Preliminary searches for spin-dependent interactions using sidebands of nuclear spin-precession signals.

Various theories beyond the Standard Model predict new particles with masses in the sub-eV range with very weak couplings to ordinary matter. A new P-odd and T-odd interaction between polarized and unpolarized nucleons proportional to s⃗⋅r̂ is one such possibility, where r⃗=rr̂ is the spatial vector connecting the nucleons, and s⃗ is the spin of the polarized nucleon. Such an interaction involving a scalar coupling gsN at one vertex and a pseudoscalar coupling gpn at the polarized nucleon vertex can be induced by the exchange of spin-0 pseudoscalar bosons. We describe a new technique to search for interactions of this form and present the first measurements of this type. We show that future improvements to this technique can improve the laboratory upper bound on the product gsNgpn by two orders of magnitude for interaction ranges at the 100 micron scale.

Demonstration of a Common DPhe7 to DNal(2')7 Peptide Ligand Antagonist Switch for Melanocortin-3 and Melanocortin-4 Receptors Identifies the Systematic Mischaracterization of the Pharmacological Properties of Melanocortin Peptides.

Melanocortin peptides containing a 3-(2-naphthyl)-d-alanine residue in position 7 (DNal(2')7), reported as melanocortin-3 receptor (MC3R) subtype-specific agonists in two separate publications, were found to lack significant MC3R agonist activity. The cell lines used at the University of Arizona for pharmacological characterization of these peptides, consisting of HEK293 cells stably transfected with human melanocortin receptor subtypes MC1R, MC3R, MC4R, or MC5R, were then obtained and characterized by quantitative polymerase chain reaction (PCR). While the MC1R cell line correctly expressed only hMCR1, the three other cell lines were mischaracterized with regard to receptor subtype expression. The demonstration that a 3-(2-naphthyl)-d-alanine residue in position 7, irrespective of the melanocortin peptide template, results primarily in the antagonism of MC3R and MC4R then allowed us to search the published literature for additional errors. The erroneously characterized DNal(2')7-containing peptides date back to 2003; thus, our analysis suggests that systematic mischaracterization of the pharmacological properties of melanocortin peptides occurred.

Rational Development of Stable PYY3-36 Peptide Y2 Receptor Agonists.

PURPOSE: The anorectic effect of PYY3-36 makes it a potential pharmacological weight loss treatment. Modifications of the endogenous peptide to obtain commercially attractive pharmacological and biophysical stability properties are examined. METHODS: Half-life extended PYY3-36 analogues were prepared and examined regarding Y2-receptor potency as well as biophysical and stability properties. RESULTS: Deamidation of asparagine in position 18 and 29 was observed upon incubation at 37°C. Asparagine in position 18 - but not position 29 - could be substituted to glutamine without detrimental effects on Y2-receptor potency. Covalent dimers were formed via the phenol impurity benzoquinone reacting with two N-terminal residues (Isoleucine-Lysine). Both residues had to be modified to suppress dimerization, which could be done without negatively affecting Y2-receptor potency or other stability/biophysical properties. Introduction of half-life extending modifications in position 30 and 35 eliminated aggregation at 37°C without negatively affecting other stability properties. Placement of a protracting moiety (fatty acid) in the receptor-binding C-terminal region reduced Y2-receptor potency substantially, whereas only minor effects of protractor position were observed on structural, biophysical or stability properties. Lipidated PYY3-36 analogues formed oligomers of various sizes depending on primary structure and solution conditions. CONCLUSIONS: By rational design, a chemically and physically stable Y2-receptor selective, half-life extended PYY3-36 peptide has been developed.

Chemical Synthesis of Phosphorylated Insulin-like Growth Factor Binding Protein 2.

Chemical protein synthesis is a powerful avenue for accessing homogeneously modified proteins. While a significant number of small modified proteins bearing native post-translational modifications and non-natural modifications have been generated to date, access to larger targets has proved challenging. Herein, we describe the use of two ligation manifolds, namely, diselenide-selenoester ligation and native chemical ligation, to assemble a 31.5 kDa phosphorylated insulin-like growth factor binding protein (IGFBP-2) that comprises 290 amino acid residues, a phosphoserine post-translational modification, and nine disulfide bonds.

In vitro prediction of in vivo pseudo-allergenic response via MRGPRX2.

In development of peptide therapeutics, rodents are commonly-used preclinical models when screening compounds for efficacy endpoints in the early stages of discovery projects. During the screening process, some peptides administered subcutaneously to rodents caused injection site reactions manifesting as localized swelling. Screening by postmortem evaluations of injection site swelling as a marker for local subcutaneous histamine release, were conducted in rats to select drug candidates without this adverse effect. Histological analysis of skin samples revealed that the injection site reactions were concurrent with mast cell degranulation, resulting in histamine release. Mast cell activation can be mediated by MRGPRX2, a GPCR that induces a pseudo-allergenic immune response. The present study demonstrates that a commercially-available cell-based MRGPRX2 assay reliably identifies compounds that induce histamine release or localized edema in ex vivo human and rodent skin samples. In vitro screening was subsequently implemented using the MRGPRX2 assay as a substitute for postmortem injection site evaluation, thus achieving a significant reduction in animal use. Thus, in cases where injection site reactions are encountered during in vivo screening, to enable faster screening during the early drug discovery process, an MRGPRX2 in vitro assay can be used as an efficient, more ethical tool with human translational value for the development of safer pharmacotherapies for patients.

UCP1-independent glucose-lowering effect of leptin in type 1 diabetes: only in conditions of hypoleptinemia.

The possibility to use leptin therapeutically for lowering glucose levels in patients with type 1 diabetes has attracted interest. However, earlier animal models of type 1 diabetes are severely catabolic with very low endogenous leptin levels, unlike most patients with diabetes. Here, we aim to test glucose-lowering effects of leptin in novel, more human-like murine models. We examined the glucose-lowering potential of leptin in diabetic models of two types: streptozotocin-treated mice and mice treated with the insulin receptor antagonist S961. To prevent hypoleptinemia, we used combinations of thermoneutral temperature and high-fat feeding. Leptin fully normalized hyperglycemia in standard chow-fed streptozotocin-treated diabetic mice. However, more humanized physiological conditions (high-fat diets or thermoneutral temperatures) that increased adiposity - and thus also leptin levels - in the diabetic mice abrogated the effects of leptin, i.e., the mice developed leptin resistance also in this respect. The glucose-lowering effect of leptin was not dependent on the presence of the uncoupling protein-1 and was not associated with alterations in plasma insulin, insulin-like growth factor 1, food intake or corticosterone but fully correlated with decreased plasma glucagon levels and gluconeogenesis. An important implication of these observations is that the therapeutic potential of leptin as an additional treatment in patients with type 1 diabetes is probably limited. This is because such patients are treated with insulin and do not display low leptin levels. Thus, the potential for a glucose-lowering effect of leptin would already have been attained with standard insulin therapy, and further effects on blood glucose level through additional leptin cannot be anticipated.

New Limit on the Permanent Electric Dipole Moment of

We report results of a new technique to measure the electric dipole moment of ^{129}Xe with ^{3}He comagnetometry. Both species are polarized using spin-exchange optical pumping, transferred to a measurement cell, and transported into a magnetically shielded room, where SQUID magnetometers detect free precession in applied electric and magnetic fields. The result from a one week measurement campaign in 2017 and a 2.5 week campaign in 2018, combined with detailed study of systematic effects, is d_{A}(^{129}Xe)=(1.4±6.6_{stat}±2.0_{syst})×10^{-28} e cm. This corresponds to an upper limit of |d_{A}(^{129}Xe)|<1.4×10^{-27} e cm (95% C.L.), a factor of 5 more sensitive than the limit set in 2001.

Diselenide-selenoester ligation for chemical protein synthesis.

Chemoselective peptide ligation methods have provided synthetic access to numerous proteins, including those bearing native post-translational modifications and unnatural labels. This protocol outlines the chemical synthesis of proteins using a recently discovered reaction (diselenide-selenoester ligation (DSL)) in a rapid, additive-free manner. After ligation, the products can be chemoselectively deselenized to produce native peptide and protein products. We describe methods for the synthesis of suitably functionalized peptide diselenide and peptide selenoester fragments via Fmoc-solid-phase peptide synthesis (SPPS) protocols, fusion of these fragments by DSL, and the chemoselective deselenization of the ligation products to generate native synthetic proteins. We demonstrate the method's utility through the total chemical synthesis of the post-translationally modified collagenous domain of the hormone adiponectin via DSL-deselenization at selenocystine (the oxidized form of selenocysteine) and the rapid preparation of two tick-derived thrombin-inhibiting proteins by DSL-deselenization at ß-selenoaspartate and γ-selenoglutamate. This method should find widespread use for the rapid synthesis of proteins, including cases in which other peptide ligation methods cannot be used (or cannot be used efficiently), e.g., at sterically hindered or deactivated acyl donors. The method's speed and efficiency may render it useful in the generation of synthetic protein libraries. Each protein discussed can be synthesized within 15 working days from resin loading and can be readily produced by practitioners with master's-level experience in organic chemistry. Each synthesis using these protocols was performed independently by two labs (one academic and one industrial), which attained comparable yields of the protein products.

Quantitative biosensor detection by chemically exchanging hyperpolarized 129Xe.

Chemical sensors informing about their local environment are of widespread use for chemical analysis. A thorough understanding of the sensor signaling is fundamental to data analysis and interpretation, and a requirement for technological applications. Here, sensors explored for the recognition and display of biomolecular and cellular markers by magnetic resonance and composed of host molecules for xenon atoms are considered. These host-guest systems are analytically powerful and also function as contrast agents in imaging applications. Using nuclear spin hyperpolarization of 129Xe and chemical exchange saturation transfer the detection sensitivity is orders of magnitude enhanced in comparison to conventional 1H NMR. The sensor signaling reflects this rather complex genesis, furthering the mere qualitative interpretation of biosensing data; to harvest the potential of the approach, however, a detailed numerical account is desired. To this end, we introduce a comprehensive expression that maps the sensor detection quantitatively by integration of the hyperpolarization generation and relaxation with the host-xenon exchange dynamics. As demonstrated for the host molecule and well-established biosensor cryptophane-A, this model reveals a distinguished maximum in sensor signaling and exerts control over experimentation by dedicated adjustments of both the amount of xenon and the duration of the saturation transfer applied in a measurement, for example to capitalize on investigations at the detection limit. Furthermore, usage of the model for data analysis makes the quantification of the sensor concentration in the nanomolar range possible. The approach is readily applicable in investigations using cryptophane-A and is straightaway adaptable to other sensor designs for extension of the field of xenon based biosensing.

Sweet Taste Receptor Activation in the Gut Is of Limited Importance for Glucose-Stimulated GLP-1 and GIP Secretion.

Glucose stimulates the secretion of the incretin hormones: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). It is debated whether the sweet taste receptor (STR) triggers this secretion. We investigated the role of STR activation for glucose-stimulated incretin secretion from an isolated perfused rat small intestine and whether selective STR activation by artificial sweeteners stimulates secretion. Intra-luminal administration of the STR agonists, acesulfame K (3.85% w/v), but not sucralose (1.25% w/v) and stevioside (2.5% w/v), stimulated GLP-1 secretion (acesulfame K: 31 ± 3 pmol/L vs. 21 ± 2 pmol/L, p < 0.05, n = 6). In contrast, intra-arterial administration of sucralose (10 mM) and stevioside (10 mM), but not acesulfame K, stimulated GLP-1 secretion (sucralose: 51 ± 6 pmol/L vs. 34 ± 4 pmol/L, p < 0.05; stevioside: 54 ± 6 pmol/L vs. 32 ± 2 pmol/L, p < 0.05, n = 6), while 0.1 mM and 1 mM sucralose did not affect the secretion. Luminal glucose (20% w/v) doubled GLP-1 and GIP secretion, but basolateral STR inhibition by gurmarin (2.5 µg/mL) or the inhibition of the transient receptor potential cation channel 5 (TRPM5) by triphenylphosphine oxide (TPPO) (100 µM) did not attenuate the responses. In conclusion, STR activation does not drive GIP/GLP-1 secretion itself, nor does it have a role for glucose-stimulated GLP-1 or GIP secretion.

Analytic framework for peptidomics applied to large-scale neuropeptide identification.

Large-scale mass spectrometry-based peptidomics for drug discovery is relatively unexplored because of challenges in peptide degradation and identification following tissue extraction. Here we present a streamlined analytical pipeline for large-scale peptidomics. We developed an optimized sample preparation protocol to achieve fast, reproducible and effective extraction of endogenous peptides from sub-dissected organs such as the brain, while diminishing unspecific protease activity. Each peptidome sample was analysed by high-resolution tandem mass spectrometry and the resulting data set was integrated with publically available databases. We developed and applied an algorithm that reduces the peptide complexity for identification of biologically relevant peptides. The developed pipeline was applied to rat hypothalamus and identifies thousands of neuropeptides and their post-translational modifications, which is combined in a resource format for visualization, qualitative and quantitative analyses.

What Is Engagement? Proactivity as the Missing Link in the HEXACO Model of Personality.

We tested the hypothesis that proactivity represents the engagement vector in the HEXACO model of personality. Questionnaire data were obtained in five studies, three of which consisted (mostly) of students: Study 1 (N = 188, Mage = 20.0, 89.4% women), Study 3 (N = 315, Mage = 20.4, 80.6% women), and Study 4 (N = 309 self-ratings, Mage = 20.0, 78.3% women; N = 307 other-ratings, Mage = 24.5, 62.2% women). Participants in the other two studies came from an ISO-certified representative community panel: Study 2 (N = 525, Mage = 51.2, 52.0% women) and Study 5 (N = 736, Mage = 42.2, 48.0% women). Proactive Personality and Proactivity were positively related to Extraversion, Conscientiousness, and Openness to Experience, but only weakly related or unrelated to Honesty-Humility, Emotionality, and Agreeableness, supporting the alignment of Proactive Personality/Proactivity with the hypothesized HEXACO engagement vector. Additionally, Proactivity explained incremental variance in self-rated job performance on top of the HEXACO facets that were most closely associated with Proactive Personality/Proactivity, that is, Social Boldness (an Extraversion facet), Diligence (a Conscientiousness facet), and Creativity (an Openness to Experience facet), but not in entrepreneurship and intrapreneurship. Proactivity is the missing engagement link in the HEXACO model of personality. The results are discussed in light of higher-order factors (e.g., general factor of personality and Alpha and Beta) of personality and bandwidth-fidelity controversies.

Melanocortin agonists stimulate lipolysis in human adipose tissue explants but not in adipocytes.

BACKGROUND: The central melanocortin system is broadly involved in the regulation of mammalian nutrient utilization. However, the function of melanocortin receptors (MCRs) expressed directly in peripheral metabolic tissues is still unclear. The objective of this study was to investigate the lipolytic capacity of MC1-5R in differentiated adipocytes versus intact white adipose tissue. RESULTS: Non-selective MCR agonist α-MSH, MC5R-selective agonist PG-901 and MC4R-selective agonist LY2112688 significantly stimulated lipolysis in intact white adipose tissue, whereas stimulation of MCRs in differentiated adipocytes failed to do so. The lipolytic response of MC5R was decreased in intact human white adipose tissue when co-treating with ß-adrenergic antagonist propranolol, suggesting that the effect may be dependent on neuronal innervation via noradrenalin release. CONCLUSION: When developing an anti-obesity therapeutic drug with selective MC4R/MC5R properties, effects on lipolysis in white adipose tissue may be physiologically relevant.

New limit on Lorentz-invariance- and CPT-violating neutron spin interactions using a free-spin-precession He3-Xe129 comagnetometer.

We report on the search for a CPT- and Lorentz-invariance-violating coupling of the He3 and Xe129 nuclear spins (each largely determined by a valence neutron) to posited background tensor fields that permeate the Universe. Our experimental approach is to measure the free precession of nuclear spin polarized He3 and Xe129 atoms in a homogeneous magnetic guiding field of about 400 nT using LTC SQUIDs as low-noise magnetic flux detectors. As the laboratory reference frame rotates with respect to distant stars, we look for a sidereal modulation of the Larmor frequencies of the colocated spin samples. As a result we obtain an upper limit on the equatorial component of the background field interacting with the spin of the bound neutron b(⊥)(n)<8.4 × 10(-34) GeV (68% C.L.). Our result improves our previous limit (data measured in 2009) by a factor of 30 and the world's best limit by a factor of 4.

Constraints on spin-dependent short-range interaction between nucleons.

We search for a spin-dependent P- and T-violating nucleon-nucleon interaction mediated by light pseudoscalar bosons such as axions or axionlike particles. We employ an ultrasensitive low-field magnetometer based on the detection of free precession of colocated 3He and 129Xe nuclear spins using SQUIDs as low-noise magnetic flux detectors. The precession frequency shift in the presence of an unpolarized mass was measured to determine the coupling of pseudoscalar particles to the spin of the bound neutron. For boson masses between 2 and 500 µeV (force ranges between 3×1(-4) m and 10(-1) m) we improved the laboratory upper bounds by up to 4 orders of magnitude.

Design, synthesis, structural and functional characterization of novel melanocortin agonists based on the cyclotide kalata B1.

BACKGROUND: Cyclotides are useful scaffolds to stabilize bioactive peptides. RESULTS: Four melanocortin analogues of kalata B1 were synthesized. One is a selective MC4R agonist. CONCLUSION: The analogues retain the native kalata B1 scaffold and introduce a designed pharmacological activity, validating cyclotides as protein engineering scaffolds. SIGNIFICANCE: A novel type of melanocortin agonist has been developed, with potential as a drug lead for treating obesity. Obesity is an increasingly important global health problem that lacks current treatment options. The melanocortin receptor 4 (MC4R) is a target for obesity therapies because its activation triggers appetite suppression and increases energy expenditure. Cyclotides have been suggested as scaffolds for the insertion and stabilization of pharmaceutically active peptides. In this study, we explored the development of appetite-reducing peptides by synthesizing MC4R agonists based on the insertion of the His-Phe-Arg-Trp sequence into the cyclotide kalata B1. The ability of the analogues to fold similarly to kalata B1 but display MC4R activity were investigated. Four peptides were synthesized using t-butoxycarbonyl peptide chemistry with a C-terminal thioester to facilitate backbone cyclization. The structures of the peptides were found to be similar to kalata B1, evaluated by Hα NMR chemical shifts. KB1(GHFRWG;23-28) had a K(i) of 29 nm at the MC4R and was 107 or 314 times more selective over this receptor than MC1R or MC5R, respectively, and had no detectable binding to MC3R. The peptide had higher affinity for the MC4R than the endogenous agonist, α-melanocyte stimulation hormone, but it was less potent at the MC4R, with an EC(50) of 580 nm for activation of the MC4R. In conclusion, we synthesized melanocortin analogues of kalata B1 that preserve the structural scaffold and display receptor binding and functional activity. KB1(GHFRWG;23-28) is potent and selective for the MC4R. This compound validates the use of cyclotides as scaffolds and has the potential to be a new lead for the treatment of obesity.

Handling a tricycle: orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin.

Gurmarin is a 35 amino acid peptide with three disulfide bridges in an inhibitor cystine knot. It is found in the plant Gymnema sylvestre, and has been identified as a sweet taste inhibitor in rodents. In this article we provide an efficient route for the synthesis of gurmarin by a controlled random oxidation strategy. We compared two oxidation procedures to form the three disulfide bridges. In the first, based on random oxidation, reduced gurmarin was synthesized using trityl for cysteine protection, and oxidized for 48 h in a Tris-HCl buffer containing cystamine and reduced glutathione to facilitate disulfide scrambling. The second was based on step-wise deprotection followed by oxidation in which the cysteine pairs are orthogonally protected with tert-Butylthio, trityl and acetamidomethyl. To verify that the native gurmarin oxidation product was obtained, thermolysin cleavage was used. Cleavage of random oxidized gurmarin showed two possible disulfide combinations; the native and a non-native gurmarin disulfide isomer. The non-native isomer was therefore synthesized using the orthogonal deprotection-oxidation strategy and the native and the non-native gurmarin isomers were analyzed using UPLC. It was found that the random oxidation procedure leads to native gurmarin in high yield. Thus, the synthetic route was simple and significantly more efficient than previously reported syntheses of gurmarin and other cysteine rich peptides. Importantly, native gurmarin was obtained by random oxidation, which was confirmed by a synthetic approach for the first time.

Characterization of murine melanocortin receptors mediating adipocyte lipolysis and examination of signalling pathways involved.

The melanocortin receptors (MCRs) belong to the G-protein coupled receptors (family A). So far, 5 different subtypes have been described (MC1R-MC5R) and of these MC2R and MC5R have been proposed to act directly in adipocytes and regulate lipolysis in rodents. Using ACTH and α-melanocyte stimulating hormone (α-MSH) generated from proopiomelanocortin (POMC), as well as synthetic MSH analogues to stimulate lipolysis in murine 3T3-L1 adipocytes it is shown that MC2R and MC5R are lipolytic mediators in differentiated 3T3-L1 adipocytes. Involvement of cAMP, phosphorylated extracellular signal-regulated kinase (ERK) 1/2, protein kinase B (PKB), adenosine 5' monophosphate activated protein kinase (AMPK) and Jun-amino-terminal kinase (JNK) in MCR mediated lipolysis were studied. Interestingly, results obtained in 3T3-L1 cells suggest that lipolysis stimulated by α-MSH, NDP-α-MSH, MT-II, SHU9119 and PG-901 is mediated through MC5R in a cAMP independent manner. Finally, we identify essential differences in MCR mediated lipolysis when using 3T3-L1 cells compared to primary adipocytes.