Sulforaphane decreases oxidative stress and inhibits NLRP3 inflammasome activation in a mouse model of ulcerative colitis.

Excessive oxidative stress and NLRP3 inflammasome activation are considered the main drivers of inflammatory bowel disease (IBD), and inhibition of inflammasomes ameliorates clinical symptoms and morphological manifestations of IBD. Herein, we examined the roles of NLRP3 activation in IBD and modulation of NLRP3 by sulforaphane (SFN), a compound with multiple pharmacological activities that is extracted from cruciferous plants. To simulate human IBD, we established a mouse colitis model by administering dextran sodium sulfate in the drinking water. SFN (25, 50 mg·kg-1·d-1, ig) or the positive control sulfasalazine (500 mg/kg, ig) was administered to colitis-affected mice for 7 days. Model mice displayed pathological alterations in colon tissue as well as classic symptoms of colitis beyond substantial tissue inflammation. Expression of NLRP3, ASC, and caspase-1 was significantly elevated in the colonic epithelium. The expression of NLRP3 inflammasomes led to activation of downstream proteins and increases in the cytokines IL-18 and IL-1ß. SFN administration either fully or partially reversed these changes, thus restoring IL-18 and IL-1ß, substantially inhibiting NLRP3 activation, and decreasing inflammation. SFN alleviated the inflammation induced by LPS and NLRP3 agonists in RAW264.7 cells by decreasing the levels of reactive oxygen species. In summary, our results revealed the pathological roles of oxidative stress and NLRP3 in colitis, and indicated that SFN might serve as a natural NLRP3 inhibitor, thereby providing a new strategy for alternative colitis treatment.

Impinging flow mediates vascular endothelial cell injury through the PKCα/ERK/PPARγ pathway in vitro.

BACKGROUND: This study aims to elucidate the mechanisms underlying endothelial injury in the context of intracranial aneurysm formation and development, which are associated with vascular endothelial injury caused by hemodynamic abnormalities. Specifically, we focus on the involvement of PKCα, an intracellular signaling transmitter closely linked to vascular diseases, and its role in activating MAPK. Additionally, we investigate the protective effects of PPARγ, a vasculoprotective factor known to attenuate vascular injury by mitigating the inflammatory response in the vessel wall. METHODS: The study employs a modified T chamber to replicate fluid flow conditions at the artery bifurcation, allowing us to assess wall shear stress effects on human umbilical vein endothelial cells (HUVECs) in vitro. Through experimental manipulations involving PKCα knockdown and Ca2+ and MAPK inhibitors, we evaluate the phosphorylation status of PKCα, NF-κB, ERK5, ERK1/2, JNK1/2/3, and P38, as well as the expression levels of PPARγ, NF-κB, and MMP2 via western blot analysis. The cellular localization of phosphorylated NF-κB was determined using immunofluorescence. RESULTS: Our results showed that impinging flow resulted in the activation of PKCα, followed by the phosphorylation of ERK5, ERK1/2, and JNK1/2/3, leading to a decrease in PPARγ expression, an increase in the expression of NF-κB and MMP2, and the induction of apoptotic injury. Inhibition of PKCα activation or knockdown of PKCα using shRNA leads to a suppression of ERK5, ERK1/2, JNK1/2/3, and P38 phosphorylation, an elevation in PPARγ expression, and a reduction in NF-κB and MMP2 expression, alleviated apoptotic injury. Furthermore, we observe that the regulation of PPARγ, NF-κB, and MMP2 expression is influenced by ERK5 and ERK1/2 phosphorylation, and activation of PPARγ effectively counteracts the elevated expression of NF-κB and MMP2. CONCLUSION: Our findings suggest that the PKCα/ERK/PPARγ pathway plays a crucial role in mediating endothelial injury under conditions of impinging flow, with potential implications for vascular diseases and intracranial aneurysm development.

An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase.

In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay to identify more AEP-type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP-type peptide ligase activity in the crude extract of all tested bamboo leaves. From a popular bamboo species, Bambusa multiplex, we identified a full-length AEP-type peptide ligase candidate (BmAEP1) via transcriptomic sequencing. After its zymogen was overexpressed in Escherichia coli and self-activated in vitro, BmAEP1 displayed high peptide ligase activity, but with considerable hydrolytic activity. After site-directed mutagenesis of its ligase activity determinants, the mutant zymogen of [G238V]BmAEP1 was normally overexpressed in E. coli, but failed to activate itself. To resolve this problem, we developed a novel protease-assisted activation approach in which trypsin was used to cleave the mutant zymogen and was then conveniently removed via ion-exchange chromatography. After the noncovalently bound cap domain was dissociated from the catalytic core domain under acidic conditions, the recombinant [G238V]BmAEP1 displayed high peptide ligase activity with much lower hydrolytic activity and could efficiently catalyze inter-molecular protein ligation and intramolecular peptide cyclization. Thus, the engineered bamboo-derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis.

Recombinant mannan-binding lectin magnetic beads increase pathogen detection in immunocompromised patients.

The microbiological diagnosis of infection for hematological malignancy patients receiving chemotherapy or allogeneic hematopoietic stem cell transplantation (allo-HSCT) patients relies primarily on standard microbial culture, especially blood culture, which has many shortcomings, such as having low positive rates, being time-consuming and having a limited pathogenic spectrum. In this prospective observational self-controlled test accuracy study, blood, cerebrospinal fluid (CSF), and bronchoalveolar lavage fluid (BALF) samples were collected from chemotherapy or allo-HSCT patients with clinical symptoms of infections who were hospitalized at Peking University First Hospital. Possible pathogens were detected by the method based on recombinant mannan-binding lectin (MBL) magnetic bead enrichment (M1 method) and simultaneously by a standard method. The analytical sensitivity of M1 method was close to that of standard culture method. Besides, the turn-around time of M1-method was significantly shorter than that of standard culture method. Moreover, the M1 method also added diagnostic value through the detection of some clinically relevant microbes missed by the standard method. M1 method could significantly increase the detection efficiency of pathogens (including bacteria and fungi) in immunocompromised patients. KEY POINTS: • The detection results of M1-method had a high coincidence rate with that of standard method • M1 method detected many pathogens which had not been found by standard clinic method.

Sulphated Fucooligosaccharide from Sargassum Horneri: Structural Analysis and Anti-Alzheimer Activity.

In the present study, sulfated polysaccharides were obtained by digestion of Sargassum horneri and preparation with enzyme-assisted extraction using three food-grade enzymes, and their anti- Alzheimer's activities were investigated. The results demonstrated that the crude sulfated polysaccharides extracted using AMGSP, CSP and VSP dose-dependently (25-100 µg·mL- 1) raised the spontaneous alternating manner (%) in the Y maze experiment of mice and reduced the escape latency time in Morris maze test. AMGSP, CSP and VSP also exhibited good anti-AChE and moderate anti-BuChE activities. CSP displayed the best inhibitory efficacy against AChE. with IC50 values of 9.77 µM. And, CSP also exhibited good inhibitory selectivity of AChE over BuChE. Next, CSP of the best active crude extract was separated by the preparation type high performance liquid phase to obtain the sulphated fucooligosaccharide section: SFcup (→3-α-L-fucp(2-SO3-)-1→4-α-L-fucp(2,3-SO3-)-1→section), SFcup showed a best inhibitory efficacy against AChE with IC50 values of 4.03 µM. The kinetic research showed that SFcup inhibited AChE through dual binding sites. Moreover, the molecular docking of SFcup at the AChE active site was in accordance with the acquired pharmacological results.

Exploring the hormetic effects of radiation on the life table parameters of Spodoptera frugiperda.

BACKGROUND: Spodoptera frugiperda, a global agricultural pest, can be effectively controlled through the sterile insect technique. However, exposure to low-dose radiation below the sterilization threshold may induce hormetic effects. Here, the biphasic aspects of the fertile progeny population of S. frugiperda were analyzed using an age-stage, two-sex life table after dosing male and female pupae with 10-350 Gy gamma radiation. RESULTS: The parental sterilizing dose for 6-day-old female and male pupae was 200 and 350 Gy, respectively. The total longevity, pre-adult survival rate, net reproduction rate, and intrinsic growth rate of the offspring population increased with decreasing radiation doses from 250 to 10 Gy. Offspring population of parents treated with low doses of 10-100 Gy showed better life table parameters compared to non-irradiated controls. Females and males fecundity irradiated with 10, 50, and 100 Gy and 10 Gy, respectively, exceeded controls, producing 2339.4, 2726.4, 2311, and 2369 eggs, as opposed to 1802.9 eggs produced by the controls. Males irradiated with 10 Gy displayed the highest intrinsic rates of increase and net reproduction rate, at 0.1709 and 682.3, respectively. Projections from the survival rate and fecundity indicated that female and male S. frugiperda populations after 10 Gy irradiation may grow considerably faster than the controls. CONCLUSION: This study explores the hormetic effects of low-dose radiation on S. frugiperda through life table analysis, while providing enhancements for utilizing substerilizing gamma dose in a modified F1 sterility technique. © 2023 Society of Chemical Industry.

Water Level Fluctuations Modulate the Microbiomes Involved in Biogeochemical Cycling in Floodplains.

Drastic changes in hydrological conditions within floodplain ecosystems create distinct microbial habitats. However, there remains a lack of exploration regarding the variations in microbial function potentials across the flooding and drought seasons. In this study, metagenomics and environmental analyses were employed in floodplains that experience hydrological variations across four seasons. Analysis of functional gene composition, encompassing nitrogen, carbon, and sulfur metabolisms, revealed apparent differences between the flooding and drought seasons. The primary environmental drivers identified were water level, overlying water depth, submergence time, and temperature. Specific modules, e.g., the hydrolysis of ß-1,4-glucosidic bond, denitrification, and dissimilatory/assimilatory nitrate reduction to ammonium, exhibited higher relative abundance in summer compared to winter. It is suggested that cellulose degradation was potentially coupled with nitrate reduction during the flooding season. Phylogenomic analysis of metagenome-assembled genomes (MAGs) unveiled that the Desulfobacterota lineage possessed abundant nitrogen metabolism genes supported by pathway reconstruction. Variation of relative abundance implied its environmental adaptability to both the wet and dry seasons. Furthermore, a novel order was found within Methylomirabilota, containing nitrogen reduction genes in the MAG. Overall, this study highlights the crucial role of hydrological factors in modulating microbial functional diversity and generating genomes with abundant nitrogen metabolism potentials.

A novel oncolytic virus-based biomarker participates in prognosis and tumor immune infiltration of glioma.

Background: Glioma is the most common central nervous malignancy. Due to its poor survival outcomes, it is essential to identify novel individualized therapy. Oncolytic virus (OV) treatment is a key therapy regulating tumor microenvironment in malignant glioma. Herein, we aim to identify the key genes after OV infection and its role in glioma. Methods: Performing an RNA-seq analysis, the differentially expressed genes (DEGs) between EV-A71-infection and mock group were screened with GFold values. DAVID online analysis was performed to identify the functional classification. Overall survival (OS) or disease-free survival (DFS) was evaluated to analyze the relation between PTBP1 expression levels and prognosis of glioma patients. Additionally, the ssGSEA and TIMER algorithms were applied for evaluating immune cell infiltration in glioma. Results: Following EV-A71 infection in glioma cells, PTBP1, one of the downregulated DEGs, was found to be associated with multiple categories of GO and KEGG enrichment analysis. We observed elevated expression levels of PTBP1 across various tumor grades of glioma in comparison to normal brain samples. High PTBP1 expression had a notable impact on the OS of patients with low-grade glioma (LGG). Furthermore, we observed an obvious association between PTBP1 levels and immune cell infiltration in LGG. Notably, PTBP1 was regarded as an essential prognostic biomarker in immune cells of LGG. Conclusion: Our research uncovered a critical role of PTBP1 in outcomes and immune cell infiltration of glioma patients, particularly in those with LGG.

Pfizer BNT 162b2 COVID-19 vaccine-induced fulminant myopericarditis: A case study.

The use of mRNA COVID-19 vaccine can on rare occasions cause life-threatening, fulminant myopericarditis. This case report demonstrates previously reported benefit of early use of venoarterial extracorporeal membrane oxygenation mechanical assistance and supports the use of intravenous highly purified immunoglobulin pharmacotherapy to help achieve a good clinical outcome.

Research trends and hotspots of exosomes in respiratory diseases.

Currently, theoretical studies on exosomes in respiratory diseases have received much attention from many scholars and have made remarkable progress, which has inestimable value and potential in future clinical and scientific research. Unfortunately, no scholar has yet addressed this field's bibliometric analysis and summary. We aim to comprehensively and profoundly study and explore the present situation and highlights of exosome research at the stage of respiratory diseases and to provide meaningful insights for the future development of this field. The WOSCC literature was gathered for the study using bibliometrics, and the data were collected and analyzed using CiteSpace, VOSviewer, Microsoft Excel, and Endnote software. The publication language is "English," and the search strategy is TS = (exosome OR exosomes OR exosomal) AND TS = (respiratory OR lung). The search time is from the beginning of the WOS construction, and the deadline is July 11, 2022, at 22:00 hours. The literature types selected were dissertation, review paper, and online published paper. The analysis includes 2456 publications in 738 journals from 76 countries, 2716 institutions, and 14,568 authors. The field's annual publications have been rising, especially in recent years. China and the US lead research, and prominent universities, including Harvard Medical School, Shanghai Jiao Tong University, and Fudan University, are essential research institutes. Takahiro Ochiya, whose research focuses on exosomes and lung cancer, and Clotilde Théry, a pioneering exosome researcher, are the most cited authors in this field. The key terms include lung cancer, non-small cell lung cancer, mesenchymal stem cells, intercellular communication, exosomal miRNAs, and oncology. Cell biology, biochemistry & biotechnology, and oncology are related fields. The final summary of research hotspots is exosomes and lung cancer, mesenchymal stem cell-derived exosomes and lung inflammation, and miRNAs in exosomes as biomarkers for respiratory illnesses. The present research situation and relevant hotspots of the area were analyzed through bibliometric studies on exosomes in respiratory diseases. The research development in this field has a considerable upside, and the exosome's function in diagnosing, treating, monitoring, and prognosis of respiratory illnesses cannot be taken lightly. Moreover, we believe the research results will bring the gospel to many patients with clinical respiratory diseases shortly.

Nanomolar range of FAM237B can activate receptor GPR83.

Our recent study confirmed that the mature neuropeptide FAM237A, also known as neurosecretory protein GL (NPGL), is an efficient agonist for GPR83. The paralog FAM237B was previously reported as a weak agonist for GPR83. In the present study, we prepared mature human FAM237B via an intein-fusion approach and demonstrated that it could cause a significant activation effect at the nanomolar range (1‒10 nM) in a NanoBiT-based ß-arrestin recruitment assay. Thus, FAM237B appears to be another endogenous agonist for GPR83 and future in vivo studies will be required to confirm this.

Construction of lncRNA-miRNA-mRNA regulatory network in severe asthmatic bronchial epithelial cells: A bioinformatics study.

Asthma is a chronic respiratory disease caused by environment-host interactions. Bronchial epithelial cells (BECs) are the first line of defense against environmental toxins. However, the mechanisms underlying the role of BECs in severe asthma (SA) are not yet fully understood. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) have been shown to play important roles in the regulation of gene expression in the pathogenesis of SA. In this study, bioinformatics was used for the first time to reveal the lncRNA-miRNA-mRNA regulatory network of BECs in SA. Five mRNA datasets of bronchial brushing samples from patients with SA and healthy controls (HC) were downloaded from the Gene Expression Omnibus (GEO) database. A combination of the Venn diagram and robust rank aggregation (RRA) method was used to identify core differentially expressed genes (DEGs). Protein-protein interaction (PPI) analysis of core DEGs was performed to screen hub genes. The miRDB, miRWalk, and ENCORI databases were used to predict the miRNA-mRNA relationships, and the ENCORI and starBase v2.0 databases were used to predict the upstream lncRNAs of the miRNA-mRNA relationships. Four core DEGs were identified: carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), interleukin-1 receptor type 2 (IL1R2), trefoil factor 3 (TFF3), and vascular endothelial growth factor A (VEGFA). These 4 core DEGs indicated that SA was not significantly associated with sex. Enrichment analysis showed that the MAPK, Rap1, Ras, PI3K-Akt and Calcium signaling pathways may serve as the principal pathways of BECs in SA. A lncRNA-miRNA-mRNA regulatory network of the severe asthmatic bronchial epithelium was constructed. The top 10 competing endogenous RNAs (ceRNAs) were FGD5 antisense RNA 1 (FGD5-AS1), metastasis associated lung adenocarcinoma transcript 1 (MALAT1), X inactive specific transcript (XIST), HLA complex group 18 (HCG18), small nucleolar RNA host gene 16 (SNHG16), has-miR-20b-5p, has-miR-106a-5p, hsa-miR-106b-5p, has-miR-519d-3p and Fms related receptor tyrosine kinase 1 (FLT1). Our study revealed a potential mechanism for the lncRNA-miRNA-mRNA regulatory network in BECs in SA.

The ghrelin receptor GHSR has two efficient agonists in the lobe-finned fish Latimeria chalumnae.

The peptide hormone ghrelin (an agonist) and LEAP2 (an antagonist) play important functions in energy metabolism via their receptor GHSR, an A-class G protein-coupled receptor. Ghrelin, LEAP2, and GHSR are widely present from fishes to mammals. However, our recent study suggested that fish GHSRs have different binding properties to ghrelin: a GHSR from the lobe-finned fish Latimeria chalumnae (coelacanth) is efficiently activated by ghrelin, but GHSRs from the ray-finned fish Danio rerio (zebrafish) and Larimichthys crocea (large yellow croaker) have lost binding to ghrelin. Do fish GHSRs use another peptide as their agonist? In the present study we tested to two fish motilins from D. rerio and L. chalumnae because motilin is distantly related to ghrelin. In ligand binding and activation assays, the fish GHSRs from D. rerio and L. crocea displayed no detectable or very low binding to all tested motilins; however, the fish GHSR from L. chalumnae bound to its motilin with high affinity and was efficiently activated by it. Therefore, it seemed that motilin is not a ligand for GHSR in the ray-finned fish D. rerio and L. crocea, but is an efficient agonist for GHSR in the lobe-finned fish L. chalumnae, one of the closest fish relatives of tetrapods. The results of present study suggested that GHSR might have two efficient agonists, ghrelin and motilin, in ancient fishes; however, this feature might be only preserved in some extant fishes with ancient evolutionary origins.

Heterophyllin B ameliorates gastric cancer tumor growth through activating ER stress.

BACKGROUND: Gastric cancer (GC) is the third leading cause of cancer-related death worldwide. Heterophyllin B (HB) has been proved to be a potential drug in cancer treatment. METHODS: In the current study, GC cells were treated with 0, 10, 25, or 50 µM of HB. Cell viability was determined by utilizing MTT assay. Flow cytometry was carried out for cell apoptosis and cell cycle analysis. The expression levels of IRE1, CHOP, GRP78 and Bcl-2 in cells and tumors were measured by Western blot and immunohistochemistry, respectively. RESULTS: Our data uncovered that HB administration significantly suppressed GC cell viability, but facilitated GC cell apoptosis and cell cycle arrest at G0/G1 phase. The effects of HB on GC cell proliferation, apoptosis and cell cycle showed dosage-dependent manner. Furthermore, expression of ER stress-associated proteins like IRE1, CHOP and GRP78 was markedly upregulated, while anti-apoptosis protein Bcl2 expression was inhibited by HB treatment in a dosage-dependent manner. Our data indicated that HB treatment facilitated caspase-3 expression in a dose-dependent manner, but had no effect on caspase-8 expression. Importantly, the inhibition of HB to GC cell apoptosis and cell cycle process and the promotion of HB to GC cell proliferation were partly rescued by inhibition of ER stress utilizing 4-PBA. In animal experiments, HB administration suppressed GC tumor growth, boosted IRE1, CHOP and GRP78 expression and inhibited Bcl-2 expression. CONCLUSION: All in all, HB treatment could effectively suppress GC cells proliferation and tumors growth and facilitate GC cells apoptosis and cell cycle arrest through activating ER stress. Our data indicated that HB may be a potential drug for GC treatment.

High-Gain Millimeter-Wave Beam Scanning Transmitarray Antenna.

In this article, a high-gain millimeter-wave transmitarray antenna (TAA) maintaining scanning ability is developed, integrating an array feed as the primary emitter. The work is achieved within a limited aperture area, avoiding the replacement or extension of the array. The addition of a set of defocused phases along the scanning direction to the phase distribution of the monofocal lens allows the converging energy to be dispersed into the scanning scope. The beam forming algorithm proposed in this article can determine the excitation coefficients of the array feed source, and is beneficial to improve the scanning capability in array-fed transmitarray antennas. A transmitarray based on the square waveguide element illuminated by an array feed is designed with a focal-to-diameter ratio (F/D) of 0.6. A 1-D scan with a scope of -5° to 5° is realized through calculation. The measured results show that the transmitarray can achieve a high gain, 37.95 dBi at 160 GHz, although a maximum 2.2 dB error appears compared with the calculation in the operating band of 150-170 GHz. The proposed transmitarray has been proven to generate scannable high-gain beams in the millimeter-wave band and is expected to demonstrate its potential in other applications.

Magnetic loofah sponge biochar facilitates microbial interspecies cooperation in surface and subsurface sediments for enhanced PAH biodegradation.

Magnetic biochar had been used for the bioremediation of polycyclic aromatic hydrocarbon (PAH)-contaminated sediments. However, the long-term remediation pattern of vertical stratification driven by the application of magnetic biochar and the assembly of microbes had received little attention. In this study, magnetic loofah sponge biochar (MagLsBC), magnetic iron oxide (MagOx) and magnetic coconut shell activated carbon (MagCoAC) were applied for the 900-day remediation of contaminated sediments. Significant (p < 0.05) PAH biodegradation was observed in both the surface and subsurface sediments with MagLsBC addition. However, enhanced PAH biodegradation was observed only in the surface sediments with MagOx and MagCoAC treatments. Magnetotactic bacteria (Magnetococcus) was dominant genera in surface sediments and indigenous PAH degradation bacteria were more abundant in subsurface sediments of MagLsBC relative to other bacterial communities. The network interaction between microbes in surface and subsurface sediments with MagLsBC treatments was a less complex and tighter than those with MagCoAC, MagOx or Control treatments. Long-distance electron transfer rates could be enhanced through cooperation between magnetotactic bacteria and indigenous degradation bacteria, thus accelerating PAH degradation in sediment with MagLsBC treatment, especially in the underlying sediment.

Splicing oxygen-rich multidentate ligands and characteristic metals to construct flame colorants: abundant structures and attractive colors.

Tetranitroethane (TNE), an energetic compound with high-nitrogen (N%, 26.7%) and oxygen (O%, 60.9%) content, is deprotonated by alkali and alkaline earth metal bases to form the corresponding metal salts of TNE which are characterized by FT-IR spectroscopy, elemental analysis, and single crystal X-ray diffraction. All the prepared energetic metal salts show excellent thermal stabilities, and the decomposition temperatures of EP-3, EP-4, and EP-5 are higher than 250 °C, due to the numerous coordination bonds of the complexes. Furthermore, the energy of formation of the nitrogen-rich salts were calculated utilizing heat of combustion. The detonation performances were calculated with the EXPLO5 software, and the impact and friction sensitivities were determined. EP-7 shows excellent energy performance (P = 30.0 GPa, VD = 8436 m s-1). EP-3, EP-4, EP-5, and EP-8 are more sensitive to mechanical stimulation. These alkali and alkaline earth metal salts of TNE show good monochromaticity by atomic emission spectroscopy (visible light), and may be used as potential flame colorants in pyrotechnics.

FAM237A, rather than peptide PEN and proCCK56-63, binds to and activates the orphan receptor GPR83.

G protein-coupled receptor 83 (GPR83) is primarily expressed in the brain and is implicated in the regulation of energy metabolism and some anxiety-related behaviours. Recently, the PCSK1N/proSAAS-derived peptide PEN, the procholecystokinin-derived peptide proCCK56-63, and family with sequence similarity 237 member A (FAM237A) were all reported as efficient agonists of GPR83. However, these results have not yet been reproduced by other laboratories and thus GPR83 is still officially an orphan receptor. The peptide PEN and proCCK56-63 share sequence similarity; however, they are completely different from FAM237A. To identify its actual ligand(s), in the present study we developed NanoLuc Binary Technology (NanoBiT)-based ligand-binding assay, fluorescent ligand-based visualization, and NanoBiT-based ß-arrestin recruitment assay for human GPR83. Using these assays, we demonstrated that mature human FAM237A could bind to GPR83 with nanomolar range affinity, and could activate this receptor and induce its internalization with nanomolar range efficiency in transfected human embryonic kidney 293T cells. However, we did not detect any interaction of PEN and proCCK56-63 with GPR83 using these assays. Thus, our results confirmed that FAM237A is an efficient agonist of GPR83, but did not support PEN and proCCK56-63 as ligands of this receptor. Clarification of their pairing paves the way for further functional studies of the brain-specific receptor GPR83 and the so far rarely studied neuropeptide FAM237A in the future.