Fragment-Based Anti-inflammatory Agent Design and Target Identification: Discovery of AF-45 as an IRAK4 Inhibitor to Treat Ulcerative Colitis and Acute Lung Injury.

UC and ALI are inflammatory diseases with limited treatment in the clinic. Herein, fragment-based anti-inflammatory agent designs were carried out deriving from cyclohexylamine/cyclobutylamine and several fragments from anti-inflammatory agents in our lab. AF-45 (IC50 = 0.53/0.60 µM on IL-6/TNF-α in THP-1 macrophages) was identified as the optimal molecule using ELISA and MTT assays from the 33 synthesized compounds. Through mechanistic studies and a systematic target search process, AF-45 was found to block the NF-κB/MAPK pathway and target IRAK4, a promising target for inflammation and autoimmune diseases. The selectivity of AF-45 targeting IRAK4 was validated by comparing its effects on other kinase/nonkinase proteins. In vivo, AF-45 exhibited a good therapeutic effect on UC and ALI, and favorable PK proprieties. Since there are currently no clinical or preclinical trials for IRAK4 inhibitors to treat UC and ALI, AF-45 provides a new lead compound or candidate targeting IRAK4 for the treatment of these diseases.

Transcriptome analysis of East Asian common octopus, Octopus sinensis, paralarvae.

BACKGROUND: The genes involved in cephalopod development and their association with hatching and survival during early life stages have been extensively studied. However, few studies have investigated the paralarvae transcriptome of the East Asian common octopus (Octopus sinen sis). OBJECTIVE: This study aimed to identify the genes related to embryonic development and hatching in O. sinensis using RNA sequencing (RNA-seq) and verify the genes most relevant to different embryonic stages. METHODS: RNA samples from hatched and 25 days post-hatching (dph) O. sinensis paralarvae were used to construct cDNA libraries. Clean reads from individual samples were aligned to the reference O. sinensis database to identify the differentially expressed genes (DEGs) between the 0- and 25-dph paralarvae libraries. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to supplement the RNA-seq data for embryogenic developmental stages. RESULTS: A total of 12,597 transcripts were annotated and 5,468 DEGs were identified between the 0- and 25-dph O. sinensis paralarvae, including 2,715 upregulated and 2,753 downregulated transcripts in the 25-dph paralarvae. Several key DEGs were related to transmembrane transport, lipid biosynthesis, monooxygenase activity, lipid transport, neuropeptide signaling, transcription regulation, and protein-cysteine S-palmitoyltransferase activity during the post-hatching development of O. sinensis paralarvae. RT-qPCR analysis further revealed that SLC5A3A, ABCC12, and NPC1 transcripts in 20 and/or 30 days post-fertilization (dpf) embryos were significantly higher (p < 0.05) than those in 10-dpf embryos. CONCLUSION: Transcriptome profiles provide molecular targets to understand the embryonic development, hatching, and survival of O. sinensis paralarvae, and enhance octopus production.

Articular Fragment Detachment and Separate Fixation for the Treatment of Comminuted Patellar Fractures with Coronal Split Articular Fragments: Articular Detachment Technique.

Background: Treatment of comminuted patellar fractures accompanied by coronal split articular and inferior pole fragments is a challenge. To treat this difficult fracture, we perform articular fragment detachment and separate fixation for coronal split articular and inferior pole fragments. We aimed to evaluate the radiological and clinical outcomes of our technique in comminuted patellar fractures at least 1 year after surgery. Methods: Between January 2019 and June 2022, 15 patients diagnosed with comminuted patellar fractures accompanied by coronal split articular and inferior pole fragments based on preoperative computed tomography underwent surgery using the articular detachment technique. The key point of this technique was anatomical reduction and subchondral fixation of the coronal split articular fragment to the superior main fragment after complete detachment of the coronal split fragment from the inferior pole. The remaining inferior pole was fixed using a separate construct. Postoperative articular gap, articular step-off, and complications, including resorption, reduction loss, and avascular necrosis of fixed articular fragments, were evaluated as radiological outcomes. Range of motion and the Lysholm scores were used to evaluate clinical outcomes. Results: Among the 15 patients, the coronal split articular fragments were fixed using Kirschner wires in 13 patients and headless screws in 2 patients. The inferior poles were fixed using separate vertical wiring in 13 patients and tension-band wiring in 2 patients. A postoperative articular gap was noted in 7 patients, with an average articular gap of 1.0 mm (range, 0.7-1.6 mm). No articular step-off was observed. Bone union and normal range of motion were achieved in all patients. On the 1-year postoperative lateral radiograph, resorption of the articular fracture site was seen in 5 patients. There was no loss of reduction or avascular necrosis of the coronal split articular fragments. The average postoperative Lysholm score at 1 year was 89.3 ± 4.1 (range, 82-95). Conclusions: The technique would be a reliable and safe option for the surgical treatment of comminuted patellar fractures accompanied by coronal split articular and inferior pole fragments in terms of anatomical reduction and stable fixation of articular fragments without risk of avascular necrosis.

A randomized controlled trial of immediate implant placement comparing hydroxyapatite nano-coated and uncoated sandblasted/acid-etched implants using a digital surgical guide.

PURPOSE: This study evaluated the implant stability, volumetric changes, and patient-reported outcome measures (PROMs) of hydroxyapatite (HA) nano-coated sandblasted/acid-etched (SLA) implants compared to uncoated SLA implants. METHODS: Forty patients were recruited and randomly allocated to HA nano-coated SLA group (test, n = 20) and uncoated SLA group (control, n = 20) using single-blinded/block randomization. Implants were immediately placed in maxillary posterior region using a digital surgical guide. Insertion torque and implant stability quotient (ISQ) were measured at implant surgery and 1, 2, 3, and 4 months postoperatively. Intraoral scans, PROMs and soft tissue inflammation data were collected, and multivariable linear regression analysis of ISQ was performed. RESULTS: In total, 48 implants (test; n = 24, control; n = 24) in 37 patients (test; n = 19, control; n = 18) were analyzed. Despite no significant between-group difference at surgery, the test group showed higher ISQ values than the control group at 2 (76.53 ± 4.17 vs. 71.32 ± 4.79, p < 0.01), 3 (77.45 ± 4.41 vs. 73.85 ± 4.69, p < 0.05), and 4 months (79.08 ± 2.96 vs. 73.43 ± 3.52, p < 0.0001) postoperatively. There were no significant differences in linear and volumetric changes, PROMs, and soft tissue inflammation analysis between two groups. The ISQ at implant surgery was influenced by age and diabetes mellitus (DM) at the implant level and DM and predicted total bone-to-implant contact area at the patient level. CONCLUSION: HA nano-coated SLA implants promoted favorable immediate implants stability during early osseointegration phase compared to uncoated SLA implants, but displayed similar dimensional changes, PROMs, and soft tissue inflammation outcomes. TRIAL REGISTRATION: Clinical Research Information Service (CRIS), KCT0006364. Registered 21 July 2021, https://cris.nih.go.kr/cris/search/detailSearch.do?seq=24221&search_page=L .

Preclinical comparison study of experimental peri-implantitis between alveolar ridge preservation and spontaneous healing sites in infected and noninfected tooth: a randomized blinded in vivo study.

BACKGROUND: This study compared the progression of experimental peri-implantitis between alveolar ridge preservation (ARP) and spontaneous healing (SH) sites in infected (IT) and noninfected tooth (NIT). METHODS: Bilateral mandibular third or fourth premolars of six beagle dogs were randomly assigned to IT and NIT groups. Before extraction, chronic dehiscence defects were created at the mesial root of mid-buccal area in IT group. Four weeks later, the mesial roots of the third and fourth premolars were extracted in all groups.ARP procedure was randomly conducted on one side of the extraction sockets using collagenated bovine bone substitutes and resorbable collagen membrane, and contralateral side was allowded spontaneous healing. After 12 weeks of healing, bone-level implants (ϕ 3.6 × 8.0 mm) were placed at the extraction sockets. Three months of ligature induced peri-implantitis and three months of spontaneous progression were allowed, with radiographs taken at each phase. Biopsies were retrieved at the implant site for histomorphometric, immunohistochemical, and polarized light-microscopic analyses. RESULTS: Radiography demonstrated that the changes in the marginal bone level during the spontaneous progression period showed no significant differences between ARP and SH sites. Only small and/or nonsignificant differences in the progression of peri-implantitis were observed between ARP and SH sites in histomorphometric, immunohistochemical, and polarized light microscopic analyses. Additionally, the IT and NIT groups exhibited similar outcomes for most parameters. CONCLUSION: ARP with xenogenic bone substitutes might provide similarly robust results as SH sites regarding the progression of experimental peri-implantitis, irrespective of the infected or noninfected nature of the site before tooth extraction.

Predictive Synthesis of Transition Metal Carbide via Thermochemical Oxocarbon Equilibrium.

Fabricating nanoscale metal carbides is a great challenge due to them having higher Gibbs free energy of formation (ΔG°) values than other metal compounds; additionally, these carbides have harsh calcination conditions, in which metal oxidation is preferred in the atmosphere. Herein, we report oxocarbon-mediated calcination for the predictive synthesis of nanoscale metal carbides. The thermochemical oxocarbon equilibrium of CO-CO2 reactions was utilized to control the selective redox reactions in multiatomic systems of Mo-C-O, contributing to the phase-forming and structuring of Mo compounds. By harnessing the thermodynamically predicted processing window, we controlled a wide range of Mo phases (MoO2, α-MoC1-x, and ß-Mo2C) and nanostructures (nanoparticle, spike, stain, and core/shell) in the Mo compounds/C nanofibers. By inducing simultaneous reactions of C-O (selective C combustion) and Mo-C (Mo carbide formation) in the nanofibers, Mo diffusion was controlled in C nanofibers, acting as a template for the nucleation and growth of Mo carbides and resulting in precise control of the phases and structures of Mo compounds. The formation mechanism of nanostructured Mo carbides was elucidated according to the CO fractions of CO-CO2 calcination. Moreover, tungsten (W) and niobium (Nb) carbides/C nanofibers have been successfully synthesized by CO-CO2 calcination. We constructed the thermodynamic map for the predictive synthesis of transition metal carbides to provide universal guideline via thermochemical oxocarbon equilibrium. We revealed that our thermochemical oxocarbon-mediated gas-solid reaction enabled the structure and phase control of nanoscale transition metal compounds to optimize the material-property relationship accordingly.

Inhibition of endocytic uptake of severe acute respiratory syndrome coronavirus 2 and endo-lysosomal acidification by diphenoxylate.

Cell culture-based screening of a chemical library identified diphenoxylate as an antiviral agent against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The observed 50% effective concentrations ranged between 1.4 and 4.9 µM against the original wild-type strain and its variants. Time-of-addition experiments indicated that diphenoxylate is an entry blocker targeting a host factor involved in viral infection. Fluorescence microscopic analysis visualized that diphenoxylate prevented SARS-CoV-2 particles from penetrating the cell membrane and also impaired endo-lysosomal acidification. Diphenoxylate exhibited a synergistic inhibitory effect on SARS-CoV-2 infection in human lung epithelial Calu-3 cells when combined with a transmembrane serine protease 2 (TMPRSS2) inhibitor, nafamostat. This synergy suggested that efficient antiviral activity is achieved by blocking both TMPRSS2-mediated early and endosome-mediated late SARS-CoV-2 entry pathways. The antiviral efficacy of diphenoxylate against SARS-CoV-2 was reproducible in a human tonsil organoids system. In a transgenic mouse model expressing the obligate SARS-CoV-2 receptor, human angiotensin-converting enzyme 2, intranasal administration of diphenoxylate (10 mg/kg/day) significantly reduced the viral RNA copy number in the lungs by 70% on day 3. This study underscores that diphenoxylate represents a promising core scaffold, warranting further exploration for chemical modifications aimed at developing a new class of clinically effective antiviral drugs against SARS-CoV-2.

Design, synthesis and bioactivity evaluation of 4-hydroxycoumarin derivatives as potential anti-inflammatory agents against acute lung injury and colitis.

Acute lung injury (ALI) and inflammatory bowel disease (IBD) are common inflammatory illnesses that seriously affect people's health. Herein, a series of 4-hydroxylcoumarin (4-HC) derivatives were designed and synthesized. The inhibitory effects of these compounds on LPS-induced interleukin-6 (IL-6) release from J774A.1 cells were then screened via ELISA assay, compound B8 showed 3 times more active than the lead compound 4-HC. The most active compound B8 had the IC50 values of 4.57 µM and 6.51 µM for IL-6 release on mouse cells J774A.1 and human cells THP-1, respectively. Furthermore, we also found that B8 could act on the MAPK pathway. Based on the target prediction results of computer virtual docking, kinase inhibitory assay was carried out, and it revealed that targeting IRAK1 was a key mechanism for B8 to exert anti-inflammatory activity. Moreover, B8 exerted a good therapeutic effect on the dextran sulfate sodium (DSS)-induced colitis model and liposaccharide (LPS)-induced ALI mouse models. The acute toxicity experiments indicated that high-dose B8 caused no adverse reactions in mice, confirming its safety in vivo. Additionally, the preliminary pharmacokinetic (PK) parameters of B8 in SD rats were also examined, revealing a bioavailability (F) of 28.72 %. In conclusion, B8 is a potential candidate of drug for the treatment of ALI and colitis.

Characterization of myoinhibitory peptide signaling system and its implication in larval metamorphosis and spawning behavior in Pacific abalone.

Myoinhibitory peptides (MIPs) affect various physiological functions, including juvenile hormone signaling, muscle contraction, larval development, and reproduction in invertebrates. Although MIPs are ligands for MIP and/or sex peptide receptors (MIP/SPRs) in diverse arthropods and model organisms belonging to Lophotrochozoa, the MIP signaling system has not yet been fully investigated in mollusks. In this study, we identified the MIP signaling system in the Pacific abalone Haliotis discus hannai (Hdh). Similar to the invertebrate MIPs, a total of eight paracopies of MIPs (named Hdh-MIP1 to Hdh-MIP8), harboring a WX5-7Wamide motif, except for Hdh-MIP2, were found in the Hdh-MIP precursor. Furthermore, we characterized a functional Hdh-MIPR, which responded to the Hdh-MIPs, except for Hdh-MIP2, possibly linked with the PKC/Ca2+ and PKA/cAMP signaling pathways. Hdh-MIPs delayed larval metamorphosis but increased the spawning behavior. These results suggest that the Hdh-MIP signaling system provides insights into the unique function of MIP in invertebrates.

The adjunctive effect of polydeoxyribonucleotide on bone formation in alveolar ridge preservation: A pre-clinical in vivo study.

AIM: This study investigated the adjunctive effect of polydeoxyribonucleotide (PDRN) on bone formation in alveolar ridge preservation (ARP) sockets. MATERIALS AND METHODS: Both mandibular second, third and fourth premolars of eight beagle dogs were randomly divided into ARP and ARP/PDRN groups. Following tooth extraction, ARP procedures were conducted using collagenized alloplastic graft material and bilayer collagen membrane soaked with normal saline (ARP group) or PDRN (ARP/PDRN group) for 10 min before application. Both groups were also randomly allocated to 2-, 4- or 12-week healing subgroups. The primary endpoint of this study was to compare histomorphometric differences between ARP and ARP/PDRN. The secondary endpoints of this study were to compare micro-CT analysis and three-dimensional volumetric measurement between the two groups. RESULTS: In the histomorphometric analysis, the ARP/PDRN group exhibited greater new bone formation at coronal, middle and total position compared with the ARP group at 2-week healing. The number of newly formed blood vessels was higher in the ARP/PDRN group than in the ARP group at 2- and 4-week healing. In micro-CT analysis, the mean new bone volume/total bone volume between ARP and ARP/PDRN was statistically significant at 2-week healing. Ridge volume alterations were significantly decreased in the ARP/PDRN group during entire healing time compared with the ARP group, especially on the buccal side. CONCLUSIONS: The application of PDRN in ARP might provide additional benefits for early bone regeneration and maintenance of buccal ridge volume.

Mass spectrometry-based ginsenoside profiling: Recent applications, limitations, and perspectives.

Ginseng, the roots of Panax species, is an important medicinal herb used as a tonic. As ginsenosides are key bioactive components of ginseng, holistic chemical profiling of them has provided many insights into understanding ginseng. Mass spectrometry has been a major methodology for profiling, which has been applied to realize numerous goals in ginseng research, such as the discrimination of different species, geographical origins, and ages, and the monitoring of processing and biotransformation. This review summarizes the various applications of ginsenoside profiling in ginseng research over the last three decades that have contributed to expanding our understanding of ginseng. However, we also note that most of the studies overlooked a crucial factor that influences the levels of ginsenosides: genetic variation. To highlight the effects of genetic variation on the chemical contents, we present our results of untargeted and targeted ginsenoside profiling of different genotypes cultivated under identical conditions, in addition to data regarding genome-level genetic diversity. Additionally, we analyze the other limitations of previous studies, such as imperfect variable control, deficient metadata, and lack of additional effort to validate causation. We conclude that the values of ginsenoside profiling studies can be enhanced by overcoming such limitations, as well as by integrating with other -omics techniques.

Dihydroartemisinin, a potential PTGS1 inhibitor, potentiated cisplatin-induced cell death in non-small cell lung cancer through activating ROS-mediated multiple signaling pathways.

Dihydroartemisinin (DHA) exerts an anti-tumor effect in multiple cancers, however, the molecular mechanism of DHA and whether DHA facilitates the anti-tumor efficacy of cisplatin in non-small cell lung cancer (NSCLC) are unclear. Here, we found that DHA potentiated the anti-tumor effects of cisplatin in NSCLC cells by stimulating reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress, C-Jun-amino-terminal kinase (JNK) and p38 MAPK signaling pathways both in vitro and in vivo. Of note, we demonstrated for the first time that DHA inhibits prostaglandin G/H synthase 1 (PTGS1) expression, resulting in enhanced ROS production. Importantly, silencing PTGS1 sensitized DHA-induced cell death by increasing ROS production and activating ER-stress, JNK and p38 MAPK signaling pathways. In summary, our findings provided new experimental basis and therapeutic prospect for the combined therapy with DHA and cisplatin in some NSCLC patients.

Validation of Korean Olfactory Questionnaire for Perioperative Olfactory Assessment in Endoscopic Endonasal Skull Base Surgery.

Objective: To analyze changes in olfactory function after endoscopic endonasal skull base surgery and compare performance of the olfactory questionnaire with those of conventional psychophysical tests. Methods: Patients were classified into 5 categories for olfactory function evaluation (normal, mild hyposmia, moderate hyposmia, severe hyposmia, and anosmia) based on a self-assessment. Patients also underwent the butanol threshold test (BTT), Cross-Cultural Smell Identification Test (CCSIT), and 11-item olfactory questionnaire. Subjects with normosmia preoperatively and who were followed up at least 6 months after surgery were analyzed. Receiver operating characteristic curves and confusion matrix analysis were performed for BTT, CCSIT, and olfactory questionnaire to compare their diagnostic abilities. The effects of age, preoperative olfaction, septal flap, tumor pathology, and tumor size on postoperative olfaction were evaluated using multivariate linear regression analysis. Results: Data from 108 patients were analyzed. Postoperative changes in the olfactory questionnaire were significantly associated with changes in the BTT and CCSIT. The area under the curve for postoperative self-olfactory function classification was highest for olfactory questionnaire (0.894), followed by BTT (0.767) and CCSIT (0.688). Patient age at the time of surgery and preoperative BTT score were significantly related to postoperative olfactory outcomes. Conclusion: The olfactory questionnaire correlated well with conventional psychosomatic olfactory function tests. In combination with clinical parameters and preoperative psychosomatic olfactory function tests, the olfactory questionnaire is suitable for assessing subjective olfactory function after endoscopic endonasal skull base surgery.

Discovery of novel osthole derivatives exerting anti-inflammatory effect on DSS-induced ulcerative colitis and LPS-induced acute lung injury in mice.

The modification based on natural products is a practical way to find anti-inflammatory drugs. In this study, 26 osthole derivatives were synthesized, and their anti-inflammatory properties were evaluated. The preliminary activity study revealed that most osthole derivatives could effectively inhibit inflammatory cytokines IL-6 secretion in LPS stimulated mouse macrophages J774A.1. Compound 7m exhibited the most effective anti-inflammatory activity (RAW264.7 IL-6 IC50: 4.57 µM, 32 times more active than osthole) in vitro with no significant influence on cell proliferation. Additionally, the mechanistic analysis demonstrated that compound 7m could block MAPK signal transduction by inhibiting the phosphorylation of JNK and p38, thereby inhibiting the release of inflammatory cytokines. Moreover, in vivo functional investigations revealed that 7m could substantially reduce DSS-induced ulcerative colitis and LPS-induced acute lung injury, with good therapeutic effects. The pharmacokinetics and acute toxicity experiments proved the safety and reliability of 7min vivo. Overall, Compound 7m could further be studied as potential anti-inflammatory candidate.

Crustacean cardioactive peptide signaling system in the gastropod mollusk Pacific abalone.

Crustacean cardioactive peptide (CCAP) signaling systems have been characterized in a diverse range of protostomes, representatively in arthropods. The cyclic CX5C-type CCAP regulates various biological activities through CCAP receptors (CCAPRs), which are orthologous to neuropeptide S receptors (NPSRs) in deuterostomes. However, the CCAPRs of the lophotrochozoa remain poorly characterized; therefore, the relationship between the CCAP, NPS, and CX4C-type oxytocin/vasopressin (OT/VP) signaling systems is unclear. In this study, we identified a CCAP precursor and two CCAPR isoforms in the Pacific abalone (Haliotis discus hannai; Hdh). The Hdh-CCAP precursor was found to harbor three CX5C-type and one CX4C-type CCAPs. The Hdh-CCAPRs displayed homology with protostome CCAPRs and deuterostome NPSRs, having characteristics of the rhodopsin-type G protein-coupled receptors. Phylogenetic analysis showed that lophotrochozoan CCAPRs, including Hdh-CCAPRs, form a monophyletic group distinct from arthropod CCAPRs. Reporter assays demonstrated that all examined Hdh-CCAPs and insect CCAP-induced intracellular Ca2+ mobilization and cAMP accumulation in Hdh-CCAPR-expressing HEK293 cells, whereas none of the CCAP peptides inhibited the forskolin-stimulated cAMP signaling pathway even at micromolar concentrations. In silico ligand-receptor docking models showed that the N-terminal FCN motifs of Hdh-CCAPs are deeply inserted inside the binding pocket of Hdh-CCAPR, forming extensive hydrophobic interactions. In mature Pacific abalone, the transcripts for Hdh-CCAP precursor and Hdh-CCAPR were highly expressed in the neural ganglia compared to the peripheral tissues. Collectively, this study characterized the first CCAP signaling system linked to both Ca2+/PKC and cAMP/PKA signal transduction pathways in gastropod mollusks and gives insights into the evolutional origins of deuterostomian NPS and OT/VP signaling systems.

Functional Characterization of Gonadotropin-Releasing Hormone and Corazonin Signaling Systems in Pacific Abalone: Toward Reclassification of Invertebrate Neuropeptides.

INTRODUCTION: The proposed evolutionary origins and corresponding nomenclature of bilaterian gonadotropin-releasing hormone (GnRH)-related neuropeptides have changed tremendously with the aid of receptor deorphanization. However, the reclassification of the GnRH and corazonin (CRZ) signaling systems in Lophotrochozoa remains unclear. METHODS: We characterized GnRH and CRZ receptors in the mollusk Pacific abalone, Haliotis discus hannai (Hdh), by phylogenetic and gene expression analyses, bioluminescence-based reporter, Western blotting, substitution of peptide amino acids, in vivo neuropeptide injection, and RNA interference assays. RESULTS: Two Hdh CRZ-like receptors (Hdh-CRZR-A and Hdh-CRZR-B) and three Hdh GnRH-like receptors (Hdh-GnRHR1-A, Hdh-GnRHR1-B, and Hdh-GnRHR2) were identified. In phylogenetic analysis, Hdh-CRZR-A and -B grouped within the CRZ-type receptors, whereas Hdh-GnRHR1-A/-B and Hdh-GnRHR2 clustered within the GnRH/adipokinetic hormone (AKH)/CRZ-related peptide-type receptors. Hdh-CRZR-A/-B and Hdh-GnRHR1-A were activated by Hdh-CRZ (pQNYHFSNGWHA-NH2) and Hdh-GnRH (pQISFSPNWGT-NH2), respectively. Hdh-CRZR-A/-B dually coupled with the Gαq and Gαs signaling pathways, whereas Hdh-GnRHR1-A was linked only with Gαq signaling. Analysis of substituted peptides, [I2S3]Hdh-CRZ and [N2Y3H4]Hdh-GnRH, and in silico docking models revealed that the N-terminal amino acids of the peptides are critical for the selectivity of Hdh-CRZR and Hdh-GnRHR. Two precursor transcripts for Hdh-CRZ and Hdh-GnRH peptides and their receptors were mainly expressed in the neural ganglia, and their levels increased in starved abalones. Injection of Hdh-CRZ peptide into abalones decreased food consumption, whereas Hdh-CRZR knockdown increased food consumption. Moreover, Hdh-CRZ induced germinal vesicle breakdown in mature oocytes. CONCLUSION: Characterization of Hdh-CRZRs and Hdh-GnRHRs and their cognate peptides provides new insight into the evolutionary route of GnRH-related signaling systems in bilaterians.

Diagnostic performance of a point-of-care test device for periodontitis using human saliva: a single-center, single-blinded, confirmatory study.

OBJECTIVE: Recently, matrix metalloproteinase-8 (MMP-8) has been used to diagnose periodontal disease in a point-of-care (POC) test in order to save time and cost relative to the traditional diagnostic workflow. This study aimed to investigate the diagnostic performance of INCLIX TRF MMP-8, a POC testing device for periodontitis using the area under the receiver operating characteristic curve, sensitivity, specificity, and predictive values. MATERIALS AND METHODS: Full-mouth periodontal examination and radiographic analysis were used for evaluating periodontal condition based on the 2018 classification of periodontal disease. A dichotomous diagnosis of clinical periodontal condition was performed using the POC device. The relationships among periodontal condition and the concentration of MMP-8, tooth loss (TL), gingival index (GI), plaque index (PI), and alveolar bone loss (ABL) were assessed by the Spearman rank correlations (rs). RESULTS: In all, 108 cases of non-periodontitis (NP) and 191 cases of periodontitis (P), including 38 cases of periodontitis stage I, 42 cases of periodontitis stage II, 99 cases of periodontitis stage III, and 11 cases of periodontitis stage IV, were enrolled in this study. Diagnostic accuracy in assessing periodontal condition with the POC device improved when it was used with participants aged ≥ 40 years. There were weak positive correlations between periodontal condition and MMP-8 and between periodontal condition and GI (rs2 = 0.1124 and rs2 = 0.0906, respectively), whereas a strong positive correlation between periodontal condition and alveolar bone loss (rs2 = 0.6877) was observed. CONCLUSION: The POC device investigated in this study is a potential tool to distinguish between NP and P in individuals ≥ 40 years of age.

6-Pentyl-α-Pyrone from Trichoderma gamsii Exert Antioxidant and Anti-Inflammatory Properties in Lipopolysaccharide-Stimulated Mouse Macrophages.

Filamentous fungi produce several beneficial secondary metabolites, including bioactive compounds, food additives, and biofuels. Trichoderma, which is a teleomorphic Hypocrea that falls under the taxonomic groups Ascomycota and Dikarya, is an extensively studied fungal genus. In an ongoing study that seeks to discover bioactive natural products, we investigated potential bioactive metabolites from the methanolic extract of cultured Trichoderma gamsii. Using liquid chromatography-mass spectrometry (LC-MS), one major compound was isolated and structurally identified as 6-pentyl-α-pyrone (6PP) based on nuclear magnetic resonance data and LC-MS analysis. To determine its antioxidant and anti-inflammatory activity, as well as the underlying mechanisms, we treated lipopolysaccharide (LPS)-stimulated Raw264.7 mouse macrophages with 6PP. We found that 6PP suppresses LPS-induced increase in the levels of nitric oxide, a mediator of oxidative stress and inflammation, and restores LPS-mediated depletion of total glutathione by stabilizing nuclear factor erythroid 2-related factor 2 (Nrf2), an antioxidative factor, and elevating heme oxygenase-1 levels. Furthermore, 6PP inhibited LPS-induced production of proinflammatory cytokines, which are, at least in part, regulated by heme oxygenase-1 (HO-1). 6PP suppressed proinflammatory responses by inhibiting the nuclear localization of nuclear factor kappa B (NF-κB), as well as by dephosphorylating the mitogen-activated protein kinases (MAPKs). These results indicate that 6PP can protect macrophages against oxidative stress and LPS-induced excessive inflammatory responses by activating the Nrf2/HO-1 pathway while inhibiting the proinflammatory, NF-κB, and MAPK pathways.

Global Profiling of Lysine Acetylation and Lactylation in Kupffer Cells.

Among the various cell types that constitute the liver, Kupffer cells (KCs) are responsible for the elimination of gut-derived foreign products. Protein lysine acetylation (Kac) and lactylation (Kla) are dynamic and reversible post-translational modifications, and various global acylome studies have been conducted for liver and liver-derived cells. However, no such studies have been conducted on KCs. In this study, we identified 2198 Kac sites in 925 acetylated proteins and 289 Kla sites in 181 lactylated proteins in immortalized mouse KCs using global acylome technology. The subcellular distributions of proteins with Kac and Kla site modifications differed. Similarly, the specific sequence motifs surrounding acetylated or lactylated lysine residues also showed differences. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to better understand the differentially expressed proteins in the studies by Kac and Kla. In the newly identified Kla, we found K82 lactylation in the high-mobility group box-1 protein in the neutrophil extracellular trap formation category using KEGG enrichment analyses. Here, we report the first proteomic survey of Kac and Kla in KCs.

Inhibitory Effects of Ehretia tinifolia Extract on the Excessive Oxidative and Inflammatory Responses in Lipopolysaccharide-Stimulated Mouse Kupffer Cells.

Ehretia tinifolia (E. tinifolia) L., an evergreen tree with substantial biological activity, including antioxidant and anti-inflammatory effects, has been used in many herbal and traditional medicines. To elucidate its antioxidant and anti-inflammatory activity and the underlying mechanisms, we applied a methanol extract of E. tinifolia (ETME) to lipopolysaccharide (LPS)-stimulated mouse immortalized Kupffer cells. ETME suppressed the LPS-induced increase in nitric oxide, a mediator for oxidative stress and inflammation, and restored LPS-mediated depletion of total glutathione level by stabilizing antioxidative nuclear factor erythroid 2-related factor 2 (Nrf2) and the subsequent increase in heme oxygenase-1 levels. Furthermore, ETME inhibited the LPS-induced production of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6. The inhibitory effects of ETME on pro-inflammatory responses were regulated by ETME-mediated dephosphorylation of mitogen-activated protein kinases (MAPKs: p38, p44/p42, and stress-associated protein kinase/c-Jun N-terminal kinase) and inhibition of nuclear localization of nuclear factor kappa B (NF-κB). These results suggest that ETME is a possible candidate for protecting Kupffer cells from LPS-mediated oxidative stress and excessive inflammatory responses by activating antioxidant Nrf2/HO-1 and inhibiting pro-inflammatory NF-κB and MAPKs, respectively.