Cognate Receptor Research Articles

Differential regulation of microglial states by colony stimulating factors.

Recent studies have emphasized the role of microglia in the progression of many neurodegenerative diseases. The colony stimulating factors, CSF-1 (M-CSF), granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF) regulate microglia through different cognate receptors. While the receptors for GM-CSF (GM-CSFR) and G-CSF (G-CSFR) are specific for their ligands, CSF-1 shares its receptor, the CSF-1 receptor-tyrosine kinase (CSF-1R), with interleukin-34 (IL-34). All four cytokines are expressed locally in the CNS. Activation of the CSF-1R in macrophages is anti-inflammatory. In contrast, the actions of GM-CSF and G-CSF elicit different activated states. We here review the roles of each of these cytokines in the CNS and how they contribute to the development of disease in a mouse model of CSF-1R-related leukodystrophy. Understanding their roles in this model may illuminate their contribution to the development or exacerbation of other neurodegenerative diseases.

Loss of PKCδ/Prkcd prevents cartilage degeneration in joints but exacerbates hyperalgesia in an experimental osteoarthritis mouse model

Pain is the prime symptom of osteoarthritis (OA) that directly affects the quality of life. Protein kinase Cδ (PKCδ/Prkcd) plays a critical role in OA pathogenesis; however, its significance in OA-related pain is not entirely understood. The present study investigated the functional role of PKCδ in OA pain sensation. OA was surgically induced in control (Prkcdfl/fl), global- (Prkcdfl/fl; ROSACreERT2), and sensory neuron-specific conditional knockout (cKO) mice (Prkcdfl/fl; NaV1.8/Scn10aCreERT2) followed by comprehensive analysis of longitudinal behavioral pain, histopathology and immunofluorescence studies. GlobalPrkcd cKO mice prevented cartilage deterioration by inhibiting matrix metalloproteinase-13 (MMP13) in joint tissues but significantly increased OA pain. Sensory neuron-specificdeletion of Prkcd in mice did not protect cartilage from degeneration but worsened OA-associated pain. Exacerbated pain sensitivity observed in global- and sensory neuron-specific cKO of Prkcd was corroborated with markedly increased specific pain mediators in knee synovium and dorsal root ganglia (DRG). These specific pain markers include nerve growth factor (NGF) and vascular endothelial growth factor (VEGF), and their cognate receptors, including tropomyosin receptor kinase A (TrkA) and vascular endothelial growth factor receptor-1 (VEGFR1). The increased levels of NGF/TrkA and VEGF/VEGFR1 were comparable in both global- and sensory neuron-specific cKO groups. These data suggest that the absence of Prkcd gene expression in the sensory neurons is strongly associated with OA hyperalgesia independent of cartilage protection. Thus, inhibition of PKCδ may be beneficial for cartilage homeostasis but could aggravate OA-related pain symptoms.

BDNF and Lactate as Modulators of Hippocampal CA3 Network Physiology.

Growing evidence supports the notion that brain-derived neurotrophic factor (BDNF) and lactate are potent modulators of mammalian brain function. The modulatory actions of those biomolecules influence a wide range of neuronal responses, from the shaping of neuronal excitability to the induction and expression of structural and synaptic plasticity. The biological actions of BDNF and lactate are mediated by their cognate receptors and specific transporters located in the neuronal membrane. Canonical functions of BDNF occur via the tropomyosin-related kinase B receptor (TrkB), whereas lactate acts via monocarboxylate transporters or the hydroxycarboxylic acid receptor 1 (HCAR1). Both receptors are highly expressed in the central nervous system, and some of their physiological actions are particularly well characterized in the hippocampus, a brain structure involved in the neurophysiology of learning and memory. The multifarious neuronal circuitry between the axons of the dentate gyrus granule cells, mossy fibers (MF), and pyramidal neurons of area CA3 is of great interest given its role in specific mnemonic processes and involvement in a growing number of brain disorders. Whereas the modulation exerted by BDNF via TrkB has been extensively studied, the influence of lactate via HCAR1 on the properties of the MF-CA3 circuit is an emerging field. In this review, we discuss the role of both systems in the modulation of brain physiology, with emphasis on the hippocampal CA3 network. We complement this review with original data that suggest cross-modulation is exerted by these two independent neuromodulatory systems.

In vitro reconstitution of herpes simplex virus 1 fusion identifies low pH as a fusion co-trigger.

HSV-1 causes lifelong, incurable infections and diseases ranging from mucocutaneous lesions to fatal encephalitis. Fusion of viral and host membranes is a critical step in HSV-1 infection of target cells that requires multiple factors on both the viral and host sides. Due to this complexity, many fundamental questions remain unanswered, such as the identity of the viral and host factors that are necessary and sufficient for HSV-1-mediated membrane fusion and the nature of the fusion trigger. Here, we developed a simplified in vitro fusion assay to examine the fusion requirements and identified low pH as a co-trigger for virus-mediated fusion in vitro. We hypothesize that low pH has a critical role in cell entry and, potentially, pathogenesis.

Characterization of erythroferrone structural domains relevant to its iron-regulatory function

Iron delivery to the plasma is closely coupled to erythropoiesis, the production of red blood cells, as this process consumes most of the circulating plasma iron. In response to hemorrhage and other erythropoietic stresses, increased erythropoietin stimulates the production of the hormone erythroferrone (ERFE) by erythrocyte precursors (erythroblasts) developing in erythropoietic tissues. ERFE acts on the liver to inhibit bone morphogenetic protein (BMP) signaling and thereby decrease hepcidin production. Decreased circulating hepcidin concentrations then allow the release of iron from stores and increase iron absorption from the diet. Guided by evolutionary analysis and Alphafold2 protein complex modeling, we used targeted ERFE mutations, deletions, and synthetic ERFE segments together with cell-based bioassays and surface plasmon resonance to probe the structural features required for bioactivity and BMP binding. We define the ERFE active domain and multiple structural features that act together to entrap BMP ligands. In particular, the hydrophobic helical segment 81 to 86 and specifically the highly conserved tryptophan W82 in the N-terminal region are essential for ERFE bioactivity and Alphafold2 modeling places W82 between two tryptophans in its ligands BMP2, BMP6, and the BMP2/6 heterodimer, an interaction similar to those that bind BMPs to their cognate receptors. Finally, we identify the cationic region 96-107 and the globular TNFα-like domain 186-354 as structural determinants of ERFE multimerization that increase the avidity of ERFE for BMP ligands. Collectively, our results provide further insight into the ERFE-mediated inhibition of BMP signaling in response to erythropoietic stress.

Authentication of a lophotrochozoan adipokinetic hormone receptor in a Gastropod, Aplysia californica

Gonadotropin-releasing hormone (GnRH) superfamily comprises multiple families of signaling peptides in both protostomes and deuterostomes. Among this superfamily, vertebrate GnRH stimulates reproduction, but other GnRH superfamily members elicit diverse pleiotropic effects. Within the GnRH superfamily members, adipokinetic hormone (AKH) and its receptor are well described in ecdysozoans but understudied in other lineages. To fill this knowledge gap, we deorphanized a putative receptor for a lophotrochozoan AKH in a gastropod mollusk, Aplysia californica, and named it Aplca-AKHR. Phylogenetic analysis revealed an orthologous relationship of Aplca-AKHR with ecdysozoan AKHRs and other putative lophotrochozoan AKHRs. Aplca-AKHR bound specifically to the previously identified Aplca-AKH with high affinity and activated the inositol phosphate pathway. Aplca-AKHR was expressed widely among central and peripheral tissues, but most prominently in several central ganglia and the heart. The expression of Aplca-AKHR was downregulated by a hyposaline challenge, consistent with a role in volume and fluid regulation previously described for its ligand, Aplca-AKH. In summary, this is the first pairing of a lophotrochozoan AKH with its cognate receptor. Expression data further support diverse central and peripheral roles, including volume and fluid control, of this ligand/receptor pair.

Molecular characterization and ontogenetic expression profiles of LPXRFa and its receptor in Japanese flounder (Paralichthys olivaceus)

Investigations concerning the LPXRFa system are rarely conducted in flatfish species. Here, we first identified and characterized lpxrfa and its cognate receptor lpxrfa-r genes in the Japanese flounder (Paralichthys olivaceus). The coding DNA sequence of lpxrfa was 579 bp in length, wich encoded a 192-aa preprohormone that can produce three mature LPXRFa peptides. The open reading frame (ORF) of lpxrfa-r was 1446 bp in size, and encoded a 481-aa LPXRFa-R protein that encompassed seven hydrophobic transmembrane domains. Subsequently, tissue distribution expression profiles of lpxrfa and lpxrfa-r transcripts were assayed by quantitative real-time PCR. The results indicated that expressions of lpxrfa transcripts were detected at the highest levels in the brain of both females and males, however, lpxrfa-r transcripts were remarkablely expressed in the brain tissue of female fish and in the testis tissue of male fish. Furthermore, transcript levels of lpxrfa and lpxrfa-r genes were investigated during early ontogenetic development, with the maximum expression levels at 30 days post-hatching. Overall, these data contribute to providing preliminary proof for the existence and structure of the LPXRFa system in Japanese flounder, and the study is just the foundation for researching physiological function of LPXRFa system in this species.

Mechanistic Insight into Poly-Reactivity of Immune Antibodies upon Acid Denaturation or Arginine Mutation in Antigen-Binding Regions.

The poly-reactivity of antibodies is defined as their binding to specific antigens as well as to related proteins and also to unrelated targets. Poly-reactivity can occur in individual molecules of natural serum antibodies, likely due to their conformation flexibility, and, for therapeutic antibodies, it plays a critical role in their clinical development. On the one hand, it can enhance their binding to target antigens and cognate receptors, but, on the other hand, it may lead to a loss of antibody function by binding to off-target proteins. Notably, poly-reactivity has been observed in antibodies subjected to treatments with dissociating, destabilizing or denaturing agents, in particular acidic pH, a common step in the therapeutic antibody production process involving the elution of Protein-A bound antibodies and viral clearance using low pH buffers. Additionally, poly-reactivity can emerge during the affinity maturation in the immune system, such as the germinal center. This review delves into the underlying potential causes of poly-reactivity, highlighting the importance of conformational flexibility, which can be further augmented by the acid denaturation of antibodies and the introduction of arginine mutations into the complementary regions of antibody-variable domains. The focus is placed on a particular antibody's acid conformation, meticulously characterized through circular dichroism, differential scanning calorimetry, and sedimentation velocity analyses. By gaining a deeper understanding of these mechanisms, we aim to shed light on the complexities of antibody poly-reactivity and its implications for therapeutic applications.

THU580 A Subset Of Migrating Cells Co-express Gonadotropin Releasing Hormone And Prokineticin Receptor 2 During Embryonic Development

Abstract Disclosure: A.E. Danku: None. C.F. Elias: None. A subset of migrating cells co-express gonadotropin releasing hormone and prokineticin receptor 2 during embryonic development Antoinette Danku and Carol F Elias. Neuroscience Graduate Program and Department of Molecular & Integrative Physiology, University to Michigan. Reproductive vitality in humans is strongly influenced by developmental foundations. The successful migration of GnRH cells and the pulsatile secretion of gonadotropin releasing hormone (GnRH) drives pubertal onset and dictates long-term fertility outcomes. During development, GnRH neurons migrate from the nasal placode to the basal forebrain to reach their destination in the medial preoptic area (MPOA). Aberrant GnRH migration causes redirection of axonal projections to atypical brain regions and dysregulated GnRH secretion. Disruption of GnRH neuronal function causes infertility or reproductive dysfunction. These etiologies are grouped as isolated gonadotropin deficiencies (IGD). Kallmann’s syndrome (KS) is a pediatric IGD that manifests as anosmia and hormonal dysfunction that can result in pubertal delay or infertility. Children with KS often have mutations in prokineticin 2 (Prok2) or the cognate receptor (ProkR2).Prok2 and ProkR2 are required for the formation of the olfactory bulb in rodents and humans. Loss-of-function mutations in these genes also disrupts GnRH cell migration, but whether the activation of ProkR2 regulates the guidance of GnRH neurons to their final destination has not been defined. Previous studies suggested that GnRH and ProkR2 are expressed in distinct cells, but how the prokineticin system may alter GnRH migration is largely unknown. To gain insights into the underlying mechanisms associated with ProkR2-GnRH interaction and migration, we conducted single nuclei RNA sequencing in nasal-forebrain junction of embryos at embryonic day 16 (E16) and fluorescent in situ hybridization in three developmental time points: E14, E16, and E18 for validation and spatial evaluation. Using these methods, we were able to isolate GnRH and ProkR2 cells and sort their cell types into clusters. While these cells differ in their expression profiles, they both express genes associated with migration at E16. Our findings suggest that ProkR2 cells in the nasal-forebrain junction migrate along with GnRH cells. Additionally, a subset of these migrating cells co-express ProkR2 and GnRH mRNA. Further studies are needed to determine whether this subset of cells take on specialized roles in GnRH cell migration and maturation. Presentation: Thursday, June 15, 2023

FRI457 Protein-coding Genetic Variation In Polycystic Ovary Syndrome

Abstract Disclosure: C. Parker: None. R. Bauer: None. C.K. Welt: None. R.S. Legro: None. M.G. Hayes: None. M. Urbanek: None. Background: Polycystic ovary syndrome (PCOS) is a multisystem endocrine disorder and is the most common form of anovulatory infertility of reproductive aged women (6-15%). PCOS is characterized by the presence of hyperandrogenemia, irregular menses, and polycystic ovarian morphology. A previous twin study has shown PCOS to be highly heritable (h2=0.72). Thus, we hypothesize that genetic variation is a major contributor to the etiology of PCOS. Objective: Our goal is to identify the genetic factors that contribute to the PCOS phenotype and how they define phenotypic subtypes of PCOS with the aim of better understanding PCOS pathology and optimizing treatment strategies for individual women. Methodology: We are performing whole exome sequencing (WES) supplemented with genomic regions mapping to PCOS GWAS loci on 772 subjects with PCOS and 300 age and ancestry-matched controls to generate a comprehensive catalog of PCOS-associated protein coding variants. We will use prediction tools to identify likely-to-be deleterious variants and in silico evidence of epigenetic modification to identify PCOS associated genes/pathways. We will then prioritize variants for molecular follow-up creating a comprehensive catalog of protein coding variants associated with PCOS. We expect the variants identified by WES to map to multiple genes and pathways including novel genes/pathways which have not previously been implicated in PCOS as well as genes/pathways that have previously been shown or predicted to play a major role in the etiology of PCOS by us or other groups. Results: To date we have obtained high quality sequencing data on 772 women with PCOS. Sequencing of 300 age and ancestry matched reproductively heathy women is being started. We have identified 81 rare protein coding genetic variants using targeted re-sequencing in three distinct pathways in an independent PCOS cohort. These genes are associated with insulin resistance, the gonadotropin signaling pathway and the Anti-Müllerian Hormone (AMH) signaling pathway. We identified 37 functionally validated deleterious variants in genes that encode AMH or its cognate receptor AMHR2, 7 rare variants that encode LMNA, 11 rare (MAF <0.1) variants that encode the insulin receptor, and 26 coding variants for the genes encoding the beta subunit of gonadotropins, LH and FSH, and their receptors, FSHR and LHCGR. Conclusion: We have identified 81 rare protein altering genetic variants in pathways that are predicted to be impaired in individuals with PCOS supporting a critical role for genetics in the etiology of PCOS. Our WES screen will allow us to generate a comprehensive catalog of likely to be deleterious PCOS associated variants. This catalog of variants will identify the genes/pathways that are mutated in PCOS leading to improved personalized phenotyping and treatment of PCOS. Presentation Date: Friday, June 16, 2023

Targeting EB3-IP3R3 Interface with Cognate Peptide Protects from Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a lung disease characterized by acute onset of noncardiogenic pulmonary edema, hypoxemia, and respiratory insufficiency. The current treatment for ARDS is mainly supportive in nature, providing a critical need for targeted pharmacological management. We addressed this medical problem by developing a pharmacological treatment for pulmonary vascular leakage, a culprit of alveolar damage and lung inflammation. Our novel therapeutic target is the microtubule accessory factor EB3 (end binding protein 3), which contributes to pulmonary vascular leakage by amplifying pathological calcium signaling in endothelial cells in response to inflammatory stimuli. EB3 interacts with IP3R3 (inositol 1,4,5-trisphosphate receptor 3) and orchestrates calcium release from endoplasmic reticulum stores. Here, we designed and tested the therapeutic benefits of a 14-aa peptide named CIPRI (cognate IP3 receptor inhibitor), which disrupted EB3-IP3R3 interaction invitro and in lungs of mice challenged with endotoxin. Treatment with CIPRI or depletion of IP3R3 in lung microvascular endothelial monolayers mitigated calcium release from endoplasmic reticulum stores and prevented a disassembly of vascular endothelial cadherin junctions in response to the proinflammatory mediator α-thrombin. Furthermore, intravenous administration of CIPRI in mice mitigated inflammation-induced lung injury, blocked pulmonary microvascular leakage, prevented activation of NFAT (nuclear factor of activated T cells) signaling, and reduced production of proinflammatory cytokines in the lung tissue. CIPRI also improved survival of mice from endotoxemia and polymicrobial sepsis. Together, these data demonstrate that targeting EB3-IP3R3 interaction with a cognate peptide is a promising strategy to address hyperpermeability of microvessels in inflammatory lung diseases.

Defective monocyte plasticity and altered cAMP pathway characterize USB1-mutated poikiloderma with neutropenia Clericuzio type.

Poikiloderma with neutropenia (PN) Clericuzio type (OMIM #604173) is a rare disease with areas of skin hyper- and hypopigmentation caused by biallelic USB1 variants. The current study was spurred by poor healing of a perianal tear wound in one affected child homozygous for c.266-1G>A (p.E90Sfster8) mutation, from a family reported previously. Treatment with G-CSF/CSF3 or GM-CSF/CSF2 transiently increased neutrophil/monocytes count with no effect on wound healing. Analysis of peripheral blood revealed a lack of non-classical (CD14+/- CD16+ ) monocytes, associated with a systemic inflammatory cytokine profile, in the two affected brothers. Importantly, despite normal expression of cognate receptors, monocytes from PN patients did not respond to M-CSF or IL-34 in vitro, as determined by cytokine secretion or CD16 expression. RNAseq of monocytes showed 293 differentially expressed genes, including significant downregulation of GATA2, AKAP6 and PDE4DIP that are associated with leucocyte differentiation and cyclic adenosine monophosphate (cAMP) signalling. Notably, the plasma cAMP was significantly low in the PN patients. Our study revealed a novel association of PN with a lack of non-classical monocyte population. The defects in monocyte plasticity may contribute to disease manifestations in PN and a defective cAMP signalling may be the primary effect of the splicing errors caused by USB1 mutation.

“Redirecting an anti-IL-1β antibody to bind a new, unrelated and computationally predicted epitope on hIL-17A”

Antibody engineering technology is at the forefront of therapeutic antibody development. The primary goal for engineering a therapeutic antibody is the generation of an antibody with a desired specificity, affinity, function, and developability profile. Mature antibodies are considered antigen specific, which may preclude their use as a starting point for antibody engineering. Here, we explore the plasticity of mature antibodies by engineering novel specificity and function to a pre-selected antibody template. Using a small, focused library, we engineered AAL160, an anti-IL-1β antibody, to bind the unrelated antigen IL-17A, with the introduction of seven mutations. The final redesigned antibody, 11.003, retains favorable biophysical properties, binds IL-17A with sub-nanomolar affinity, inhibits IL-17A binding to its cognate receptor and is functional in a cell-based assay. The epitope of the engineered antibody can be computationally predicted based on the sequence of the template antibody, as is confirmed by the crystal structure of the 11.003/IL-17A complex. The structures of the 11.003/IL-17A and the AAL160/IL-1β complexes highlight the contribution of germline residues to the paratopes of both the template and re-designed antibody. This case study suggests that the inherent plasticity of antibodies allows for re-engineering of mature antibodies to new targets, while maintaining desirable developability profiles.

The importance of the PapR7 C-terminus and amide protons in mediating quorum sensing in Bacilluscereus

The opportunistic human pathogen Bacillus cereus controls the expression of key infection-promoting phenotypes using bacterial quorum sensing (QS). QS signal transduction within the species is controlled by an autoinducing peptide, PapR7, and its cognate receptor, PlcR, indicating that the PlcR:PapR interface is a prime target for QS inhibitor development. The C-terminal region of the peptide (PapR7; ADLPFEF) has been successfully employed as a scaffold to develop potent QS modulators. Despite the noted importance of the C-terminal carboxylate and amide protons in crystallographic data, their role in QS activity has yet to be explored. In this study, an N-methyl scan of PapR7 was conducted in conjunction with a C-terminal modification of previously identified B. cereus QS inhibitors. The results indicate that the amide proton at Glu6 and the C-terminal carboxylate are important for effective QS inhibition of the PlcR regulon. Through β-galactosidase and hemolysis assays, a series of QS inhibitors were discovered, including several capable of inhibiting QS with nanomolar potency. These inhibitors, along with the structure–activity data reported, will serve as valuable tools for disrupting the B. cereus QS pathway towards developing novel anti-infective strategies.

Surface growth of Pseudomonas aeruginosa reveals a regulatory effect of 3-oxo-C12-homoserine lactone in the absence of its cognate receptor, LasR.

The bacterium Pseudomonas aeruginosa colonizes and thrives in many environments, in which it is typically found in surface-associated polymicrobial communities known as biofilms. Adaptation to this social behavior is aided by quorum sensing (QS), an intercellular communication system pivotal in the expression of social traits. Regardless of its importance in QS regulation, the loss of function of the master regulator LasR is now considered a conserved adaptation of P. aeruginosa, irrespective of the origin of the strains. By investigating the QS circuitry in surface-grown cells, we found an accumulation of QS signal 3-oxo-C12-HSL in the absence of its cognate receptor and activator, LasR. The current understanding of the QS circuit, mostly based on planktonic growing cells, is challenged by investigating the QS circuitry of surface-grown cells. This provides a new perspective on the beneficial aspects that underline the frequency of LasR-deficient isolates.

A tactile twist: decoding the phenomena of mechanical itch and alloknesis.

Itch is a sensation in the skin which provokes the desire to scratch. In the past few decades there has been a significant elucidation of the immune and neural pathways which underly the sensation of itch. An interesting divergence in the itch pathway relates to the type of stimulation used to evoke an itchy sensation. Commonly, chemical mediators of itch such as histamine are injected into the skin where they activate their cognate receptors on sensory neurons. Another way to evoke itch, particularly in patients with chronic itch, is to use light mechanical stimulation. Investigation into these pathways utilizing the mouse model have shown that the neuronal pathways which underly chemical itch are distinct from those which mediate itch in response to mechanical stimulation. Specific populations of primary sensory neurons, spinal interneurons and transmission neurons have been identified which suggests a labeled line for itch transmission. Additionally, Piezo channels, which underly mechanosensation, were discovered to play an important role in the mechanical itch pathway. Given these novel findings relating to the mechanical itch pathway, the purpose of this review is to summarize the reports from human subjects and animal studies to highlight the advances in our understanding of mechanical itch and alloknesis.

Gonadotropin Receptor Cross-Talk and Altered Functions in Gonadal and Non-Gonadal Tissues

Reproduction depends on the responses of gonadotropins through their specific receptors. The gonadotropin family has three members; Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), and Human Chorionic Gonadotropin (hCG). These glycoprotein hormones comprise two subunits, an identical α-subunit and a hormone-specific-β subunit. Their cognate receptors (FSHR and LHCGR) are two adrenergic receptor-like family A/rhodopsin-like G-Protein Coupled Receptors (GPCRs) with structurally distinct ligand binding domains. The hCG binds to LHCGR but has a longer half-life and higher affinity to LHCGR. The expression of FSHR and LHCGR is observed in both gonadal and nongonadal cells. In this review, we will be emphasizing the differential expression of gonadotropin receptors in different cells of the human body, their specific responses through cross-talk, and how a defect in the expression and activity of FSHR and LHCGR may alter the responses of FSH and LH/hCG leading to diseases like PCOS, cancer and metabolic disorders.

MAb-Functionalized Biomimetic MamC-Mediated-Magnetoliposomes as Drug Delivery Systems for Cancer Therapy.

In cancer therapy, new therapeutic nanoformulations able to mediate targeted chemotherapy are required. Recently, biomimetic magnetic nanoparticles (BMNPs) mediated by MamC, a magnetosome protein from Magnetococcus marinus MC-1, have proven, in vitro and in vivo, to be effective drug nanocarriers (following the application of an external gradient magnetic field) and to allow combination with hyperthermia. However, these nanoassemblies require further optimization to improve cytocompatibility, stability and active targeting ability. Herein, we describe the production of the magnetoliposomes (LP) embedding BMNPs functionalized (or not) with doxorubicin (DOXO), [LP(+/-DOXO-BMNPs)], and their surface modification with the DO-24 mAb, which targets the human Met/HGF receptor's ectodomain (overexpressed in many cancers). Nanoformulations were extensively characterized using TEM, DLS, FTIR and when tested in vitro, the lipid coating increased the colloidal stability and their biocompatibility, favoring the cellular uptake in cells overexpressing the cognate receptor. Indeed, the magnetoliposomes mAb-LP(+/-DOXO-BMNPs) exerted a specific active targeting ability by the presence of the mAb that preserved its immunocompetence. Both LP(BMNPs) and mAb-LP(BMNPs) were not toxic to cells, while +/-mAb-LP(DOXO-BMNPs) nanoformulations were indeed cytotoxic. Therefore, this study represents a proof of concept for the development of promising drug carriers for cancer therapy based on local chemotherapy directed by mAbs.

Novel RXFP3 negative allosteric modulator RLX-33 reduces alcohol self-administration in rats.

There is much interest in identifying novel pharmacotherapeutic targets that improve clinical outcomes for the treatment of alcohol use disorder (AUD). One promising target for therapeutic intervention is the relaxin family peptide 3 (RXFP3) receptor, a cognate receptor for neuropeptide relaxin-3, which has previously been implicated in regulating alcohol drinking behavior. Recently, we developed the first small-molecule RXFP3-selective negative allosteric modulator (NAM) RLX-33. Therefore, the goal of the present work was to characterize the impact of this novel NAM on affective-related behaviors and alcohol self-administration in rats. First, the effects of RLX-33 were tested on alcohol and sucrose self-administration in Wistar and alcohol-preferring P rats to determine the dose-response profile and specificity for alcohol. Then, we assessed the effects of systemic RLX-33 injection in Wistar rats in a battery of behavioral assays (open-field test, elevated zero maze, acoustic startle response test, and prepulse inhibition) and tested for alcohol clearance. We found that the lowest effective dose (5 mg/kg) reduced alcohol self-administration in both male and female Wistar rats, while in alcohol-preferring P rats, this effect was restricted to males, and there were no effects on sucrose self-administration or general locomotor activity. The characterization of affective and metabolic effects in Wistar rats generally found few locomotor, affective, or alcohol clearance changes, particularly at the 5 mg/kg dose. Overall, these findings are promising and suggest that RXFP3 NAM has potential as a pharmacological target for treating AUD.

Neuropeptide B (NPB) and NPB receptor 2b (NPBWR2b) in the ricefield eel Monopterus albus: expression and potential involvement in the regulation of gonadotropins.

The neuropeptide B/W signaling system is composed of neuropeptide B (NPB), neuropeptide W (NPW), and two cognate receptors, NPBWR1 and NPBWR2, which are involved in diverse physiological processes, including the central regulation of neuroendocrine axes in vertebrates. The components of this signaling system are not well conserved during vertebrate evolution, implicating its functional diversity. The present study characterized the ricefield eel neuropeptide B/W system, generated a specific antiserum against the neuropeptide B/W receptor, and examined the potential roles of the system in the regulation of adenohypophysial functions. The ricefield eel genome contains npba, npbb, and npbwr2b but lacks the npw, npbwr1, and npbwr2a genes. The loss of npw and npbwr1 probably occurred at the base of ray-finned fish radiation and that of npbwr2a species specifically in ray-finned fish. Npba and npbb genes are produced through whole-genome duplication (WGD) in ray-finned fish. The ricefield eel npba was expressed in the brain and some peripheral tissues, while npbb was predominantly expressed in the brain. The ricefield eel npbwr2b was also expressed in the brain and in some peripheral tissues, such as the pituitary, gonad, heart, and eye. Immunoreactive Npbwr2b was shown to be localized to Lh and Fsh cells but not to Gh or Prl cells in the pituitary of ricefield eels. Npba upregulated the expression of fshb and cga but not lhb mRNA in pituitary fragments of ricefield eels cultured in vitro. The results of the present study suggest that the NPB system of ricefield eels may be involved in the neuroendocrine regulation of reproduction.