Efficacy of KPR-5714, a selective transient receptor potential melastatin 8 antagonist, on chronic psychological stress-induced bladder overactivity in male rats.

AIMS: Chronic psychological stress aggravates lower urinary tract symptoms. Among others, water avoidance stress is a chronic psychological stressor that plays a causal role in the exacerbation and development of bladder dysfunction in rats. In this report, the effects of KPR-5714, which is a selective transient receptor potential melastatin 8 (TRPM8) antagonist, on bladder overactivity induced by water avoidance stress were examined. METHODS: Male rats were subjected to water avoidance stress for 2 h per day for 10 consecutive days. The effects of water avoidance stress on voiding behavior using metabolic cages and histological bladder changes were investigated in rats. The involvement of bladder C-fiber afferent on voiding frequency in rats exposed to water avoidance stress was assessed using capsaicin. The effects of KPR-5714 on storage dysfunction in rats subjected to water avoidance stress were examined. RESULTS: In voiding behavior measurements, water avoidance stress-induced storage dysfunction, causing a decrease in the mean voided volume and increasing voiding frequency. A comparison of bladders from normal rats and rats exposed to water avoidance stress showed no histological differences. Water avoidance stress-induced bladder overactivity was completely inhibited by pretreatment with capsaicin. KPR-5714 showed a tendency to increase the mean voided volume and significantly decreased the voiding frequency without affecting the total voided volume in these rats. CONCLUSION: The results suggest that KPR-5714 is a promising option for treating chronic psychological stress-induced bladder overactivity.

[Embolization Using a Debranched Left Common Carotid Artery Stump for Gutter Endoleak After Thoracic Endovascular Aortic Repair with the Double-debranching and Chimney Technique].

An 88-year-old male underwent thoracic endovascular aortic repair (TEVAR) with the double-debranching and chimney technique for arch aortic aneurysm. When the aforementioned procedure was performed, the left common carotid artery was closed and transected, and the left subclavian artery was embolized and bypassed, respectively. However, postoperatively, the gutter endoleak persisted, and the aneurysm enlarged;therefore, requiring additional surgery. A skin incision was made on the left side of the neck, and the closed and dissected left common carotid artery stump was detected. A sheath was placed at the stump and an angiographic catheter and guidewire were used to retrograde cannulate the gutter beside the chimney graft, and coil embolization was performed. No endoleak was observed at postoperatively and 6-month follow up computed tomography( CT). We believe that embolization from a deblanched left common carotid artery stump is useful for endoleaks after TEVAR employing the chimney and debranching technique.

Direct cleavage during the first mitosis is a sign of abnormal fertilization in cattle.

Direct cleavage, a type of abnormal cleavage in which one zygote divides into three or more blastomeres, has been reported in mammals. The incidence of direct cleavage increases in zygotes with three or more pronuclei (multi-PN) and those showing abnormal pronuclei migration. However, there are few reports on the relationship between pronuclei and direct cleavage, and the effects of these relationships on subsequent embryogenesis have not been clarified. It is difficult to observe pronuclei under visible light, especially in bovine zygotes, because of abundant dark lipid droplets in the cytoplasm. We visualized pronuclei by removing lipid droplets from bovine zygotes and analyzed the relationship between the number of pronuclei and direct cleavage using time-lapse cinematography. The direct cleavage rate of multi-PN zygotes was 78.6%, which was significantly higher than that of zygotes with one pronucleus (1 PN, 0.0%) and two pronuclei (2 PN, 8.2%). Observation of pronuclei migration in 2 PN zygotes showed that 3.1% of 2 PN zygotes had non-apposed pronuclei. The direct cleavage rate of zygotes with non-apposed pronuclei was 66.7%, which was significantly higher than that of zygotes with apposed pronuclei (6.4%). Among multi-PN zygotes, the proportions of zygotes with apposed pronuclei and non-apposed pronuclei were 37.5% and 64.3%, respectively. The direct cleavage rate of multi-PN zygotes with non-apposed pronuclei was 100.0%, which was significantly higher than that of zygotes with apposed pronuclei (40.0%). Three-dimensional live-cell imaging of bovine zygotes injected with the mRNA-encoding histone H2B-mCherry showed that the direct cleavage rates of 2 PN and multi-PN zygotes bypassing syngamy were 63.2% and 75.5%, respectively. These rates were significantly higher than that of 2 PN and multi-PN zygotes that underwent syngamy (5.6% and 20.0%, respectively). Regardless of the number of pronuclei, a high frequency of direct cleavage was observed in zygotes in which the pronuclei did not migrate inward the cytoplasm and bypassed syngamy. These results suggest that abnormal fertilization such as multi-PN and migration error of pronuclei in cattle is the primary reason for direct cleavage during the first mitosis. Assessment of direct cleavage during the first mitosis allows exclusion of embryos with abnormal fertilization and may contribute to in vitro produced embryo transfer success.

Zamamiphidins B and C, Manzamine-Related Alkaloids from an Amphimedon sp. Marine Sponge Collected in Okinawa.

Zamamiphidins B (1) and C (2), two new manzamine-related alkaloids with an unprecedented fused diazahexacyclic ring system, were isolated from an Amphimedon sp. marine sponge collected in Okinawa. The structures of zamamiphidins B (1) and C (2) including the relative configurations were elucidated on the basis of spectroscopic data.

KPR-5714, a selective transient receptor potential melastatin 8 antagonist, improves voiding dysfunction in rats with bladder overactivity but does not affect voiding behavior in normal rats.

AIMS: Transient receptor potential melastatin 8 (TRPM8) has a role in the abnormal sensory transduction of the bladder and is involved in the pathophysiology of hyperactivity bladder disorders. The aim of this study is to examine the effects of KPR-5714, a novel and selective TRPM8 antagonist, on voiding dysfunction induced by bladder afferent hyperactivity via mechanosensitive C-fibers in rats. METHODS: The effects of intragastric administration of KPR-5714 on bladder overactivity induced by intravesical instillation of 10 mM ATP were investigated using cystometry in conscious female rats. We examined the effects of oral administration of KPR-5714 on voiding behavior using a metabolic cage in normal male rats and rats with an intratesticular injection of 3% acetic acid. RESULTS: In cystometry measurements, the intercontraction interval was decreased by intravesical ATP instillation. KPR-5714 (0.1, 0.3, and 1 mg/kg) dose-dependently prolonged the shortened intercontraction interval provoked by ATP. In voiding behavior measurements, intratesticular injection of acetic acid decreased the mean voided volume and increased voiding frequency. KPR-5714 (0.1 and 0.3 mg/kg) dose-dependently increased the mean voided volume and decreased voiding frequency without affecting the total voided volume in these rats. However, KPR-5714 (1 and 10 mg/kg) did not influence the voiding behavior in normal rats. CONCLUSION: The present results suggest that KPR-5714 improves voiding dysfunction by inhibiting the enhanced activity of mechanosensitive bladder C-fibers in rats with bladder overactivity and shows no significant change in voiding behavior in normal rats.

Cholestenone functions as an antibiotic against Helicobacter pylori by inhibiting biosynthesis of the cell wall component CGL.

Helicobacter pylori, a pathogen responsible for gastric cancer, contains a unique glycolipid, cholesteryl-α-D-glucopyranoside (CGL), in its cell wall. Moreover, O-glycans having α1,4-linked N-acetylglucosamine residues (αGlcNAc) are secreted from gland mucous cells of gastric mucosa. Previously, we demonstrated that CGL is critical for H. pylori survival and that αGlcNAc serves as antibiotic against H. pylori by inhibiting CGL biosynthesis. In this study, we tested whether a cholesterol analog, cholest-4-en 3-one (cholestenone), exhibits antibacterial activity against H. pylori in vitro and in vivo. When the H. pylori standard strain ATCC 43504 was cultured in the presence of cholestenone, microbial growth was significantly suppressed dose-dependently relative to microbes cultured with cholesterol, and cholestenone inhibitory effects were not altered by the presence of cholesterol. Morphologically, cholestenone-treated H. pylori exhibited coccoid forms. We obtained comparable results when we examined the clarithromycin-resistant H. pylori strain "2460." We also show that biosynthesis of CGL and its derivatives cholesteryl-6-O-tetradecanoyl-α-D-glucopyranoside and cholesteryl-6-O-phosphatidyl-α-D-glucopyranoside in H. pylori is remarkably inhibited in cultures containing cholestenone. Lastly, we asked whether orally administered cholestenone eradicated H. pylori strain SS1 in C57BL/6 mice. Strikingly, mice fed a cholestenone-containing diet showed significant eradication of H. pylori from the gastric mucosa compared with mice fed a control diet. These results overall strongly suggest that cholestenone could serve as an oral medicine to treat patients infected with H. pylori, including antimicrobial-resistant strains.

Morphokinetic analysis of pronuclei using time-lapse cinematography in bovine zygotes.

The morphokinetics of pronuclei (PN) are considered crucial factors affecting embryogenesis in mammals. Whereas, since bovine zygotes contain a large number of cytosolic lipid droplets, detailed observation of PN has not been performed. In this study, we visualized PN using time-lapse cinematography (TLC) with light microscopy for the first time in delipidated bovine zygotes. The proportions of 0 PN, 1PN, 2PN, and multi-PN in delipidated bovine zygotes were 10.1%, 6.5%, 72.7%, and 10.8%, respectively. Abnormal fertilization, including 1 PN and multi-PN, was observed in 15.6% of blastocysts. The times from IVF to PN appearance, PN fading, and first cleavage in 2 PN bovine zygotes that developed into blastocysts were 10.4, 25.5, and 27.6 h, respectively, which were similar to PN morphokinetics in humans. The 2 PN zygotes showed that the prolonged time from IVF to the appearance of PN and from the fading of PN to the first cleavage negatively affected blastocyst formation. The time from appearance to fading of PN in multi-PN zygotes that developed into blastocysts was longer than that in multi-PN zygotes that did not develop into blastocysts. Besides, among zygotes that developed into blastocysts, the time from appearance to fading of PN in multi-PN zygotes was longer than that in 2 PN and 1 PN zygotes. These results suggest that PN morphokinetic abnormalities are associated with subsequent embryonic development. Observation of PN in bovine zygotes by using non-invasive visible light TLC by delipidation could be a powerful tool to clarify the relationship between PN morphokinetics and developmental competence.

Efficacy of the combination of KPR-5714, a novel transient receptor potential melastatin 8 (TRPM8) antagonist, and ß3-adrenoceptor agonist or anticholinergic agent on bladder dysfunction in rats with bladder overactivity.

Transient receptor potential melastatin 8 (TRPM8) channels may contribute to the pathophysiological bladder afferent hyperactivity, thus a TRPM8 antagonist would be a promising therapeutic target for the bladder hypersensitive disorders including urinary urgency in overactive bladder (OAB). We aimed to investigate a pharmacological effect of KPR-5714, a novel selective TRPM8 antagonist, on TRPM8 channels, M3 receptors and ß3-adrenoceptors using the transfected cells of each gene at first. Then, combination effects of KPR-5714 and mirabegron, a ß3-adrenoceptor agonist, or tolterodine tartrate, an anticholinergic agent, were studied on rhythmic bladder contractions (RBCs) in normal rats and bladder function in frequent-voiding rats. In vitro measurements showed that KPR-5714 acts on neither ß3-adrenoceptor nor M3 receptor. In normal rats, KPR-5714 and mirabegron significantly reduced the frequency of RBCs, and a combined administration showed an additive effect. In rats with cerebral infarction, KPR-5714 and mirabegron significantly reduced the voiding frequency, and a combined administration showed an additive effect. In rats exposed to cold temperature, KPR-5714 and tolterodine tartrate significantly reduced the voiding frequency accompanied by the increased mean voided volume, and a combined administration showed additive effects. The present study demonstrated that the combined administration of KPR-5714 and mirabegron or tolterodine tartrate showed the additive effects on bladder dysfunction in different animal models, suggesting that the combination therapy of TRPM8 antagonist and ß3-adrenoceptor agonist or anticholinergic agent can be the potential treatment option for obtaining additive effects in comparison with monotherapy for OAB.

Identification of N-acyl-N-indanyl-α-phenylglycinamides as selective TRPM8 antagonists designed to mitigate the risk of adverse effects.

Transient receptor potential melastatin 8 (TRPM8), a temperature-sensitive ion channel responsible for detecting cold, is an attractive molecular target for the treatment of pain and other disorders. We have previously discovered a selective TRPM8 antagonist, KPR-2579, which inhibited bladder afferent hyperactivity induced by acetic acid instillation into the bladder. However, additional studies have revealed potential adverse effects with KPR-2579, such as the formation of a reactive metabolite, CYP3A4 induction, and convulsions. In this report, we describe the optimization of α-phenylglycinamide derivatives to mitigate the risk of these adverse effects. The optimal compound 13x exhibited potent inhibition against icilin-induced wet-dog shakes and cold-induced frequent voiding in rats, with a wide safety margin against the potential side effects.

Abundance of cell-free mitochondrial DNA in spent culture medium associated with morphokinetics and blastocyst collapse of expanded blastocysts.

PURPOSE: This retrospective observational study investigated relationships between the abundance of cell-free mitochondrial DNA (cf-mtDNA) in spent culture medium (SCM) of human-expanded blastocysts and their morphokinetics to address the question of whether the abundance of cf-mtDNA in SCM could predict the quality of blastocysts. METHODS: Embryos (n = 53) were individually cultured in a time-lapse incubator until they reached the expanded blastocyst stage (5 or 6 days), following which copy numbers of cf-mtDNA in SCM (20 µL) of expanded blastocysts were determined using real-time PCR. RESULTS: The duration between start of blastulation to expanded blastocyst (tEB-tSB) and between that of the blastocyst stage to expanded blastocyst (tEB-tB) significantly and positively correlated with the abundance of cf-mtDNA in the SCM (tEB-tSB: r = .46; P < .01; tEB-tB: r = .47; P < .01). The abundance of cf-mtDNA in the SCM was significantly greater in blastocysts with blastocyst collapse (BC), than without BC, and significantly and positively correlated with the number of BC. CONCLUSIONS: The abundance of cf-mtDNA in the SCM was associated with expansion duration and BC. Thus, cf-mtDNA abundance in the SCM serves as a marker to predict the quality of expanded blastocysts.

Integrated omics unveil the secondary metabolic landscape of a basal dinoflagellate.

BACKGROUND: Some dinoflagellates cause harmful algal blooms, releasing toxic secondary metabolites, to the detriment of marine ecosystems and human health. Our understanding of dinoflagellate toxin biosynthesis has been hampered by their unusually large genomes. To overcome this challenge, for the first time, we sequenced the genome, microRNAs, and mRNA isoforms of a basal dinoflagellate, Amphidinium gibbosum, and employed an integrated omics approach to understand its secondary metabolite biosynthesis. RESULTS: We assembled the ~ 6.4-Gb A. gibbosum genome, and by probing decoded dinoflagellate genomes and transcriptomes, we identified the non-ribosomal peptide synthetase adenylation domain as essential for generation of specialized metabolites. Upon starving the cells of phosphate and nitrogen, we observed pronounced shifts in metabolite biosynthesis, suggestive of post-transcriptional regulation by microRNAs. Using Iso-Seq and RNA-seq data, we found that alternative splicing and polycistronic expression generate different transcripts for secondary metabolism. CONCLUSIONS: Our genomic findings suggest intricate integration of various metabolic enzymes that function iteratively to synthesize metabolites, providing mechanistic insights into how dinoflagellates synthesize secondary metabolites, depending upon nutrient availability. This study provides insights into toxin production associated with dinoflagellate blooms. The genome of this basal dinoflagellate provides important clues about dinoflagellate evolution and overcomes the large genome size, which has been a challenge previously.

Agesasines A and B, Bromopyrrole Alkaloids from Marine Sponges Agelas spp.

Exploration for specialized metabolites of Okinawan marine sponges Agelas spp. resulted in the isolation of five new bromopyrrole alkaloids, agesasines A (1) and B (2), 9-hydroxydihydrodispacamide (3), 9-hydroxydihydrooroidin (4), and 9E-keramadine (5). Their structures were elucidated on the basis of spectroscopic analyses. Agesasines A (1) and B (2) were assigned as rare bromopyrrole alkaloids lacking an aminoimidazole moiety, while 3-5 were elucidated to be linear bromopyrrole alkaloids with either aminoimidazolone, aminoimidazole, or N-methylated aminoimidazole moieties.

Borderline Microenvironment Fibrosis Is a Novel Poor Prognostic Marker of Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: The tumor microenvironment (TME) balances tumor growth and suppression through humoral factors and cell-cell interactions. In oral squamous cell carcinoma (OSCC), TMEs have been associated with prognosis of cancer patients and are evaluated by microscopy; however, these methods of evaluation vary among studies. MATERIALS AND METHODS: To evaluate the TME, borderline microenvironment fibrosis (bMF) was evaluated histologically in 236 OSCC cases and used to determine the clinicopathological status. RESULTS: bMF was observed in 47% (110 in 236 cases) of OSCC cases and associated with higher T category, N category, stage, histological grade and mode of invasion. bMF-positive was related to overall survival (OS) and progression-free survival (PFS). Multivariate analysis revealed that bMF-positive was an independent factor for OS in all cases [n=226; HR=1.683 (1.018-2.781); p=0.042], especially in T1+T2 cases [n=186; HR=1.926 (1.079-3.440); p=0.024], and PFS in all cases [n=226; HR=2.254 (1.397-3.637); p=0.001]. CONCLUSION: bMF may act as a novel biomarker for OSCC.

Invasive liver abscess syndrome accompanied by spondylodiscitis: a case report and review of the literature.

A 65-year-old man who had diabetes mellitus was referred to our hospital due to fever and back pain. Laboratory findings showed liver dysfunction and C-reactive protein (CRP) elevation. Enhanced computed tomography (CT) showed multiple liver abscesses, hepatic vein and inferior vena cava thrombosis, and spondylodiscitis in the fourth lumbar vertebrae. Based on several detections of Klebsiella pneumoniae (K. pneumoniae) in blood culture, he was diagnosed as having invasive liver abscess syndrome (ILAS), which is characterized by liver abscess and metastatic infection caused by K. pneumoniae. Despite the prompt improvement of liver abscess and thrombosis, after administering antibiotics and anticoagulant, spondylodiscitis worsened. Additionally, iliopsoas abscess emerged on repeated CT imaging. Lumbar laminectomy was needed as a radical treatment. We reviewed 12 cases of ILAS representing spondylodiscitis, and surgical treatments for spondylodiscitis were required in 7 of these cases (58%). When encountering patients with liver abscess and spondylodiscitis, we should consider the possibility of invasive disseminated K. pneumoniae infection, which is hard to treat with antibiotics alone and sometimes requires surgical treatments for spondylodiscitis.

The manzamine alkaloids.

The manzamine alkaloids are absolutely one of the most fascinating marine natural products. The representative manzamine alkaloids, manzamines A-C, were isolated from a marine sponge Haliclona sp. collected off Cape Manzamo, Okinawa, Japan. The manzamine alkaloids are a unique class of alkaloids possessing a characteristic heterocyclic system, and exhibit a diverse range of bioactivities including cytotoxicity, antimicrobial activity, antimalarial activity, antiviral and antiinflammatory activities, antiinsecticidal activity, and proteasome inhibitory activity. About 100 manzamine alkaloids have been isolated from more than 16 species of marine sponges belonging to 5 families. The unusual ring systems, an intriguing suggested biogenetic pathway, and promising biological activities of manzamine alkaloids have attracted great interest as challenging targets for the total synthesis. This review is the continuation of the previous review published in volume 60 of The Alkaloids and covers isolation, structure elucidation, biosynthesis and biogenesis, chemical synthesis, and biological activity of manzamine alkaloids reported from 2003 to 2018.

Prognostic value of HLA class I expression in patients with oral squamous cell carcinoma.

Human leukocyte antigen (HLA) class Ⅰ molecules play a central role in anticancer immunity, but their prognostic value in oral squamous cell carcinoma (OSCC) remains unclear. We examined HLA class I expression in 2 distinct tumor compartments, namely, the tumor center and invasive front, and evaluated the association between its expression pattern and histopathological status in 137 cases with OSCC. Human leukocyte antigen class Ⅰ expression was graded semiquantitatively as high, low, and negative. At the invasive front of the tumor, HLA class I expression was high in 72 cases (52.6%), low in 44 cases (32.1%), and negative in 21 cases (15.3%). The HLA class I expression in the tumor center was high in 48 cases (35.0%), low in 58 cases (42.4%), and negative in 31 cases (22.6%). The 5-year overall survival and disease-specific survival rates were good in cases with high HLA class I expression at the invasive front; however, there was no significant difference in survival based on HLA class I expression in the tumor center. In addition, high HLA class I expression was correlated with high CD8+ T cell density, whereas negative HLA class I expression was correlated with low CD8+ T cell density at the invasive front. These results suggest that it is easier for CD8+ T cells to recognize presented peptides in the case of high HLA class Ⅰ expression at the tumor invasive front and could be a prognostic factor for OSCC.

KPR-5714, a Novel Transient Receptor Potential Melastatin 8 Antagonist, Improves Overactive Bladder via Inhibition of Bladder Afferent Hyperactivity in Rats.

Transient receptor potential (TRP) melastatin 8 (TRPM8) is a temperature-sensing ion channel mainly expressed in primary sensory neurons (Aδ-fibers and C-fibers in the dorsal root ganglion). In this report, we characterized KPR-5714 (N-[(R)-3,3-difluoro-4-hydroxy-1-(2H-1,2,3-triazol-2-yl)butan-2-yl]-3-fluoro-2-[5-(4-fluorophenyl)-1H-pyrazol-3-yl]benzamide), a novel and selective TRPM8 antagonist, to assess its therapeutic potential against frequent urination in rat models with overactive bladder (OAB). In calcium influx assays with HEK293T cells transiently expressing various TRP channels, KPR-5714 showed a potent TRPM8 antagonistic effect and high selectivity against other TRP channels. Intravenously administered KPR-5714 inhibited the hyperactivity of mechanosensitive C-fibers of bladder afferents and dose-dependently increased the intercontraction interval shortened by intravesical instillation of acetic acid in anesthetized rats. Furthermore, we examined the effects of KPR-5714 on voiding behavior in conscious rats with cerebral infarction and in those exposed to cold in metabolic cage experiments. Cerebral infarction and cold exposure induced a significant decrease in the mean voided volume and increase in voiding frequency in rats. Orally administered KPR-5714 dose-dependently increased the mean voided volume and decreased voiding frequency without affecting total voided volume in these models. This study demonstrates that KPR-5714 improves OAB in three different models by inhibiting exaggerated activity of mechanosensitive bladder C-fibers and suggests that KPR-5714 may provide a new and useful approach to the treatment of OAB. SIGNIFICANCE STATEMENT: TRPM8 is involved in bladder sensory transduction and plays a role in the abnormal activation in hypersensitive bladder disorders. KPR-5714, as a novel and selective TRPM8 antagonist, may provide a useful treatment for the disorders related to the hyperactivity of bladder afferent nerves, particularly in overactive bladder.

Ma'edamines C and D, New Bromotyrosine Alkaloids Possessing a Unique Tetrasubstituted Pyridinium Moiety from an Okinawan Marine Sponge Suberea sp.

Two new bromotyrosine alkaloids, ma'edamines C and D, were isolated from an Okinawan marine sponge Suberea sp. The structures of ma'edamines C and D were elucidated on the basis of spectroscopic data. Ma'edamines C and D were the first natural products possessing a unique tetrasubstituted pyridinium moiety such as N-alkyl-3,5-diethyl-2-propylpyridinium and N-alkyl-3,5-diethyl-4-propylpyridinium, respectively. Ma'edamines C and D exhibited moderate cytotoxicity against murine leukemia cell line L1210 in vitro.

Selective Neck Dissection and Survival in Pathologically Node-Positive Oral Squamous Cell Carcinoma.

The most important prognostic factor in oral squamous cell carcinoma (OSCC) is neck metastasis, which is treated by neck dissection. Although selective neck dissection (SND) is a useful tool for clinically node-negative OSCC, its efficacy for neck node-positive OSCC has not been established. Sixty-eight OSCC patients with pN1⁻3 disease who were treated with curative surgery using SND and/or modified-radical/radical neck dissection (MRND/RND) were retrospectively reviewed. The neck control rate was 94% for pN1⁻3 patients who underwent SND. The five-year overall survival (OS) and disease-specific survival (DSS) in pN1-3 OSCC patients were 62% and 71%, respectively. The multivariate analysis of clinical and pathological variables identified the number of positive nodes as an independent predictor of SND outcome (OS, hazard ratio (HR) = 4.98, 95% confidence interval (CI): 1.48⁻16.72, p < 0.01; DSS, HR = 6.44, 95% CI: 1.76⁻23.50, p < 0.01). The results of this retrospective study showed that only SND for neck node-positive OSCC was appropriate for those with up to 2 lymph nodes that had a largest diameter ≤3 cm without extranodal extension (ENE) of the neck and adjuvant radiotherapy. However, the availability of postoperative therapeutic options for high-risk OSCC, including ENE and/or multiple positive lymph nodes, needs to be further investigated.