A conserved peptide-binding pocket in HyNaC/ASIC ion channels.

The only known peptide-gated ion channels-FaNaCs/WaNaCs and HyNaCs-belong to different clades of the DEG/ENaC family. FaNaCs are activated by the short neuropeptide FMRFamide, and HyNaCs by Hydra RFamides, which are not evolutionarily related to FMRFamide. The FMRFamide-binding site in FaNaCs was recently identified in a cleft atop the large extracellular domain. However, this cleft is not conserved in HyNaCs. Here, we combined molecular modeling and site-directed mutagenesis and identified a putative binding pocket for Hydra-RFamides in the extracellular domain of the heterotrimeric HyNaC2/3/5. This pocket localizes to only one of the three subunit interfaces, indicating that this trimeric ion channel binds a single peptide ligand. We engineered an unnatural amino acid at the putative binding pocket entrance, which allowed covalent tethering of Hydra RFamide to the channel, thereby trapping the channel in an open conformation. The identified pocket localizes to the same region as the acidic pocket of acid-sensing ion channels (ASICs), which binds peptide ligands. The pocket in HyNaCs is less acidic, and both electrostatic and hydrophobic interactions contribute to peptide binding. Collectively, our results reveal a conserved ligand-binding pocket in HyNaCs and ASICs and indicate independent evolution of peptide-binding cavities in the two subgroups of peptide-gated ion channels.

A Neuropeptide Signaling Network That Regulates Developmental Timing and Systemic Growth in Drosophila.

Animals sense chemical cues such as nutritious and noxious stimuli through the chemosensory system and adapt their behavior, physiology, and developmental schedule to the environment. In the Drosophila central nervous system, chemosensory interneurons that produce neuropeptides called Hugin (Hug) peptides receive signals from gustatory receptor neurons and regulate feeding behavior. Because Hug neurons project their axons to the higher brain region within the protocerebrum where dendrites of multiple neurons producing developmentally important neuropeptides are extended, it has been postulated that Hug neurons regulate development through the neuroendocrine system. In this study, we show that Hug neurons interact with a subset of protocerebrum neurons that produce prothoracicotropic hormone (PTTH) and regulate the onset of metamorphosis and systemic growth. Loss of the hug gene and silencing of Hug neurons caused a delay in larval-to-prepupal transition and an increase in final body size. Furthermore, deletion of Hug receptor-encoding genes also caused developmental delay and body size increase, and the phenotype was restored by expressing Hug receptors in PTTH-producing neurons. These results indicate that Hug neurons regulate developmental timing and body size via PTTH-producing neurons. This study provides a basis for understanding how chemosensation is converted into neuroendocrine signaling to control insect growth and development.

Rabphilin-3A negatively regulates neuropeptide release, through its SNAP25 interaction.

Neuropeptides and neurotrophins are stored in and released from dense core vesicles (DCVs). While DCVs and synaptic vesicles (SVs) share fundamental SNARE/SM proteins for exocytosis, a detailed understanding of DCV exocytosis remains elusive. We recently identified the RAB3-RIM1 pathway to be essential for DCV, but not SV exocytosis, highlighting a significant distinction between the SV and DCV secretory pathways. Whether RIM1 is the only RAB3 effector that is essential for DCV exocytosis is currently unknown. In this study, we show that rabphilin-3A (RPH3A), a known downstream effector of RAB3A, is a negative regulator of DCV exocytosis. Using live-cell imaging at single-vesicle resolution with RPH3A deficient hippocampal mouse neurons, we show that DCV exocytosis increased threefold in the absence of RPH3A. RAB3A-binding deficient RPH3A lost its punctate distribution, but still restored DCV exocytosis to WT levels when re-expressed. SNAP25-binding deficient RPH3A did not rescue DCV exocytosis. In addition, we show that RPH3A did not travel with DCVs, but remained stationary at presynapses. RPH3A null neurons also had longer neurites, which was partly restored when ablating all regulated secretion with tetanus neurotoxin. Taken together, these results show that RPH3A negatively regulates DCV exocytosis, potentially also affecting neuron size. Furthermore, RAB3A interaction is required for the synaptic enrichment of RPH3A, but not for limiting DCV exocytosis. Instead, the interaction of RPH3A with SNAP25 is relevant for inhibiting DCV exocytosis.

Alterations of pleiotropic neuropeptide-receptor gene couples in Cetacea.

BACKGROUND: Habitat transitions have considerable consequences in organism homeostasis, as they require the adjustment of several concurrent physiological compartments to maintain stability and adapt to a changing environment. Within the range of molecules with a crucial role in the regulation of different physiological processes, neuropeptides are key agents. Here, we examined the coding status of several neuropeptides and their receptors with pleiotropic activity in Cetacea. RESULTS: Analysis of 202 mammalian genomes, including 41 species of Cetacea, exposed an intricate mutational landscape compatible with gene sequence modification and loss. Specifically for Cetacea, in the 12 genes analysed we have determined patterns of loss ranging from species-specific disruptive mutations (e.g. neuropeptide FF-amide peptide precursor; NPFF) to complete erosion of the gene across the cetacean stem lineage (e.g. somatostatin receptor 4; SSTR4). CONCLUSIONS: Impairment of some of these neuromodulators may have contributed to the unique energetic metabolism, circadian rhythmicity and diving response displayed by this group of iconic mammals.

Isolation and characterization of NGFFYamide neuropeptide from Patiria pectinifera pyloric caeca extract.

Neuropeptides are small molecules that mediate intercellular signaling and regulate physiological processes. Starfish possess various myoactive neuropeptides, including starfish myorelaxant peptide (SMP) and a calcitonin-type peptide with apical muscle relaxing properties. In this study, we report the purification of a neuropeptide from starfish (Patiria pectinifera) pyloric caeca extract using high-performance liquid chromatography (HPLC) and an in vitro bioassay to screen for fractions and peptides with relaxing effects on P. pectinifera apical muscle preparations. A series of HPLC steps using reversed-phase and cation-exchange columns yielded a purified peptide with muscle-relaxing effects. The purified peptide's structure was determined by LC-MS and Edman degradation, revealing a pentapeptide with an amidated C-terminus (NGFFYamide) and a molecular mass of 646.2930 Da. This is the first report of NGFFYamide purification from P. pectinifera through biochemical methods. The nucleotide sequence encoding the NGFFYamide precursor was determined, showing the presence of a conserved neurophysin domain in the C-terminal region. RT-qPCR results confirmed high expression in radial nerves cord, consistent with previous findings on NG peptides in echinoderms. In vitro pharmacological studies on muscle preparations from P. pectinifera and Asterias amurensis revealed differential relaxing activity of NGFFYamide on apical muscles, while its effects on tube foot preparations were similar in both species. NGFFYamide also induced potent contraction in P. pectinifera cardiac stomach. Comparison of three NG peptides (NGFFYamide, NGFFFamide, and NGIWYamide) on P. pectinifera cardiac stomach revealed varying potency, suggesting class-specific receptor interactions. Tachyphylaxis was observed in P. pectinifera apical muscle but not in A. amurensis, warranting further investigation. Based on these results, it is plausible that NGFFYamide could be involved in regulating locomotion and feeding behavior in P. pectinifera, consistent with findings in Asterias rubens.

An evolutionarily conserved pathway mediated by neuroparsin-A regulates reproductive plasticity in ants.

Phenotypic plasticity displayed by an animal in response to different environmental conditions is supposedly crucial for its survival and reproduction. The female adults of some ant lineages display phenotypic plasticity related to reproductive role. In pharaoh ant queens, insemination induces substantial physiological/behavioral changes and implicates remarkable gene regulatory network (GRN) shift in the brain. Here, we report a neuropeptide neuroparsin A (NPA) showing a conserved expression pattern associated with reproductive activity across ant species. Knock-down of NPA in unmated queen enhances ovary activity, whereas injection of NPA peptide in fertilized queen suppresses ovary activity. We found that NPA mainly affected the downstream gene JHBP in the ovary, which is positively regulated by NPA and suppression of which induces elevated ovary activity, and shadow which is negatively regulated by NPA. Furthermore, we show that NPA was also employed into the brain-ovary axis in regulating the worker reproductive changes in other distantly related species, such as Harpegnathos venator ants.

Ion transport peptide and ion transport peptide-like regulate ecdysis behavior and water transport during ecdysis in Gryllus bimaculatus.

Ion transport peptide (ITP) and ITP-like (ITPLs) are pleiotropic bioactive peptides in insects. Although the contribution of these peptides to ecdysis has been studied, the precise regulatory mechanisms remain poorly understood. Here, we characterized the functions of itp and itpl variants in the two-spotted cricket, Gryllus bimaculatus. Reverse transcription-quantitative PCR and whole-mount in situ hybridization revealed that itp was expressed in the brain and terminal abdominal ganglion, whereas itpl variants were expressed in all ganglia of the central nervous system. Simultaneous knockdown of itp and itpls disrupted ecdysis behavior and water transport from the gut into the hemolymph during molting. Nevertheless, knockdown of itpls without influencing itp expression did not significantly affect ecdysis behavior but caused a reduction in hemolymph mass. Although water transport into the hemolymph is considered necessary for the swelling required to split the old cuticle layers during molting, a rescue experiment by injection of water or cricket Ringer's solution into the hemolymph of knockdown crickets did not recover the normal phenotype. Therefore, we propose that ITP/ITPL control ecdysis behavior probably not by regulating water transport from the gut into the hemolymph in crickets.

Anthropogenic electromagnetic radiation alters the transcription levels of the genes encoding the SIFamide and myoinhibitory peptide and their receptors in Ixodes ricinus synganglion.

The research of the influences of man-made electromagnetic fields on tick physiology has been very sparse and long neglected since the pioneer studies published in 1996 and 2000. Once multiple behavioral tests confirmed an attraction and possible perception of electromagnetic fields in ticks, a new interest in this topic erupted in recent years. In this study, qRT-PCR is utilized to determine the changes in the mRNA transcript levels of neuropeptides SIFamide and myoinhibitory peptide (mip and sifa) and their representative receptors (mip-r1 and sifa-r1) in the synganglia of the tick Ixodes ricinus irradiated by 900 MHz radiofrequency electromagnetic field. It was determined that 40 V/m intensity has a significant suppressory effect on the transcript levels of all genes after at least 60 minutes of constant exposure in both sexes. Commonly occurring intensity of radiation in urban areas (2 V/m) produced an elevation in mRNA levels after various timespans in every gene. A significant decrease of transcript abundances was detected in females after one hour of exposure to 2 V/m. Results of this study widen the knowledge of EMF-induced alterations in the neurophysiology of I. ricinus, the most commonly distributed hard tick in Europe.

Discovery of a neuropeptide that acts as an autotomy-promoting factor.

One of the most remarkable adaptations to survive attacks from predators is to detach an appendage-a process known as autotomy. This occurs in a variety of animals, including lizards (tail), crabs (legs), and starfish (arms). There has been extensive investigation of the evolution, ecology, and biomechanical impact of autotomy,1,2,3 but little is known about neural mechanisms controlling autotomy in animals. However, evidence for the existence of a peptide that acts as an autotomy-promoting factor in starfish has been reported.4 While investigating in vivo effects of a sulfakinin/cholecystokinin-type neuropeptide (ArSK/CCK1) in the starfish Asterias rubens,5,6 we observed that this peptide triggered arm autotomy in some animals. Furthermore, when injection of ArSK/CCK1 was combined with mechanical clamping of an arm, autotomy of the clamped arm occurred in 85% of animals tested, with 46% also autotomizing one or more other arms. In contrast, no autotomy was observed in clamped animals that were injected with water (control). To examine the physiological relevance of these findings, we analyzed expression of ArSK/CCK1 in the autotomy plane, a specialized region at the base of the arms in A. rubens.7,8 In accordance with its in vivo effects, nerve fibers expressing ArSK/CCK1 were revealed in the tourniquet muscle, a band of muscle that mediates constriction of the arm during and after autotomy. We conclude that ArSK/CCK1 acts as an autotomy-promoting factor in starfish and as such it is the first neuropeptide to be identified as a regulator of autotomy in animals.

Allatotropin (AT) related peptides L-ATRP and D2-ATRP diastereomers activate an endogenous receptor and suppress heart rate in the Pacific abalone Haliotis discus hannai.

Allatotropin (AT) has been identified in many insects and plays important roles in the regulation of their intestinal contraction, heart rate, ion transport, and digestive enzyme secretion. However, information on AT-related bioinformatics in other animal phyla is scarce. In this study, we cloned a full-length cDNA encoding the AT-related peptide receptor (ATRPR) of the abalone Haliotis discus hannai (Hdh) and further characterized Hdh-ATRPR with its potential ligands, Hdh-ATRPs. In luciferase reporter and Ca2+ mobilization assays, Hdh-ATRPs, including a D-type Phe at the second amino acid position, Hdh-D2-ATRP, activated Hdh-ATRPR in a dose-dependent manner, whereas all-L-type Hdh-ATRP was a more potent ligand than Hdh-D2-ATRP. Furthermore, Hdh-ATRPs induced ERK1/2 phosphorylation in Hdh-ATRPR-expressing HEK293 cells, which was dose-dependently abolished by the PKC inhibitor Gö6983. The heart rate decreased significantly within 10 min when Hdh-D2-ATRP was injected into the adduct muscle sinus of abalone (0.2 or 1.0 µg/g body weight), while the abalone injected with a high concentration of Hdh-D2-ATRP (1.5 µg/g body weight) were sublethal within 5 h. Thus, Hdh-ATRP signaling is primarily linked to the Gαq/PKC and is possibly associated with heart rate regulation in abalone.

Reconsidering Neurogenetic Indication in the Human Brain: Broad Expression of Doublecortin Transcript in the Hippocampal and Cortical Cell Populations.

INTRODUCTION: Neurogenesis in the adult brain may play an important role in memory and cognition; however, knowledge of neurogenic markers in the human brain remains limited. We compared the single-nucleus transcriptome of the hippocampus with that of other cortical regions to identify hippocampus-specific neurogenic markers. METHODS: We analyzed 26,189 nuclei from four human brains collected within 16 h of death. Clustering and annotation were performed to examine differential expression, gene ontology, and intercellular communication. DCX expression was validated by ddPCR. RESULTS: Immature markers such as DCX, CALB2, NES, SOX2, PAX6, DPYSL3, and TUBB3 were expressed in both hippocampus and prefrontal cortex, with higher levels in the prefrontal cortex. ddPCR confirmed higher expression of DCX in the prefrontal cortex. DCX was involved in both neurogenesis and neuroprotection pathways. CONCLUSION: Neurogenic markers are not definitive indicators of adult neurogenesis as their roles are more complex than previously understood.

Neuritin Controls Axonal Branching in Serotonin Neurons: A Possible Mediator Involved in the Regulation of Depressive and Anxiety Behaviors via FGF Signaling.

Abnormal neuronal morphological features, such as dendrite branching, axonal branching, and spine density, are thought to contribute to the symptoms of depression and anxiety. However, the role and molecular mechanisms of aberrant neuronal morphology in the regulation of mood disorders remain poorly characterized. Here, we show that neuritin, an activity-dependent protein, regulates the axonal morphology of serotonin neurons. Male neuritin knock-out (KO) mice harbored impaired axonal branches of serotonin neurons in the medial prefrontal cortex and basolateral region of the amygdala (BLA), and male neuritin KO mice exhibited depressive and anxiety-like behaviors. We also observed that the expression of neuritin was decreased by unpredictable chronic stress in the male mouse brain and that decreased expression of neuritin was associated with reduced axonal branching of serotonin neurons in the brain and with depressive and anxiety behaviors in mice. Furthermore, the stress-mediated impairments in axonal branching and depressive behaviors were reversed by the overexpression of neuritin in the BLA. The ability of neuritin to increase axonal branching in serotonin neurons involves fibroblast growth factor (FGF) signaling, and neuritin contributes to FGF-2-mediated axonal branching regulation in vitro. Finally, the oral administration of an FGF inhibitor reduced the axonal branching of serotonin neurons in the brain and caused depressive and anxiety behaviors in male mice. Our results support the involvement of neuritin in models of stress-induced depression and suggest that neuronal morphological plasticity may play a role in controlling animal behavior.

Functional expression and characterization of CAPA receptor in the digestive tract and life stages of Drosophila suzukii, and differential activities with insect PRXamide peptides.

Spotted-wing drosophila, Drosophila suzukii (Matsumura), is an invasive vinegar fly that is a major threat to the small fruits industries globally. Insect capa genes encode multiple neuropeptides, including CAPA-periviscerokinin (CAPA-PVK) peptides, that are specifically known to cause diuresis or anti-diuresis in various organisms. Here we identified and characterized a corresponding G protein-coupled receptor (GPCR) of the D. suzukii CAPA-PVK peptides: CAPA receptor (CAPA-R). To better characterize the behavior of D. suzukii CAPA-R, we used insect cell-based functional expression assays to evaluate responses of CAPA-R against D. suzukii CAPA-PVKs, CAPA-PVKs from five species in Insecta, one species from Mollusca, modified CAPA-PVK peptides, and some PRXamide family peptides: pyrokinin (PK), diapause hormone (DH), and ecdysis-triggering hormone (ETH). Functional studies revealed that the D. suzukii CAPA-R is strongly activated by both of its own natural D. suzukii CAPA-PVKs, and interestingly, it was strongly activated by other CAPA-PVK peptides from Frankliniella occidentallis (Thysanoptera), Solenopsis invicta (Hymenoptera), Helicoverpa zea (Lepidoptera) and Plutella xylostella (Lepidoptera). However, D. suzukii CAPA-R was not activated by Mollusca CAPA-PVK or the other PRXamide peptides. Gene expression analyses showed that the CAPA-R was highly expressed in the Malpighian tubules and moderately in hindgut compared to other digestive organs or the rest of body, supporting diuretic/antidiuretic functionality. When compared across life stages of D. suzukii, expression of CAPA-R was approximately 1.5x greater in the third instar than the other stages and minimally detected in the eggs, 4-day old pupae and 3-day old adults. Our results functionally characterized the D. suzukii CAPA-R and a few short peptides were identified as potential biological targets to exploit the CAPA-R for D. suzukii management.

A new type II CHH neuropeptide involves ovarian development in the peppermint shrimp, Lysmata vittata.

Type II crustacean hyperglycemic hormone (CHH) neuropeptides play diverse roles in crustaceans. In the hermaphrodite shrimp Lysmata vittata, two transcripts of type II CHHs (molt-inhibiting hormone/gonad-inhibiting hormone, MIH/GIH1 and MIH/GIH2) were identified by transcriptome sequencing, and MIH/GIH1 was later named Lvit-GIH1 for its inhibitory effect on ovarian development. Based on the high similarity of MIH/GIH2 to Lvit-GIH1, we named tentatively MIH/GIH2 as Lvit-GIH2 and explored the role of Lvit-GIH2 in ovarian development. The open reading frame (ORF) of Lvit-GIH2 was 333 bp in length, encoding a precursor consisted of a 32-aa signal peptide and a 78-aa mature peptide, which shared high sequence similarity with the type II subfamily peptides in crustaceans. Notably, Lvit-GIH2 was widely expressed in multiple tissues. The qRT-PCR findings indicated a rising trend in the expression of Lvit-GIH2 from the male phase to the euhermaphrodite phase. Both RNA interference and addition of GIH2 recombinant proteins (rGIH2) experiments showed that Lvit-GIH2 suppressed Lvit-Vg expression in hepatopancreas and Lvit-VgR expression in ovary. To further investigate the role of Lvit-GIH2 in ovarian development, the RNA-sequence analysis was performed to examine the changes in ovary after addition of rGIH2. The results showed that the pathways (Cysteine and methionine metabolism, Apoptosis-multiple species, etc.) and the genes (17bHSD8, IGFR, CHH, etc.) related to ovarian development were negatively regulated by rGIH2. In brief, Lvit-GIH2 might inhibit the ovarian development in L. vittata.

Neuritin accelerates Schwann cell dedifferentiation via PI3K/Akt/mTOR signalling pathway during Wallerian degeneration.

Neuritin, also known as candidate plasticity gene 15 (CPG15), was first identified as one of the activity-dependent gene products in the brain. Previous studies have been reported that Neuritin induces neuritogenesis, neurite arborization, neurite outgrowth and synapse formation, which are involved in the development and functions of the central nervous system. However, the role of Neuritin in peripheral nerve injury is still unknown. Given the importance and necessity of Schwann cell dedifferentiation response to peripheral nerve injury, we aim to investigate the molecular mechanism of Neuritin steering Schwann cell dedifferentiation during Wallerian degeneration (WD) in injured peripheral nerve. Herein, using the explants of sciatic nerve, an ex vivo model of nerve degeneration, we provided evidences indicating that Neuritin vividly accelerates Schwann cell dedifferentiation. Moreover, we found that Neuritin promotes Schwann cell demyelination as well as axonal degeneration, phagocytosis, secretion capacity. In summary, we first described Neuritin acts as a positive regulator for Schwann cell dedifferentiation and WD after peripheral nerve injury.

Characterization of corazonin signaling in a molluscan model species, Lymnaea stagnalis.

In recent years, new concepts have emerged regarding the nomenclature, functions, and relationships of different peptide families of the gonadotropin-releasing hormone (GnRH) superfamily. One of the main driving forces for this originated from the emerging evidence that neuropeptides previously called molluscan GnRH are multifunctional and should be classified as corazonin (CRZ). However, research articles still appear that use incorrect nomenclature and attribute the same function to molluscan CRZs as vertebrate GnRHs. The aim of the present study was to further support the recent interpretation of the origin and function of the GnRH superfamily. Towards this goal, we report the characterization of CRZ signaling system in the molluscan model species, the great pond snail (Lymnaea stagnalis). We detected a CRZ-receptor-like sequence (Lym-CRZR) by homology-searching in the Lymnaea transcriptomes and the deduced amino acid sequence showed high sequence similarity to GnRH receptors and CRZ receptors. Molecular phylogenetic tree analysis demonstrated that Lym-CRZR is included in the cluster of molluscan CRZRs. Lym-CRZR transiently transfected into HEK293 cells was found to be localized at the plasma membrane, confirming that it functions as a membrane receptor, like other G protein-coupled receptors. The signaling assays revealed that the previously identified Lym-CRZ neuropeptide stimulated intracellular Ca2+ mobilization in a dose-dependent manner, but not cyclic AMP production, in HEK293 cells transfected with Lym-CRZR. Finally, we demonstrated a wide tissue distribution of Lym-CRZR. These results suggest that Lym-CRZ is a multifunctional peptide and provide further insights into the evolution of the GnRH neuropeptide superfamily. The present study also supports the notion that previously termed molluscan "GnRH" should be classified as "CRZ".

Proteome-wide neuropeptide identification using NeuroPeptide-HMMer (NP-HMMer).

Neuropeptides are essential neuronal signaling molecules that orchestrate animal behavior and physiology via actions within the nervous system and on peripheral tissues. Due to the small size of biologically active mature peptides, their identification on a proteome-wide scale poses a significant challenge using existing bioinformatics tools like BLAST. To address this, we have developed NeuroPeptide-HMMer (NP-HMMer), a hidden Markov model (HMM)-based tool to facilitate neuropeptide discovery, especially in underexplored invertebrates. NP-HMMer utilizes manually curated HMMs for 46 neuropeptide families, enabling rapid and accurate identification of neuropeptides. Validation of NP-HMMer on Drosophila melanogaster, Daphnia pulex, Tribolium castaneum and Tenebrio molitor demonstrated its effectiveness in identifying known neuropeptides across diverse arthropods. Additionally, we showcase the utility of NP-HMMer by discovering novel neuropeptides in Priapulida and Rotifera, identifying 22 and 19 new peptides, respectively. This tool represents a significant advancement in neuropeptide research, offering a robust method for annotating neuropeptides across diverse proteomes and providing insights into the evolutionary conservation of neuropeptide signaling pathways.

Unlocking opioid neuropeptide dynamics with genetically encoded biosensors.

Neuropeptides are ubiquitous in the nervous system. Research into neuropeptides has been limited by a lack of experimental tools that allow for the precise dissection of their complex and diverse dynamics in a circuit-specific manner. Opioid peptides modulate pain, reward and aversion and as such have high clinical relevance. To illuminate the spatiotemporal dynamics of endogenous opioid signaling in the brain, we developed a class of genetically encoded fluorescence sensors based on kappa, delta and mu opioid receptors: κLight, δLight and µLight, respectively. We characterized the pharmacological profiles of these sensors in mammalian cells and in dissociated neurons. We used κLight to identify electrical stimulation parameters that trigger endogenous opioid release and the spatiotemporal scale of dynorphin volume transmission in brain slices. Using in vivo fiber photometry in mice, we demonstrated the utility of these sensors in detecting optogenetically driven opioid release and observed differential opioid release dynamics in response to fearful and rewarding conditions.

Identification of ETH receptor and its possible roles in the mud crab Scylla paramamosain.

Ecdysis-triggering hormone (ETH) is a neuropeptide hormone characterized by a conserved KxxKxxPRx amide structure widely identified in arthropods. While its involvement in the regulation of molting and reproduction in insects is well-established, its role in crustaceans has been overlooked. This study aimed to de-orphanise a receptor for ETH in the mud crab Scylla paramamosain and explore its potential impact on ovarian development. A 513-amino-acid G protein-coupled receptor for ETH (SpETHR) was identified in S. paramamosain, exhibiting a dose-dependent activation by SpETH with an EC50 value of 75.18 nM. Tissue distribution analysis revealed SpETH was in the cerebral ganglion and thoracic ganglion, while SpETHR was specifically expressed in the ovary, hepatopancreas, and Y-organ of female crabs. In vitro experiments demonstrated that synthetic SpETH (at a concentration of 10-8 M) significantly increased the expression of SpVgR in the ovary and induced ecdysone biosynthesis in the Y-organ. In vivo experiments showed a significant upregulation of SpEcR in the ovary and Disembodied and Shadow in the Y-organ after 12 h of SpETH injection. Furthermore, a 16-day administration of SpETH significantly increased 20E titers in hemolymph, gonadosomatic index (GSI) and oocyte size of S. paramamosain. In conclusion, our findings suggest that SpETH may play stimulatory roles in ovarian development and ecdysone biosynthesis by the Y-organ.

Analysis of Neuropeptides in the Intestinal Mucus of Patients with Ulcerative Colitis Using RNA Sequencing.

INTRODUCTION: Inflammation in ulcerative colitis (UC) originates in the colorectal mucosa. Transcriptome sequencing analysis of the colorectal mucosa allows the identification of potential neuropeptides related to local neurotransmission. The intestinal mucus lining the surface of the mucosa may harbor biomarkers of mucosal inflammation; however, this has not been sufficiently investigated, given the difficulty in obtaining human samples. We previously reported the feasibility of obtaining mucin samples for proteomic analysis by brushing during colonoscopy. Herein, we aimed to investigate the composition of the intestinal mucus and detect neuropeptides characteristic of UC. METHODS: Mucus and mucosal samples were collected from patients with UC from the colorectum in areas showing remission or active UC using a brush catheter and biopsy forceps during colonoscopy. RNA sequencing findings of mucus samples of active and remission areas were compared. RNA and protein expression levels of significantly upregulated neuropeptides were analyzed. RESULTS: Of the neuropeptides associated with UC, somatostatin (SST) was significantly elevated in areas of remission, according to RNA sequencing results of mucus and expression levels in mucus RNA and proteins. Conversely, SST expression in the mucosa was increased in the inflamed areas. Flow cytometry revealed that the fluorescence intensity of SST-positive cells in the remission zone was higher in the mucus than in the mucosa. CONCLUSION: SST expression in the mucus is considered to be an important factor associated with UC activity.