Chitosan-carbon nanofiber based disposable bioelectrode for electrochemical detection of oxytocin.

Bioelectrodes with low carbon footprint can provide an innovative solution to the surmounting levels of e-waste. Biodegradable polymers offer green and sustainable alternatives to synthetic materials. Here, a chitosan-carbon nanofiber (CNF) based membrane has been developed and functionalized for electrochemical sensing application. The surface characterization of the membrane revealed crystalline structure with uniform particle distribution, and surface area of 25.52 m2/g and pore volume of 0.0233 cm3/g. The membrane was functionalized to develop a bioelectrode for the detection of exogenous oxytocin in milk. Electrochemical impedance spectroscopy was employed to determine oxytocin in a linear concentration range of 10 to 105 ng/mL. The developed bioelectrode showed an LOD of 24.98 ± 11.37 pg/mL and sensitivity of 2.77 × 10-10 Ω / log ng mL-1/mm2 for oxytocin in milk samples with 90.85-113.34 percent recovery. The chitosan-CNF membrane is ecologically safe and opens new avenues for environment-friendly disposable materials for sensing applications.

The oxytocin signaling complex reveals a molecular switch for cation dependence.

Oxytocin (OT) and vasopressin (AVP) are conserved peptide signaling hormones that are critical for diverse processes including osmotic homeostasis, reproduction, lactation and social interaction. OT acts through the oxytocin receptor (OTR), a magnesium-dependent G protein-coupled receptor that is a therapeutic target for treatment of postpartum hemorrhage, dysfunctional labor and autism. However, the molecular mechanisms that underlie OTR activation by OT and the dependence on magnesium remain unknown. Here we present the wild-type active-state structure of human OTR bound to OT and miniGq/i determined by cryo-EM. The structure reveals a unique activation mechanism adopted by OTR involving both the formation of a Mg2+ coordination complex between OT and the receptor, and disruption of transmembrane helix 7 (TM7) by OT. Our functional assays demonstrate the role of TM7 disruption and provide the mechanism of full agonism by OT and partial agonism by OT analogs. Furthermore, we find that the identity of a single cation-coordinating residue across vasopressin family receptors determines whether the receptor is cation-dependent. Collectively, these results demonstrate how the Mg2+-dependent OTR is activated by OT, provide essential information for structure-based drug discovery efforts and shed light on the molecular determinants of cation dependence of vasopressin family receptors throughout the animal kingdom.

Vortex Fluidic Mediated Oxidative Sulfitolysis of Oxytocin.

In peptide production, oxidative sulfitolysis can be used to protect the cysteine residues during purification, and the introduction of a negative charge aids solubility. Subsequent controlled reduction aids in ensuring correct disulfide bridging. In vivo, these problems are overcome through interaction with chaperones. Here, a versatile peptide production process has been developed using an angled vortex fluidic device (VFD), which expands the viable pH range of oxidative sulfitolysis from pH 10.5 under batch conditions, to full conversion within 20 min at pH 9-10.5 utilising the VFD. VFD processing gave 10-fold greater conversion than using traditional batch processing, which has potential in many applications of the sulfitolysis reaction.

[Oxytocin and its receptor: molecular and therapeutic approaches]. / Approches moléculaires et thérapeutiques des interactions entre l'ocytocine et son récepteur.

It is known since the fifties that oxytocin is a neurohormone synthesized in the brain and released in blood circulation to trigger uterus contraction during delivery. It is also involved in milk ejection during breast-feeding. Over the past 25 years, many other central and peripheral functions have been discovered, in particular for attachment between child and parents as well as between individuals and interaction between a human being and its social group. Over this period, we have studied the functional supramolecular architecture of the hormone bound to its receptor. This information was used to design pharmacological probes and drug candidates. This led to the discovery of the first non-peptide oxytocin receptor full agonist. This molecule, LIT-001, restores social interaction in an animal model of autism and paves the way for a treatment of this neurodevelopmental disorder.

Title: Approches moléculaires et thérapeutiques des interactions entre l'ocytocine et son récepteur. Abstract: L'ocytocine est une neurohormone connue à l'origine pour son rôle dans les contractions de l'utérus au moment de l'accouchement et les contractions des glandes mammaires pour permettre l'éjection du lait lors de la tétée. Depuis les 25 dernières années, de multiples autres effets centraux et périphériques ont été identifiés, notamment dans les processus d'attachement entre parents et enfant, entre adultes et entre un individu et son groupe social. Nous avons abordé au cours de cette période la question fondamentale de l'architecture structurale et fonctionnelle du complexe formé par l'ocytocine et son récepteur et l'application de ce savoir à la conception de candidats médicaments. Ceci a conduit à la découverte du premier agoniste non peptidique de l'ocytocine, le LIT-001, restaurant l'interaction sociale dans un modèle animal d'autisme.

An oxytocin/vasopressin-related neuropeptide modulates social foraging behavior in the clonal raider ant.

Oxytocin/vasopressin-related neuropeptides are highly conserved and play major roles in regulating social behavior across vertebrates. However, whether their insect orthologue, inotocin, regulates the behavior of social groups remains unknown. Here, we show that in the clonal raider ant Ooceraea biroi, individuals that perform tasks outside the nest have higher levels of inotocin in their brains than individuals of the same age that remain inside the nest. We also show that older ants, which spend more time outside the nest, have higher inotocin levels than younger ants. Inotocin thus correlates with the propensity to perform tasks outside the nest. Additionally, increasing inotocin pharmacologically increases the tendency of ants to leave the nest. However, this effect is contingent on age and social context. Pharmacologically treated older ants have a higher propensity to leave the nest only in the presence of larvae, whereas younger ants seem to do so only in the presence of pupae. Our results suggest that inotocin signaling plays an important role in modulating behaviors that correlate with age, such as social foraging, possibly by modulating behavioral response thresholds to specific social cues. Inotocin signaling thereby likely contributes to behavioral individuality and division of labor in ant societies.

The effect of trehalose, antioxidants, and acetate buffer concentration on oxytocin stability.

Oxytocin is a cyclic nonapeptide used to induce labor and prevent bleeding after childbirth. Due to its instability, storage and transport of oxytocin formulations can be problematic in hot/tropical climates. The aim of this study was to investigate the effect of trehalose and select antioxidants (uric acid, butylated hydroxytoluene, and l-ascorbic acid) on oxytocin stability in solution. The effect of buffer composition and acetate buffer concentration was also studied. Acetate buffer was found to work better than citrate/phosphate buffer for the oxytocin stability. Lower acetate buffer concentrations (0.025 M or less) were also found to yield improved oxytocin stability compared to higher concentrations. Although known degradation pathways of oxytocin include oxidation, the antioxidants uric acid and butylated hydroxytoluene had negligible effect on the oxytocin stability while l-ascorbic acid led to significantly faster degradation. Despite trehalose's reputation as a great stabilizer for biomolecules, it also had small to negligible effect on oxytocin stability at concentrations up to 1 M in acetate buffer. These results were surprising given the present literature on trehalose as a stabilizer for various biomolecules, including proteins and lipids.

Non-covalently embedded oxytocin in alkanethiol monolayer as Zn2+ selective biosensor.

Peptides are commonly used as biosensors for analytes such as metal ions as they have natural binding preferences. In our previous peptide-based impedimetric metal ion biosensors, a monolayer of the peptide was anchored covalently to the electrode. Binding of metal ions resulted in a conformational change of the oxytocin peptide in the monolayer, which was measured using electrochemical impedance spectroscopy. Here, we demonstrate that sensing can be achieved also when the oxytocin is non-covalently integrated into an alkanethiol host monolayer. We show that ion-binding cause morphological changes to the dense host layer, which translates into enhanced impedimetric signals compared to direct covalent assembly strategies. This biosensor proved selective and sensitive for Zn2+ ions in the range of nano- to micro-molar concentrations. This strategy offers an approach to utilize peptide flexibility in monitoring their response to the environment while embedded in a hydrophobic monolayer.

Region-specific sex modulation of central oxytocin receptor by gut microbiota: An ontogenic study.

Oxytocin (OT) is a developmentally important neuropeptide recognized to play a dominant role in social functioning and stress-related behaviors, in a sex-dependent manner. Nonetheless, the underlining factors driving OT and OT receptor (OTR) early brain development remain unclear. Recent evidence highlight the critical influence of gut microbiota and its bidirectional interaction with the brain on neurodevelopment via the gut microbiota-brain axis. Therefore, we aimed to determine the impact of gut microbiota on the OTR system of the rat brain at different developmental stages in a pilot study. Quantitative OTR [125 I]-OVTA autoradiographic binding was carried out in the forebrain of male and female conventional (CON) and germ-free (GF) rats at postnatal days (PND) 8, 22, and 116-150. OTR binding was also assessed in the eyes of PND 1 and PND 4 GF female rats. Significant "microbiota × sex × region" interaction and age-dependent effects on OTR binding were demonstrated. Microbiota status influenced OTR levels in males but not females with higher levels of OTR observed in GF versus CON rats in the cingulate, prelimbic, and lateral/medial/ventral orbital cortex, and septum across all age groups, while sex differences were observed in GF, but not in CON rats. Interestingly, OTRs present in the eyes of CON rats were abolished in GF rats. This is the first study to uncover a sex-specific role of gut microbiota on the central OTR system, which may have implications in understanding the developmental neuroadaptations critical for behavioral regulation and the etiology of certain neurodevelopmental disorders.

Structurally related peptide impurity identification and accurate quantification for synthetic oxytocin by liquid chromatography-high-resolution mass spectrometry.

Oxytocin (OXT) is an important peptide that is mainly used as a therapeutic drug to induce labor or strengthen uterine contractions, or to control bleeding after childbirth. OXT has also been reported as a biomarker linked to emotion, and as a potential biomarker for cancer diagnosis. The accurate purity characterization of OXT calibrators is critical for quality control of pharmaceuticals and the development of reference measurement systems for this analyte in laboratory medicine. OXT possesses the particular analytical measurement challenge of a disulfide bond. Accurate value assignment of the purity of oxytocin calibrators can be carried out by applying the mass balance approach or alternative approaches such as amino acid analysis, quantitative nuclear magnetic resonance spectrometry, and nitrogen determination. In order to avoid biases, all these approaches require a correction for structurally related peptide impurities. Structurally related peptide impurities present in a synthetic OXT material have been identified and quantified by a newly developed and in-house-validated liquid chromatography-high-resolution mass spectrometry (LC-hrMS) method. This method was adopted for the measurement of the study material used for an international comparison evaluating the competencies of laboratories to perform peptide characterization. Eighteen structurally related impurities were identified, confirmed, and accurately quantified in the OXT study material by using LC-hrMS. The study material contained a total mass fraction of 31.1 mg/g structurally related OXT impurities with an associated expanded uncertainty of 1.7 mg/g.

Effect of reduction and alkylation treatment in three different assays used for the measurement of oxytocin in saliva of pigs.

Oxytocin is a hormone that is increasingly being used for welfare evaluation in animals. Although several types of samples have been used for oxytocin measurement, saliva can be a suitable option for pigs producing less stress than blood sampling. In this study, 3 different methods for oxytocin measurements, 2 based on alphaLISA technology (one with a monoclonal and other with a polyclonal antibody) and one commercially available kit, were compared in saliva of pigs. These methods were used in saliva samples obtained from female pigs at 3 different days during gestation and lactation, with and without a reduction/alkylation (R/A), which is a procedure for breaking the links between oxytocin and proteins of the sample. The assays showed a different behavior after the R/A procedure, with no significant changes in the oxytocin results in case of the alphaLISA monoclonal method, a significant decrease with the alphaLISA polyclonal method, and a significant increase with the commercial kit. Although all assays showed a similar tendency in detecting the changes in oxytocin during gestation and lactation, they showed changes of different magnitude and statistical signification. This report indicates that different assays can measure different forms of oxytocin present in saliva and can have a different behavior after R/A of the sample and when are used to measure oxytocin in gestation and lactation.

Storage conditions of oxytocin in a tropical climate in a low-income country.

OBJECTIVE: To establish the storage conditions of oxytocin in a health facility in a low-income country with a tropical climate, as suboptimal storage may lead to ineffectiveness of drugs essential to prevent and treat postpartum hemorrhage. METHODS: At Mulago National Referral Hospital (28 000-33 000 deliveries/year) in Kampala, Uganda, temperature logging Safe-Rx cards were placed in boxes of oxytocin and in every known storage location. The route of the boxes through the hospital was tracked for 54 days, and storage conditions were observed. RESULTS: Oxytocin was stored within the recommended temperature range (2°C-8°C) 24% of the time. The average temperature measured within the oxytocin boxes was 18.2°C, with a minimum of -2.3°C and maximum of 30.4°C. Six out of twelve known storage places had a refrigerator, but not one location stored medication at the recommended temperature constantly. The average temperature in the storage places ranged from 9.5°C to 27.6°C, with a minimum temperature of 2.3°C and maximum of 30.9°C. CONCLUSION: Oxytocin is not stored in the recommended temperature range for the majority of time. The presence of refrigerators does not ensure adherence to advised temperature storage conditions.

New small molecule fluorescent probes for G protein-coupled receptors: valuable tools for drug discovery.

G protein-coupled receptors (GPCRs) are essential signaling proteins and tractable therapeutic targets. To develop new drug candidates, GPCR drug discovery programs require versatile, sensitive pharmacological tools for ligand binding and compound screening. With the availability of new imaging modalities and proximity-based ligand binding technologies, fluorescent ligands offer many advantages and are increasingly being used, yet labeling small molecules remains considerably more challenging relative to peptides. Focusing on recent fluorescent small molecule studies for family A GPCRs, this review addresses some of the key challenges, synthesis approaches and structure-activity relationship considerations, and discusses advantages of using high-resolution GPCR structures to inform conjugation strategies. While no single approach guarantees successful labeling without loss of affinity or selectivity, the choice of fluorophore, linker type and site of attachment have proved to be critical factors that can significantly affect their utility in drug discovery programs, and as discussed, can sometimes lead to very unexpected results.

Is Oxytocin "Nature's Medicine"?

Oxytocin is a pleiotropic, peptide hormone with broad implications for general health, adaptation, development, reproduction, and social behavior. Endogenous oxytocin and stimulation of the oxytocin receptor support patterns of growth, resilience, and healing. Oxytocin can function as a stress-coping molecule, an anti-inflammatory, and an antioxidant, with protective effects especially in the face of adversity or trauma. Oxytocin influences the autonomic nervous system and the immune system. These properties of oxytocin may help explain the benefits of positive social experiences and have drawn attention to this molecule as a possible therapeutic in a host of disorders. However, as detailed here, the unique chemical properties of oxytocin, including active disulfide bonds, and its capacity to shift chemical forms and bind to other molecules make this molecule difficult to work with and to measure. The effects of oxytocin also are context-dependent, sexually dimorphic, and altered by experience. In part, this is because many of the actions of oxytocin rely on its capacity to interact with the more ancient peptide molecule, vasopressin, and the vasopressin receptors. In addition, oxytocin receptor(s) are epigenetically tuned by experience, especially in early life. Stimulation of G-protein-coupled receptors triggers subcellular cascades allowing these neuropeptides to have multiple functions. The adaptive properties of oxytocin make this ancient molecule of special importance to human evolution as well as modern medicine and health; these same characteristics also present challenges to the use of oxytocin-like molecules as drugs that are only now being recognized. SIGNIFICANCE STATEMENT: Oxytocin is an ancient molecule with a major role in mammalian behavior and health. Although oxytocin has the capacity to act as a "natural medicine" protecting against stress and illness, the unique characteristics of the oxytocin molecule and its receptors and its relationship to a related hormone, vasopressin, have created challenges for its use as a therapeutic drug.

Stability of Oxytocin Preparations in Malawi and Rwanda: Stabilizing Effect of Chlorobutanol.

Oxytocin is used for the prevention and treatment of postpartum hemorrhage, the leading cause of maternal mortality in low- and middle-income countries. Because of the high instability of oxytocin, most products are labeled for storage at 2-8°C. Some other products are on the market which are labeled for non-refrigerated storage, but independent evaluations of their stability hardly exist. In the present study, seven brands (nine batches) of oxytocin were purchased from wholesalers and medical stores in Malawi and Rwanda and investigated by accelerated stability testing according to the ICH/WHO guidelines. Two oxytocin brands approved by a stringent regulatory authority (SRA) or by the WHO Prequalification of Medicines program and purchased in Europe were used as comparison. All investigated brands which were either produced in countries with SRAs, or were WHO-prequalified products, were labeled for storage at 2-8°C, and all of them passed stability testing with very good results. Even exposure to 25°C or 30°C for several months hardly affected their oxytocin content. However, two other investigated brands were labeled for non-refrigerated storage, and both of them had been produced in countries without SRAs. These two preparations showed not higher but lower stability than the brands labeled for storage at 2-8°C, and, for both of them, noncompliance with pharmacopoeial specifications was found after accelerated stability testing. At 40°C, and in forced degradation studies at 80°C, chlorobutanol showed a remarkable stabilizing effect on oxytocin, which may deserve further investigation. The results of the present study support the policy "Buy Quality Oxytocin, Keep It Cool."

Hormonal reactivity during martial arts practice among high-risk youths.

Martial arts have become a popular afterschool activity for youths across the globe. Accumulating data suggest that these activities may confer substantial cognitive and psychological benefits, and recent efforts have been made to introduce martial arts training into educational and rehabilitation settings. However, few studies have examined the potential mechanisms that may underlie these benefits. The current study evaluated the reactivity of two hormones, oxytocin (OT) and cortisol (CT), thought to be respectively involved in regulating mammalian social behaviors and responsivity to stress, to a session of intensive martial arts training in samples of at high-risk and low-risk (in regular educational establishments) youths. OT and CT were measured at baseline, during peak training, and following a cool down period. Analyses revealed that high-risk youths had lower OT but similar CT baseline levels, compared to low-risk youths, prior to the martial arts session. A significant group by time interaction indicated that whereas the OT levels among low-risk youths returned to baseline levels following training, OT levels among high-risk youths remained elevated. Finally, unlike low-risk youths for whom CT levels continued to increase throughout the training session, high-risk youths showed no significant CT reactivity. This study suggests that some of the beneficial effects of martial arts may be related to hormonal processes, especially increases in OT levels, and highlights the differing effects that training may have in different populations.

Mechanistic Insights into an Unusual Side-Chain-Mediated N-Cα Bond Cleavage under Collision-Induced Dissociation Conditions in the Disulfide-Containing Peptide Conopressin.

Conopressin, a nonapeptide disulfide CFIRNCPKG amide present in cone snail venom, undergoes a facile cleavage at the Cys6-Pro7 peptide bond to yield a disulfide bridged b6 ion. Analysis of the mass spectral fragmentation pattern reveals the presence of a major fragment ion, which is unambiguously assigned as the tripeptide sequence IRN amide. The sequence dependence of this unusual fragmentation process has been investigated by comparing it with the fragmentation patterns of related peptides, oxytocin (CYIQNCPLG amide), Lys-vasopressin (CYFQNCPKG amide), and a series of synthetic analogues. The results establish the role of the Arg4 residue in facilitating the unusual N-Cα bond cleavage at Cys6. Structures are proposed for a modified disulfide bridged fragment containing the Cys1 and Cys6 residues. Gas-phase molecular dynamics simulations provide evidence for the occurrence of conformational states that permit close approach of the Arg4 side chain to the Cys6 Cß methylene protons.

Oxytocin in saliva of pigs: an assay for its measurement and changes after farrowing.

Oxytocin is a hormone of interest in reproduction, but also in the field of psychology and behavior, being considered as a biomarker of positive emotions. Saliva can be a noninvasive way to measure oxytocin, which is very useful in species such as the pig where blood collection can produce a high degree of stress. In this study, a new assay for oxytocin measurement was developed, analytically validated, and used to measure possible changes in oxytocin in saliva of female pigs at different days after farrowing. The assay showed an adequate accuracy and precision and does not need a previous extraction step. In addition, oxytocin concentrations were significantly higher at day 1 of lactation than at day 9 after farrowing, but levels increased at day 20 again. This assay can contribute to a wider use of oxytocin measurements in pigs as it is a noninvasive sampling procedure that minimizes stress.

Evaluating changes in salivary oxytocin and cortisol following positive reinforcement training in two adult male western lowland gorillas (Gorilla gorilla gorilla).

Positive reinforcement training (PRT) is associated with increases in species-typical behavior and decreases in stereotypic and abnormal behavior in participating animals. Physiological changes following PRT, for example, increases in oxytocin (OXT) and/or decreases in cortisol (CORT), may facilitate these behavioral changes. This study evaluated salivary OXT and salivary CORT concentrations in two adult male western lowland gorillas (Gorilla gorilla gorilla) following PRT with their primary animal care staff. Following PRT, no change in OXT was observed. CORT decreased in one subject following PRT. Changes in endogenous OXT are related to affiliative interactions and interact with strongly bonded conspecifics. PRT may not activate the oxytocinergic system because PRT is not a species-specific affiliative interaction and/or animal care staff are not viewed as conspecifics. Regardless, PRT may still be viewed as a positive interaction resulting in stress reduction via a decrease in CORT. Relationships are unique, thus these results only apply to these two gorillas and one animal caregiver. Larger population-level studies are needed to understand overall trends in human-animal interactions, and ultimately human-animal relationships. Further evaluation of physiological changes following human-animal interactions should be informative for understanding the human-animal relationship in zoos.

I8-arachnotocin-an arthropod-derived G protein-biased ligand of the human vasopressin V2 receptor.

The neuropeptides oxytocin (OT) and vasopressin (VP) and their G protein-coupled receptors OTR, V1aR, V1bR, and V2R form an important and widely-distributed neuroendocrine signaling system. In mammals, this signaling system regulates water homeostasis, blood pressure, reproduction, as well as social behaviors such as pair bonding, trust and aggression. There exists high demand for ligands with differing pharmacological profiles to study the physiological and pathological functions of the individual receptor subtypes. Here, we present the pharmacological characterization of an arthropod (Metaseiulus occidentalis) OT/VP-like nonapeptide across the human OT/VP receptors. I8-arachnotocin is a full agonist with respect to second messenger signaling at human V2R (EC50 34 nM) and V1bR (EC50 1.2 µM), a partial agonist at OTR (EC50 790 nM), and a competitive antagonist at V1aR [pA2 6.25 (558 nM)]. Intriguingly, I8-arachnotocin activated the Gαs pathway of V2R without recruiting either ß-arrestin-1 or ß-arrestin-2. I8-arachnotocin might thus be a novel pharmacological tool to study the (patho)physiological relevance of ß-arrestin-1 or -2 recruitment to the V2R. These findings furthermore highlight arthropods as a novel, vast and untapped source for the discovery of novel pharmacological probes and potential drug leads targeting neurohormone receptors.

Leu8 and Pro8 oxytocin agonism differs across human, macaque, and marmoset vasopressin 1a receptors.

Oxytocin (OXT) is an important neuromodulator of social behaviors via activation of both oxytocin receptors (OXTR) and vasopressin (AVP) 1a receptors (AVPR1a). Marmosets are neotropical primates with a modified OXT ligand (Pro8-OXT), and this ligand shows significant coevolution with traits including social monogamy and litter size. Pro8-OXT produces more potent and efficacious responses at primate OXTR and stronger behavioral effects than the consensus mammalian OXT ligand (Leu8-OXT). Here, we tested whether OXT/AVP ligands show differential levels of crosstalk at primate AVPR1a. We measured binding affinities and Ca2+ signaling responses of AVP, Pro8-OXT and Leu8-OXT at human, macaque, and marmoset AVPR1a. We found that AVP binds with higher affinity than OXT across AVPR1a, and marmoset AVPR1a show a 10-fold lower OXT binding affinity compared to human and macaque AVPR1a. Both Leu8-OXT and Pro8-OXT produce a less efficacious response than AVP at human AVPR1a and higher efficacious response than AVP at marmoset AVPR1a. These data suggest that OXT might partially antagonize endogenous human AVPR1a signaling and enhance marmoset AVPR1a signaling. These findings aid in further understanding inconsistencies observed following systemic intranasal administration of OXT and provide important insights into taxon-specific differences in nonapeptide ligand/receptor coevolution and behavior.