Programmed Death-Ligand (PD-L1), Epidermal Growth Factor (EGF), Relaxin, and Matrix Metalloproteinase-3 (MMP3): Potential Biomarkers of Malignancy in Canine Mammary Neoplasia.

Gene expression has been suggested as a putative tool for prognosis and diagnosis in canine mammary neoplasia (CMNs). In the present study, 58 formalin-fixed paraffin-embedded (FFPE) paraffined canine mammary neoplasias from 27 different bitches were included. Thirty-seven tumours were classified as benign, whereas thirty-one were classified as different types of canine carcinoma. In addition, mammary samples from three healthy bitches were also included. The gene expression for vascular endothelial growth factor-α (VEGFα), CD20, progesterone receptor (PGR), hyaluronidase-1 (HYAL-1), programmed death-ligand 1 (PD-L1), epidermal growth factor (EGF), relaxin (RLN2), and matrix metalloproteinase-3 (MMP3) was assessed through RT-qPCR. All the assessed genes yielded a higher expression in neoplastic mammary tissue than in healthy tissue. All the evaluated genes were overexpressed in neoplastic mammary tissue, suggesting a role in the process of tumorigenesis. Moreover, PD-L1, EGF, relaxin, and MMP3 were significantly overexpressed in malignant CMNs compared to benign CMNs, suggesting they may be useful as malignancy biomarkers.

Specific expression and blood kinetics for relaxin 2, lipocalin 2, and tissue factor pathway inhibitor 2 at the canine placenta and pregnant bloods.

In general, humoral factors released from the placenta influence pregnancy progression, but the involvement of the canine placenta is often unidentified. We investigated specific genes in canine placentas and analyzed the blood dynamics of the translated proteins. Furthermore, RNAs are known to be released from placentas embedding in exosomes, a type of extracellular vesicles. Here, the presence of cell-free RNAs in pregnant serums was also confirmed. RNA specimens were purified from the normal healthy dog placentas and applied to RNA-Seq analysis. Expressions of frequent genes were confirmed by RT-PCR using placentas from other individuals and breeds. Relaxin (RLN) 2, lipocalin (LCN) 2, and tissue factor pathway inhibitor (TFPI) 2 were selected as high-expressed and placenta-specific genes. By western blot, the three factors were clearly detected in the pregnant serums. Quantitative analysis revealed that the amount of RLN2 increased significantly from non-pregnancy to day 41 of pregnancy. Regarding LCN2 and TFPI2, the protein serum levels elevated during pregnancy, but the statistical differences were not detected. Exosomes were found in all pregnant serums; however, the percentage was less than 6% in total extracellular vesicles. The cell-free RNA related to RLN2 was detected, but no elevation was confirmed during pregnancy. We found specific genes in the canine placenta and the transition of their translated protein into the blood. These factors may become useful tools for research on canine pregnancy and monitoring of reproductive management. Exosomes and cell-free RNA could not be found to be valid in canine reproduction.

Investigating the role of the relaxin-3/RXFP3 system in neuropsychiatric disorders and metabolic phenotypes: A candidate gene approach.

The relaxin-3/RXFP3 system has been implicated in the modulation of depressive- and anxiety-like behaviour in the animal literature; however, there is a lack of human studies investigating this signalling system. We seek to bridge this gap by leveraging the large UK Biobank study to retrospectively assess genetic risk variants linked with this neuropeptidergic system. Specifically, we conducted a candidate gene study in the UK Biobank to test for potential associations between a set of functional, candidate single nucleotide polymorphisms (SNPs) pertinent to relaxin-3 signalling, determined using in silico tools, and several outcomes, including depression, atypical depression, anxiety and metabolic syndrome. For each outcome, we used several rigorously defined phenotypes, culminating in subsample sizes ranging from 85,881 to 386,769 participants. Across all outcomes, there were no associations between any candidate SNP and any outcome phenotype, following corrections for multiple testing burden. Regression models comprising several SNPs per relevant candidate gene as exploratory variables further exhibited no prediction of outcome. Our findings corroborate conclusions from previous literature about the limitations of candidate gene approaches, even when based on firm biological hypotheses, in the domain of genetic research for neuropsychiatric disorders.

Integrative stemness characteristics associated with prognosis and the immune microenvironment in lung adenocarcinoma.

BACKGROUND: To comprehensively analyze the stemness characteristics related to prognosis and the immune microenvironment in lung adenocarcinoma (LUAD). METHODS: The OCLR machine learning method was used to calculate the stemness index (mRNAsi) of the LUAD samples. DEGs common between the low mRNAsi, normal, and high mRNAsi groups were screened and the immune-stemness genes were obtained. Then the PPI network was created and enrichment analyses were performed. Moreover, different subtypes based on immune-stemness genes associated with prognosis were identified, and the relationships between LUAD stemness and TIME variables were systematically analyzed, followed by TMB analysis. RESULTS: Patients in the high mRNAsi groups with poor prognosis were screened along with 144 immune-stemness genes. IL-6, FPR2, and RLN3 showed a higher degree in the PPI network. A total of 26 immune-stemness genes associated with prognosis were screened. Two clusters were obtained (cluster 1 and cluster 2). Survival analysis revealed that patients in cluster 2 had a poor prognosis. A total of 12 immune cell subpopulations exhibited significant differences between cluster 1 and cluster 2 (P < 0.05). A total of 10 immune checkpoint genes exhibited significantly higher expression in cluster 1 (P < 0.05) than in cluster 2. Further, the TMB value in cluster 2 was higher than that in cluster 1 (P < 0.05). CONCLUSION: Immune-stemness genes, including L-6, FPR2, and RLN3, might play significant roles in LUAD development via cytokine-cytokine receptor interaction, neuroactive ligand‒receptor interaction, and the JAK‒STAT pathway. Immune-stemness genes were related to tumor-infiltrating immune cells, TMB, and expression of immune checkpoint gene.

Comparative Genomic Characterization of Relaxin Peptide Family in Cattle and Buffalo.

Relaxin family peptides significantly regulate reproduction, nutrient metabolism, and immune response in mammals. The present study aimed to identify and characterize the relaxin family peptides in cattle and buffalo at the genome level. The genomic and proteomic sequences of cattle, buffalo, goat, sheep, horse, and camel were accessed through the NCBI database, and BLAST was performed. We identified four relaxin peptides genes (RLN3, INSL3, INSL5, and INSL6) in Bos taurus, whereas three relaxin genes (RLN3, INSL3, and INSL6) in Bubalus bubalis. Evolutionary analysis revealed the conserved nature of relaxin family peptides in buffalo and cattle. Physicochemical properties revealed that relaxin proteins were thermostable, hydrophilic, and basic peptides except for INSL5 which was an acidic peptide. Three nonsynonymous mutations (two in RLN3 at positions A16 > T and P29 > A, and one in INSL6 at position R32 > Q) in Bos taurus, whereas two nonsynonymous mutations (one in RLN3 at positions G105 > w and one in INSL3 at position G22 > R) in Bubalus bubalis, were identified. INSL3 had one indel (insertion) at position 55 in Bos taurus. Gene duplication analysis revealed predominantly segmental duplications (INSL5/RLN3 and INSL6/INSL3 gene pairs) that helped expand this gene family, whereas Bubalus bubalis showed primarily tandem duplication (INSL3/RLN3). Our study concluded that relaxin family peptides remained conserved during the evolution, and gene duplications might help to adapt and enrich specific functions like reproduction, nutrient metabolism, and immune response. Further, the nonsynonymous mutations identified potentially affect these functions in buffalo.

Relaxin Affects Airway Remodeling Genes Expression through Various Signal Pathways Connected with Transcription Factors.

Fibrosis is one of the parameters of lung tissue remodeling in asthma. Relaxin has emerged as a natural suppressor of fibrosis, showing efficacy in the prevention of a multiple models of fibrosis. Therefore, the aim of this study was to analyze the aptitudes of relaxin, in the context of its immunomodulatory properties, in the development of airway remodeling. WI-38 and HFL1 fibroblasts, as well as epithelial cells (NHBE), were incubated with relaxin. Additionally, remodeling conditions were induced with two serotypes of rhinovirus (HRV). The expression of the genes contributing to airway remodeling were determined. Moreover, NF-κB, c-Myc, and STAT3 were knocked down to analyze the pathways involved in airway remodeling. Relaxin decreased the mRNA expression of collagen I and TGF-ß and increased the expression of MMP-9 (p < 0.05). Relaxin also decreased HRV-induced expression of collagen I and α-SMA (p < 0.05). Moreover, all the analyzed transcription factors­NF-κB, c-Myc, and STAT3­have shown its influence on the pathways connected with relaxin action. Though relaxin requires further study, our results suggest that this natural compound offers great potential for inhibition of the development, or even reversing, of factors related to airway remodeling. The presented contribution of the investigated transcription factors in this process additionally increases its potential possibilities through a variety of its activity pathways.

The Relaxin-3 Receptor, RXFP3, Is a Modulator of Aging-Related Disease.

During the aging process our body becomes less well equipped to deal with cellular stress, resulting in an increase in unrepaired damage. This causes varying degrees of impaired functionality and an increased risk of mortality. One of the most effective anti-aging strategies involves interventions that combine simultaneous glucometabolic support with augmented DNA damage protection/repair. Thus, it seems prudent to develop therapeutic strategies that target this combinatorial approach. Studies have shown that the ADP-ribosylation factor (ARF) GTPase activating protein GIT2 (GIT2) acts as a keystone protein in the aging process. GIT2 can control both DNA repair and glucose metabolism. Through in vivo co-regulation analyses it was found that GIT2 forms a close coexpression-based relationship with the relaxin-3 receptor (RXFP3). Cellular RXFP3 expression is directly affected by DNA damage and oxidative stress. Overexpression or stimulation of this receptor, by its endogenous ligand relaxin 3 (RLN3), can regulate the DNA damage response and repair processes. Interestingly, RLN3 is an insulin-like peptide and has been shown to control multiple disease processes linked to aging mechanisms, e.g., anxiety, depression, memory dysfunction, appetite, and anti-apoptotic mechanisms. Here we discuss the molecular mechanisms underlying the various roles of RXFP3/RLN3 signaling in aging and age-related disorders.

Changes in microRNA Expression Profiles in Diabetic Cardiomyopathy Rats Following H3 Relaxin Treatment.

ABSTRACT: MicroRNAs (miRNAs) are noncoding RNAs that play an important role in the mechanisms of diabetic cardiomyopathy (DCM); however, whether human recombinant relaxin-3 (H3 relaxin) inhibits myocardial injury in DCM rats and the underlying mechanisms involving miRNAs remain unknown. miRNA expression profiles were detected using miRNA microarray and bioinformatics analyses of myocardial tissues from control, DCM, and H3 relaxin-administered DCM groups, and the regulatory mechanisms of the miRNAs were investigated. A total of 5 miRNAs were downregulated in the myocardial tissues of DCM rats and upregulated in H3 relaxin-treated DCM rats, and 1 miRNA (miRNA let-7d-3p) was increased in the myocardial tissue of DCM rats and decreased in H3 relaxin-treated DCM rats as revealed by miRNA microarray and validated by real-time polymerase chain reaction. Important signaling pathways were found to be triggered by the differentially expressed miRNAs, including metabolism, cancer, Rap1, PI3K-Akt, and MAPK signaling pathways. The study revealed that H3 relaxin improved glucose uptake in DCM rats, potentially via the regulation of miRNA let-7d-3p.

The putative role of the relaxin-3/RXFP3 system in clinical depression and anxiety: A systematic literature review.

The relaxin-3/RXFP3 system is one of several neuropeptidergic systems putatively implicated in regulating the behavioural alterations that characterise clinical depression and anxiety, making it a potential target for clinical translation. Accordingly, this systematic review identified published reports on the role of relaxin-3/RXFP3 signalling in these neuropsychiatric disorders and their behavioural endophenotypes, evaluating evidence from animal and human studies to ascertain any relationship. We searched PubMed, EMBASE, PsycINFO and Google Scholar databases up to February 2021, finding 609 relevant records. After stringent screening, 51 of these studies were included in the final synthesis. There was considerable heterogeneity in study designs and some inconsistency across study outcomes. However, experimental evidence is consistent with an ability of relaxin-3/RXFP3 signalling to promote arousal and suppress depressive- and anxiety-like behaviour. Moreover, meta-analyses of six to eight articles investigating food intake revealed that acute RXFP3 activation had strong orexigenic effects in rats. This appraisal also identified the lack of high-quality clinical studies pertinent to the relaxin-3/RXFP3 system, a gap that future research should attempt to bridge.

Structural Insights into the Unique Modes of Relaxin-Binding and Tethered-Agonist Mediated Activation of RXFP1 and RXFP2.

Our poor understanding of the mechanism by which the peptide-hormone H2 relaxin activates its G protein coupled receptor, RXFP1 and the related receptor RXFP2, has hindered progress in its therapeutic development. Both receptors possess large ectodomains, which bind H2 relaxin, and contain an N-terminal LDLa module that is essential for receptor signaling and postulated to be a tethered agonist. Here, we show that a conserved motif (GDxxGWxxxF), C-terminal to the LDLa module, is critical for receptor activity. Importantly, this motif adopts different structures in RXFP1 and RXFP2, suggesting distinct activation mechanisms. For RXFP1, the motif is flexible, weakly associates with the LDLa module, and requires H2 relaxin binding to stabilize an active conformation. Conversely, the GDxxGWxxxF motif in RXFP2 is more closely associated with the LDLa module, forming an essential binding interface for H2 relaxin. These differences in the activation mechanism will aid drug development targeting these receptors.

Endurance exercise improves avoidance learning and spatial memory, through changes in genes of GABA and relaxin-3, in rats.

Different exercise patterns, neurotransmitters, and some genes have numerous effects on learning and memory. This research aims to investigate the long-term effects of submaximal aerobic exercise on spatial memory (SM), passive avoidance learning (PAL), levels of serum relaxin-3, gamma-aminobutyric acid (GABA), RLN3 gene, and glutamic acid decarboxylase (GAD65/67 genes) in the brainstem of adult male Wistar rats. Fifty male Wistar rats were randomly divided into five groups: aerobic exercise groups, performed on a treadmill running (TR), for 5 weeks (Ex5, n = 10), 10 weeks (Ex10, n = 10), involuntary running wheel group for 5 weeks (IRW5, n = 10), sham (Sh, n = 10) and control (Co, n = 10). Consequently, SM, PAL, serum relaxin-3, GABA, and GAD65/67 and RLN3 genes were measured by ELISA and PCR. Ex5, Ex10 and IRW5 improved significantly SM (p ≤ 0.05), PAL (p ≤ 0.001) and decreased significantly relaxin-3 (p ≤ 0.001). RLN3 in the brain also decreased. However, it was not significant. GABA and GAD65/GAD67 increased significantly (p ≤ 0.05) in Ex5, Ex10 compared to Sh and Co. Aerobic exercise enhanced SM and PAL in Ex compared to Co and Sh. However, duration and type of exercise affected the level of enhancement. The serum relaxin-3 and RLN3 gene displayed reverse functions compared to GABA and GAD65/67 genes in Ex. Therefore, the changes of neurotransmitters in serum relaxin-3, GABA, and their genes: RLN3 and GAD65/67 respectively, influenced learning and memory meaningfully.

Relaxin increased blood pressure and sympathetic activity in paraventricular nucleus of hypertensive rats via enhancing oxidative stress.

Relaxin, an ovarian polypeptide hormone, is found in the hypothalamic paraventricular nucleus (PVN) which is an important central integrative site for the control of blood pressure and sympathetic outflow. The aim of this study was to determine if superoxide anions modulate the effects of relaxin in the PVN. Experiments were performed in normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Relaxin mRNA and protein, and its receptor, relaxin family peptide receptor 1 (RXFP1) levels in PVN were 3.24, 3.17, and 3.64 times higher in SHRs than in WKY rats, respectively. Microinjection of relaxin-2 into the PVN dose-dependently increased mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA) and heart rate (HR) in both WKY rats and SHRs, although the effects on MAP (16.87 ±â€¯1.99 vs. 8.97 ±â€¯1.48 mm Hg in 100 nmol), RSNA (22.60 ±â€¯2.15 vs. 11.77 ±â€¯1.43 % in 100 nmol) and HR (22.85 ±â€¯3.13 vs. 12.62 ±â€¯2.83 beats/min in 100 nmol) were greater in SHRs. Oxidative stress level was enhanced after relaxin-2 microinjection into the PVN. Pretreatment with superoxide anion scavengers or NADPH oxidase inhibitor blocked, and superoxide dismutase inhibitor potentiated the effects of relaxin-2 on MAP, RSNA and HR. RXFP1 knockdown significantly attenuated the blood pressure of SHRs, and inhibited the increases of atrial natriuretic peptide, brain natriuretic peptide, collagen I, collagen III and fibronectin in the heart of SHRs. These results demonstrated that relaxin is expressed in the PVN, and contributes to hypertension and sympathetic overdrive via oxidative stress. Down-regulation of RXFP1 in the PVN could attenuate hypertension and cardiac remodeling.

Relaxin gene delivery modulates macrophages to resolve cancer fibrosis and synergizes with immune checkpoint blockade therapy.

Cancer fibrosis serves as a major therapeutic barrier in desmoplastic tumors. Relaxin (RLN; a systemic hormone) is efficacious to decrease fibrosis, but the in vivo mechanism of action is not clear. Considering the localization of relaxin family peptide receptor type 1 (RXFP1), the receptor for RLN, on macrophages, we hypothesize that macrophages can be modulated by RLN to ameliorate cancer fibrosis. Using KPC mouse model of pancreatic ductal adenocarcinoma (PDAC), here, we report locally expressed RLN with targeted gene delivery induces increased F4/80+CD206+ macrophages originating from Ly6C+ monocytes, promoting fibrosis depletion and cytotoxic T cell infiltration. Moreover, RLN gene delivery synergizes with PD-L1 blockade for tumor inhibition by enhancing T cell-mediated tumor cell killing and macrophage phagocytosis. Collectively, our results reveal previously unidentified insights into the modulation of macrophages to regulate tumor-associated fibrosis, providing a feasible strategy to reverse the immunosuppressive environment and improve the therapeutic outcome of checkpoint immunotherapies.

Identification of Potent and Long-Acting Single-Chain Peptide Mimetics of Human Relaxin-2 for Cardiovascular Diseases.

The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (e.g., 54). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with sub-nanomolar activity, high subcutaneous bioavailability, extended half-lives, and in vivo efficacy (e.g., 64).

Hepatic macrophages act as a central hub for relaxin-mediated alleviation of liver fibrosis.

Relaxin is an antifibrotic peptide hormone previously assumed to directly reverse the activation of hepatic stellate cells for liver fibrosis resolution. Using nanoparticle-mediated delivery, here we show that, although relaxin gene therapy reduces liver fibrosis in vivo, in vitro treatment fails to induce quiescence of the activated hepatic stellate cells. We show that hepatic macrophages express the primary relaxin receptor, and that, on relaxin binding, they switch from the profibrogenic to the pro-resolution phenotype. The latter releases exosomes that promote the relaxin-mediated quiescence of activated hepatic stellate cells through miR-30a-5p. Building on these results, we developed lipid nanoparticles that preferentially target activated hepatic stellate cells in the fibrotic liver and encapsulate the relaxin gene and miR-30a-5p mimic. The combinatorial gene therapy achieves synergistic antifibrosis effects in models of mouse liver fibrosis. Collectively, our findings highlight the key role that macrophages play in the relaxin-primed alleviation of liver fibrosis and demonstrate a proof-of-concept approach to devise antifibrotic strategies through the complementary application of nanotechnology and basic science.

BRAFV600E, hypothyroidism, and human relaxin in thyroid carcinogenesis.

PURPOSE: BRAFV600E, a major driver of thyroid cancer, evaluated in the context of thyroid hormones and human relaxin. METHODS: Immunohistochemical expressions of BRAFV600E, TSH, TSH receptor (TSHR), T4, T3 receptor (T3R), RLNH2, and its receptor, RXFP1, were evaluated in thyroid tumors from a retrospective U.S. population of 481 cancer cases diagnosed in 1983-2004. RESULTS: BRAFV600E was expressed in 52% of all thyroid tumors; expression of other markers ranged from 25% for T4 to 98% for RLNH2. Tumors predominantly exhibited hypothyroid-like conditions characterized by elevated TSH and TSHR and reduced T4. BRAFV600E prevalence was significantly higher in tumors expressing TSH, TSHR, T3R, and RXFP1 and lower in tumors expressing T4. The proportion of BRAFV600E mutation in classic papillary tumors significantly increased from 56 to 72% over the 21-year period of diagnoses, while expression of RXFP1, TSH, TSHR, and T3R decreased in non-tumor. Racial/ethnic differences were observed in thyroid hormone marker expression. Non-tumor expression of TSH, TSHR, and T3R were each associated with shorter overall survival, but did not remain significant after adjustment for demographic and clinical factors. CONCLUSIONS: Our study provides the first evidence of the potential interaction of BRAFV600E mutation, relaxin, and thyroid hormones in thyroid carcinogenesis. Moreover, our results suggest that hypothyroidism, influenced by RLNH2 activity, may underlie the development of the majority of thyroid cancers and mediate the role of BRAFV600E in thyroid carcinogenesis. BRAFV600E mutation is increasing in papillary thyroid cancers and may be contributing to the rising incidence of this malignancy.

A novel G protein-coupled receptor for starfish gonadotropic hormone, relaxin-like gonad-stimulating peptide.

Gonadotropic hormones play important regulatory roles in reproduction. Relaxin-like gonad-stimulating peptide (RGP) is a gonadotropin-like hormone in starfish. However, a receptor for RGP remains to be identified. Here, we describe the identification of an authentic receptor for RGP (RGPR) in the starfish, Patiria pectinifera. A binding assay using radioiodinated P. pectinifera RGP (PpeRGP) revealed that RGPR was expressed in ovarian follicle cells. A RGPR candidate was identified by homology-searching of transcriptome data of P. pectinifera follicle cells. Based on the contig sequences, a putative 947-amino acid PpeRGPR was cloned from follicle cells. Like the vertebrate relaxin family peptide receptors (RXFP 1 and 2), PpeRGPR was a G protein-coupled receptor that harbored a low-density lipoprotein-receptor class A motif and leucine-rich repeat sequences in the extracellular domain of the N-terminal region. Sf9 cells transfected with Gαq16-fused PpeRGPR activated calcium ion mobilization in response to PpeRGP, but not to RGP of another starfish Asterias amurensis, in a dose-dependent fashion. These results confirmed the species-specific reactivity of RGP and the cognate receptor. Thus, the present study provides evidence that PpeRGPR is a specific receptor for PpeRGP. To the best of our knowledge, this is the first report on the identification of a receptor for echinoderm RGP.

Mapping Cell Types and Efferent Pathways in the Ascending Relaxin-3 System of the Nucleus Incertus.

Relaxin-3 (Rln3) is an insulin-family peptide neurotransmitter expressed primarily in neurons of the nucleus incertus (NI) of the pontine tegmentum, with smaller populations located in the deep mesencephalon (DpMe) and periaqueductal gray (PAG). Here, we have used targeted recombination at the Rln3 gene locus to generate an Rln3Cre transgenic mouse line, and characterize the molecular identity and axonal projections of Rln3-expressing neurons. Expression of Cre recombinase in Rln3Cre mice, and the expression of Cre-mediated reporters, accurately reflect the expression of Rln3 mRNA in all brain regions. In the NI, Rln3 mRNA is expressed in a subset of a larger population of tegmental neurons that express the neuropeptide neuromedin-b (NMB). These Rln3-expressing and NMB-expressing neurons also express the GABAergic marker GAD2 but not the glutamatergic marker Slc17a6 (VGluT2). Cre-mediated anterograde tracing with adeno-associated viruses (AAVs) shows that the efferents of the Rln3-expressing neurons in the DpMe and PAG are largely confined to the brain regions in which they originate, while the NI-Rln3 neurons form an extensive ascending system innervating the limbic cortex, septum, hippocampus, and hypothalamus. Viral anterograde tracing also reveals the potential synaptic targets of NI-Rln3 neurons in several brain regions, and the distinct projections of Rln3-expressing and non-expressing neurons in the pontine tegmentum. Rabies virus (RV)-mediated transsynaptic retrograde tracing demonstrates a probable synaptic link between NI-Rln3 neurons and GABAergic neurons in the septum, with implications for the modulation of neural activity in the septo-hippocampal system. Together, these results form the basis for functional studies of the NI-Rln3 system.

Hydrophobic interactions of relaxin family peptide receptor 3 with ligands identified using a NanoBiT-based binding assay.

Relaxin family peptide receptor 3 (RXFP3) is a G protein-coupled receptor implicated in the regulation of food intake and stress response upon activation by the neuropeptide relaxin-3. In recent studies, interactions of RXFP3 with some natural or synthetic ligands have been investigated. In the present study, we identified the hydrophobic interactions of human RXFP3 with the chimeric agonist R3/I5 and the chimeric antagonist R3(ΔB23-27)R/I5 using a newly developed NanoBiT-based homogenous binding assay. We first demonstrated that the conserved large aliphatic B15Ile and B19Ile were important for the binding of the agonist and antagonist to RXFP3, because alanine replacement significantly decreased their receptor-binding potency. Thereafter, we demonstrated that the conserved large aliphatic Leu246 and Leu248 in extracellular loop 2 were important for RXFP3 binding to the agonist and antagonist, because alanine replacement significantly decreased the binding affinity of RXFP3 for both ligands. Finally, we deduced probable hydrophobic interactions based on the ability of RXFP3 mutants to distinguish the wild-type and mutant ligands: Leu246 of RXFP3 interacted with B15Ile of both ligands, while Leu248 of RXFP3 interacted with both B15Ile and B19Ile of the agonist and antagonist. The present results not only provided new insights into the interaction mechanism of RXFP3 with agonists and antagonists, but also demonstrated usefulness of the NanoBiT-based homogenous binding assay to study the interaction mechanism of certain receptors with their ligands.

Serelaxin activates eNOS, suppresses inflammation, attenuates developmental delay and improves cognitive functions of neonatal rats after germinal matrix hemorrhage.

Germinal matrix hemorrhage (GMH) is a detrimental form of neonatal CNS injury. Following GMH-mediated eNOS inhibition, inflammation arises, contributing to GMH-induced brain injury. We investigated the beneficial effects of Serelaxin, a clinical tested recombinant Relaxin-2 protein, on brain injury after GMH in rats. We investigated whether effects of Serelaxin are mediated by its ability to activate the GMH-suppressed eNOS pathway resulting in attenuation of inflammatory marker overproduction. GMH was induced by intraparenchymal injection of bacterial collagenase (0.3U). Seven day old Sprague-Dawley rat pups (P7) were used (n = 63). GMH animals were divided in vehicle or serelaxin treated (3 µg once, 30 µg once, 30 µg multiple, i.p., starting 30 after GMH and then daily). Sham operated animals were used. We monitored the developmental profile working memory and spatial function (T-maze and open field test respectively). At day 28, all rats underwent MRI-scans for assessment of changes in cortical thickness and white matter loss. Effects of Serelaxin on eNOS pathway activation and post-GMH inflammation were evaluated. We demonstrated that Serelaxin dose-dependently attenuated GMH-induced developmental delay, protected brain and improved cognitive functions of rats after GMH. That was associated with the decreased post-GMH inflammation, mediated at least partly by amelioration of GMH-induced eNOS inhibition.