxybenzoic acid (3-HBA) as an agonist for both HCA2 and HCA1, whereas 3,5-dihydroxybenzoic acid (3,5-DHBA) is a specific agonist for only HCA1 (EC(50) ∼150 μM). 3,5-DHBA inhibits lipolysis in wild-type mouse adipocytes but not in HCA1-deficient adipocytes. Therefore, 3,5-DHBA is a useful tool for the in vivo study of HCA1 function and offers a base for further HCA1 agonist design. Because 3-HBA and 3,5-DHBA are polyphenolic acids found in many natural products, such as fruits, berries, and coffee, it is intriguing to speculate that other heretofore undiscovered natural substances may have therapeutic benefits.

Orexin receptor antagonists are being investigated as therapeutic agents for insomnia and addictive disorders. In this study the interactions between the orexin receptors (orexin 1 receptor and orexin 2 receptor), orexin peptides, and small molecule orexin antagonists were explored. To study these phenomena, a variety of mutant orexin receptors was made and tested using receptor binding and functional assays. Domains of the two orexin receptors were exchanged to show the critical ligand binding domains for orexin peptides and representative selective orexin receptor antagonists. Results from domain exchanges between the orexin receptors suggest that transmembrane domain 3 is crucially important for receptor interactions with small molecule antagonists. These data also suggest that the orexin peptides occupy a larger footprint, interacting with transmembrane domain 1, the amino terminus and transmembrane domain 5 as well as transmembrane domain 3. Transmembrane domain 3 has been shown to be an important part of the small molecule binding pocket common to rhodopsin and β2-adrenergic receptors. Additional orexin receptor 2 point mutations were made based on the common arrangement of receptor transmembrane domains shown in the G-protein coupled receptor crystal structure literature and the impact of orexin 2 receptor residue threonine 135 on the ligand selectivity of the 2 orexin receptors. These data support a model of the orexin receptor binding pocket in which transmembrane domains 3 and 5 are prominent contributors to ligand binding and functional activity. The data also illustrate key contact points for ligand interactions in the consensus small molecule pocket of these receptors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The successful cloning and functional expression of the histamine H(3) receptor in the late 1990 s has greatly facilitated our efforts to identify small molecule, non-imidazole based compounds to permit the evaluation of H(3) antagonists in models of CNS disorders. High-throughput screening identified several series of lead compounds, including a series of imidazopyridines, which led to JNJ-6379490, a compound with high affinity for the human H(3) receptor. Analysis of structural features common to several series of non-imidazole H(3) receptor ligands resulted in a pharmacophore model. This model led to the design of JNJ-5207852, a diamine-based H(3) antagonist with good in vitro and in vivo efficacy but with an undesirable long half-life. However, further modifications of the template provided an understanding of the effect of structural modifications on pharmacokinetic properties, ultimately affording several additional series of compounds including JNJ-10181457, a compound with an improved pharmacokinetic profile. These compounds allowed in vivo pharmacological evaluation to show that H(3) antagonists promote wakefulness, but unlike modafinil and classical psychostimultants, they do not increase locomotor activity or produce any alteration of the EEG power spectral activity in rats. H(3) antagonists also increase extracellular acetylcholine and norepinephrine but not dopamine in rat frontal cortex and show efficacy in various models of learning-memory deficit. In addition, cFos immunoreactivity studies show H(3) antagonists activate neuronal cells in restricted rat brain regions in contrast to widespread activation after modafinil or amphetamine treatment. Therefore, H(3) antagonists are promising clinical candidates for the treatment of excessive day time sleepiness and/or cognitive disorders.

Experiments were performed to characterize the pharmacology of SCH 206272 [(R,R)-1'[5-[(3,5-dichlorobenzoyl)methylamino]-3-(3,4-dichlorophenyl)-4(Z)-(methoxyimino)pentyl]-N-methyl-2-oxo-[1,4'bipiperidine]-3-acetamide] as a potent and selective antagonist of tachykinin (NK) NK(1), NK(2), and NK(3) receptors. SCH 206272 inhibited binding at human tachykinin NK(1), NK(2), and NK(3) receptors (K(i) = 1.3, 0.4, and 0.3 nM, respectively) and antagonized [Ca(2+)](i) mobilization in Chinese hamster ovary (CHO) cells expressing the cloned human tachykinin NK(1), NK(2), or NK(3) receptors. SCH 206272 inhibited relaxation of the human pulmonary artery (pK(b) = 7.7 +/- 0.3) induced by the tachykinin NK(1) receptor agonist, [Met-O-Me] substance P and contraction of the human bronchus (pK(b = 8.2 +/- 0.3) induced by the tachykinin NK(2) receptor agonist, neurokinin A. In isolated guinea pig tissues, SCH 206272 inhibited substance P-induced enhancement of electrical field stimulated contractions of the vas deferens, (pK(b = 7.6 +/- 0.2), NKA-induced contraction of the bronchus (pK(b) = 7.7 +/- 0.2), and senktide-induced contraction of the ileum. In vivo, oral SCH 206272 (0.1-10 mg/kg, p.o.) inhibited substance P-induced airway microvascular leakage and neurokinin A-induced bronchospasm in the guinea pig. In a canine in vivo model, SCH 206272 (0.1-3 mg/kg, p.o.) inhibited NK(1) and NK(2) activities induced by exogenous substance P and neurokinin A. Furthermore, in guinea pig models involving endogenously released tachykinins, SCH 206272 inhibited hyperventilation-induced bronchospasm, capsaicin-induced cough, and airway microvascular leakage induced by nebulized hypertonic saline. These data demonstrate that SCH 206272 is a potent, orally active tachykinin NK(1), NK(2), and NK(3) receptor antagonist. This compound may have beneficial effects in diseases thought to be mediated by tachykinins, such as cough, asthma, and chronic obstructive pulmonary disease., (Copyright 2002 Elsevier Science B.V.)

The characterization and anatomical distribution of 5-hydroxytryptamine (5-HT)(7) receptor binding sites in brain tissue has been hampered by the lack of a specific radioligand. In the present autoradiographic study, we took advantage of 5-HT(1A) knockout and 5-HT(1A/1B) double-knockout mice to revisit the pharmacological characterization and anatomical localization of 5-HT(7) binding sites in mouse brain using [(3)H]5-carboxamidotryptamine (5-CT) and [(3)H]8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT). The distribution pattern of [(3)H]5-CT binding sites (2 nM) in the brain of mice lacking the 5-HT(1A/1B) receptor was scarce and confined to the septum, globus pallidus, thalamus, hypothalamus, amygdala, cortex, and substantia nigra. The low densities of [(3)H]5-CT binding sites detected in septum, thalamus, hypothalamus, amygdala, and cortex were displaced by 10 microM of the selective 5-HT(7) receptor antagonist (R)-3-(2-(2-(4-methylpiperidin-1-yl) ethyl)pyrrolidine-1-sulfonyl) phenol (SB-269970). The SB-269970-insensitive [(3)H]5-CT binding sites detected in globus pallidus and substantia nigra of 5-HT(1A/1B) knockout mice were displaced by N-[3-(2-dimethylamino)ethoxy-4-methoxy-phenyl]-2'-methyl-4'- (5-methyl-1,2,4-oxadiazol-3-yl)-(1,1'-biphenyl)-4-carboxamide hydrochloride (SB-216641) (1 microM), demonstrating the 5-HT(1D) nature of these binding sites. In contrast to the low densities of [(3)H]5-CT binding sites, high-to-moderate densities of [(3)H]8-OH-DPAT binding sites (10 nM) were found throughout the brain of 5-HT(1A) and 5-HT(1A/1B) knockout mice (olfactory system, septum, thalamus, hypothalamus, amygdala, CA3 field of the hippocampus, cortical mantle, and central gray). These [(3)H]8-OH-DPAT binding sites were displaced by 10 microM SB-269970, risperidone, and methiothepin but not by pindolol, N-tert-butyl-3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenylpropanamide (WAY- 100135), or citalopram. We conclude that despite its high affinity for the 5-HT(7) receptor in tissue homogenates, [(3)H]5-CT is not a good tracer for measuring 5-HT(7) receptor binding sites autoradiographically. Also, the lower affinity ligand [(3)H]8-OH-DPAT is a much better tracer for autoradiographic studies at the 5-HT(7) receptor binding sites.

Histamine is a multifunctional hormone that regulates smooth muscle contraction in the airways, acid secretion in the gut, and neurotransmitter release in the central nervous system through three well characterized receptor subtypes, H(1), H(2), H(3), respectively. As part of a directed effort to discover novel G-protein-coupled receptors through homology searching of genomic databases, we identified a partial clone (GPCR105) that had significant homology to the recently identified histamine H(3) receptor cDNA. Expression of the full-length human GPCR105 in cells confers the ability to bind [(3)H]histamine with high affinity (K(D) = 5 nM). GPCR105 is pharmacologically similar to the histamine H(3) receptor in that it binds many of the known H(3) agonists and antagonists, albeit with a different rank order of affinity/potency. GPCR105 does not bind (i.e., K(D) > 10 microM) all tested H(1) and H(2) receptor antagonists such as diphenhydramine, loratadine, ranitidine, and cimetidine, but has modest affinity for the H(2) receptor agonist, dimaprit (377 nM). Whereas the H(3) receptor is expressed almost exclusively in nervous tissues, GPRC105 is expressed primarily in bone marrow and eosinophils. Together, these data demonstrate that GPCR105 is a novel histamine receptor structurally and pharmacologically related to the H(3) receptor. However, its unique expression profile and physiological role suggest that GPCR105 is a fourth histamine receptor subtype (H(4)) and may be a therapeutic target for the regulation of immune function, particularly with respect to allergy and asthma.

Purpose: The purpose of this study was to determine the complication rates associated with the use of the endotracheal tube (ET) and the use of the esophageal obturator airway/esophageal gastric tube airway (EOA/EGTA) during the treatment of patients with prehospital cardiac arrest., Methods: A descriptive, quasi-experimental study of 509 consecutive adults, cardiac arrest patients was conducted. Patients were examined prospectively for airway intervention type and complications. Some patients were examined at their final destinations (field, morgue, funeral home), while other patients were examined by EMS providers in the field when airway adjuncts were switched. Also, airways were evaluated for complications by emergency physicians at destination emergency departments., Results: The airway in use at the time of examination was the esophageal obturator airway (EOA) or esophageal gastric tube airway (EGTA) in 208 patients (40.1%); the ET (endotracheal tube) in 232 patients (45.6%); and an oral or nasopharyngeal airway in 47 patients (9.2%). Twenty-two patients (4.3%) had both an EOA/EGTA and an ET tube in place at the time of the examination. The survival rates were similar between the EOA/EGTA and the ET groups (28% and 32%, respectively). The complication rates overall also were similar, but the serious or potentially lethal complication rate was 3.3 times more common with the use of the EOA/EGTA than with the ET tube (8.7% versus 2.6%, respectively)., Conclusions: The complication rate for the EOA/EGTA is unacceptably high, and careful thought must be given to its continued use.(ABSTRACT TRUNCATED AT 250 WORDS)