Planktonic sea urchin larvae change their swimming direction in response to strong photoirradiation.

To survive, organisms need to precisely respond to various environmental factors, such as light and gravity. Among these, light is so important for most life on Earth that light-response systems have become extraordinarily developed during evolution, especially in multicellular animals. A combination of photoreceptors, nervous system components, and effectors allows these animals to respond to light stimuli. In most macroscopic animals, muscles function as effectors responding to light, and in some microscopic aquatic animals, cilia play a role. It is likely that the cilia-based response was the first to develop and that it has been substituted by the muscle-based response along with increases in body size. However, although the function of muscle appears prominent, it is poorly understood whether ciliary responses to light are present and/or functional, especially in deuterostomes, because it is possible that these responses are too subtle to be observed, unlike muscle responses. Here, we show that planktonic sea urchin larvae reverse their swimming direction due to the inhibitory effect of light on the cholinergic neuron signaling>forward swimming pathway. We found that strong photoirradiation of larvae that stay on the surface of seawater immediately drives the larvae away from the surface due to backward swimming. When Opsin2, which is expressed in mesenchymal cells in larval arms, is knocked down, the larvae do not show backward swimming under photoirradiation. Although Opsin2-expressing cells are not neuronal cells, immunohistochemical analysis revealed that they directly attach to cholinergic neurons, which are thought to regulate forward swimming. These data indicate that light, through Opsin2, inhibits the activity of cholinergic signaling, which normally promotes larval forward swimming, and that the light-dependent ciliary response is present in deuterostomes. These findings shed light on how light-responsive tissues/organelles have been conserved and diversified during evolution.

Multiplex Intraoperative Rapid Immunohistochemistry with Noncontact Antibody Mixing for Distinguishing the Histologic Phenotype of Lung Cancer.

INTRODUCTION: Determining a surgical strategy for early-stage lung cancer requires an accurate histologic diagnosis. Immunohistochemistry (IHC) enables reliable diagnosis of histological types but requires more time and more tumor tissue slides than hematoxylin and eosin staining. We aimed to assess the clinical validity of a new rapid multiplex IHC technique utilizing alternating current (AC) mixing for intraoperative lung cancer diagnosis. METHODS: Forty-three patients who underwent radical resection of lung cancers were enrolled in a retrospective observational study. Frozen sections were prepared from lung tumor samples, and rapid IHC employing AC mixing was implemented alongside a multiplex IHC protocol targeting thyroid transcription factor-1 + cytokeratin 5, desmoglein 3 + Napsin A, and p63 + tripartite motif containing 29. We then evaluated the concordance between intraoperative diagnoses derived from rapid multiplex IHC and final pathology. RESULTS: The concordance rate between the pathological diagnosis made with added rapid multiplex IHC and the final pathology was 93.0% (Cohen's 𝜅 coefficient = 0.860 and 95% CI: 0.727-0.993). When considering only adenocarcinoma and squamous cell carcinoma, the diagnoses were in agreement for all cases. CONCLUSIONS: We suggest rapid multiplex IHC as a promising tool for determining surgical strategies for lung tumors.

Direct TGF-ß signaling via alk4/5/7 pathway is involved in gut bending in sea urchin embryos.

BACKGROUND: Precise gastrulation is essential for formation of functional bodies in cnidarians and bilaterians. Previously, by using an alk4/5/7-specific inhibitor, we showed that transforming growth factor-beta (TGF-ß)-alk4/5/7 signaling pathway is important for correct gut bending in sea urchin embryos. However, it is still unclear where functional TGF-ß signals are received in embryos for correct gut bending because details of the spatiotemporal expression pattern of alk4/5/7 have not been reported. RESULTS: We revealed that alk4/5/7 are expressed from the 2-cell to early pluteus stage throughout the entire body, including the invaginating gut. To investigate whether TGF-ß signals directly received in endoderm are required for correct gut bending, we made chimeras in which alk4/5/7 translation was inhibited only in endomesoderm lineage. As a result, the gut of the chimeric embryos did not bend precisely, in contrast to the control chimeras. CONCLUSION: We conclude that direct TGF-ß signaling to the endoderm via alk4/5/7 pathway regulates correct gut bending. However, TGF-ß-alk4/5/7 pathway is not related to mouth opening because the mouth is formed without TGF-ß signaling to the endoderm. This research contributes to understanding the mechanisms leading to the proper positioning of the end of the archenteron for forming a through-gut, which is commonly needed for bilaterians.

Tertiary lymphoid structures are associated with favorable survival outcomes in patients with endometrial cancer.

Immunotherapy has experienced remarkable growth recently. Tertiary lymphoid structures (TLSs) and B cells may play a key role in the immune response and have a survival benefit in some solid tumors, but there have been no reports about their role in endometrial cancer (EC). We investigated the clinicopathological and pathobiological characteristics of the tumor microenvironment (TME) in EC. Patients with EC at Kyoto University Hospital during 2006-2011 were retrospectively included. In 104 patients with EC who met study inclusion criteria, 81 (77.9%) had TLSs, which consisted of areas rich in CD20+ B cells, CD8+ T cells, CD4+ T cells, and CD38+ plasma cells. The absence of TLS was independently associated with tumor progression (HR, 0.154; 95% CI, 0.044-0.536; P = 0.003). Patients with TLSs that included CD23+ germinal centers had better PFS. All tumor infiltrating lymphocytes were counted in the intratumor site. The number of CD20+ B cells was significantly larger in patients with TLSs than in those without TLS (P < 0.001). CD20+ B cells numbers were positively correlated with other TLSs. The larger number of CD20+ B cell was associated with better PFS (P = 0.015). TLSs and B cell infiltration into tumors are associated with favorable survival outcomes in patients with EC. They may represent an active immune reaction of the TME in endometrial cancer.

TrBase: A genome and transcriptome database of Temnopleurus reevesii.

Sea urchins have a long history as model organisms in biology, but their use in genetics is limited because of their long breeding cycle. In sea urchin genetics, genome editing technology was first established in Hemicentrotus pulcherrimus, whose genome has already been published. However, because this species also has a long breeding cycle, new model sea urchins that are more suitable for genetics have been sought. Here, we report a draft genome of another Western Pacific species, Temnopleurus reevesii, which we established as a new model sea urchin recently since this species has a comparable developmental process to other model sea urchins but a short breeding cycle of approximately half a year. The genome of T. reevesii was assembled into 28,742 scaffold sequences with an N50 length of 67.6 kb and an estimated genome size of 905.9 Mb. In the assembled genome, 27,064 genes were identified, 23,624 of which were expressed in at least one of the seven developmental stages. To provide genetic information, we constructed the genome database TrBase (https://cell-innovation.nig.ac.jp/Tree/). We also constructed the Western Pacific Sea Urchin Genome Database (WestPac-SUGDB) (https://cell-innovation.nig.ac.jp/WPAC/) with the aim of establishing a portal site for genetic information on sea urchins in the West Pacific. This site contains genomic information on two species, T. reevesii and H. pulcherrimus, and is equipped with homology search programs for comparing the two datasets. Therefore, TrBase and WestPac-SUGDB are expected to contribute not only to genetic research using sea urchins but also to comparative genomics and evolutionary research.

Early temperament as a predictor of language skills at 40 months.

BACKGROUND: Mastering language involves the development of expressive and receptive skills among children. While it has been speculated that early temperament plays a role in the acquisition of language, the actual mechanism has not yet been explored. We investigated whether temperament at 18 months predicted expressive or receptive language skills at 40 months. METHODS: A representative sample of 901 children and their mothers who were enrolled and followed-up longitudinally in the Hamamatsu Birth Cohort for Mothers and Children study was included in the analysis. Child temperament was measured at 18 months using the Japanese version of the Early Childhood Behavior Questionnaire. Expressive and receptive language skills were measured at 40 months using the Mullen Scales of Early Learning. RESULTS: The multiple regression analysis, adjusting for potential confounders, suggested that higher motor activation (fidgeting) at 18 months was associated with lower expressive and receptive language skills at 40 months. Higher perceptual sensitivity was associated with higher expressive and receptive language skills at 40 months. CONCLUSIONS: Specific temperament at 18 months of age predicted the development of the child's expressive and receptive language skills at 40 months.

Vertical transmission of methicillin-resistant Staphylococcus aureus at delivery and its clinical impact: An observational, prospective cohort study.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) infection has a significant clinical impact on both pregnant women and neonates. The aim of this study was to assess accurately the vertical transmission rate of MRSA and its clinical impacts on both pregnant mothers and neonates. MATERIAL AND METHODS: We conducted a prospective observational cohort study of 898 pregnant women who were admitted to our department and 905 neonates from August 2016 to December 2017. MRSA was cultured from nasal and vaginal samples taken from the mothers at enrollment and from nasal and umbilical surface swabs taken from neonates at the time of delivery. We examined the vertical transmission rate of MRSA in mother-neonate pairs. We used multivariable logistic regression to identify risk factors for maternal MRSA colonization and maternal/neonatal adverse outcomes associated with maternal MRSA colonization. RESULTS: The prevalence of maternal MRSA colonization was 6.1% (55 of 898) at enrollment. The independent risk factors were multiparity and occupation (healthcare provider) (odds ratio [OR] 2.35, 95% confidence interval [CI] 1.25-4.42 and OR 2.58, 95% CI 1.39-4.79, respectively). The prevalence of neonatal MRSA colonization at birth was 12.7% (7 of 55 mother-neonate pairs) in the maternal MRSA-positive group, whereas it was only 0.12% (one of 843 pairs) in the maternal MRSA-negative group (OR 121, 95% CI 14.6-1000). When maternal vaginal samples were MRSA-positive, vertical transmission was observed in four of nine cases (44.4%) in this study. Skin and soft tissue infections developed more frequently in neonates in the maternal MRSA-positive group than in the MRSA-negative group (OR 7.47, 95% CI 2.50-22.3). CONCLUSIONS: The prevalence of MRSA in pregnant women was approximately 6%. Vertical transmission caused by maternal vaginal MRSA colonization was observed in four of nine cases (44.4%). Although our study includes a limited number of maternal MRSA positive cases, the vertical transmission of MRSA may occur in up to 44% of neonates of mothers with vaginal MRSA colonization. Maternal MRSA colonization may be associated with increased development of skin and soft tissue infections in neonates via vertical transmission.

A novel classification of glucose profile in pregnancy based on continuous glucose monitoring data.

AIM: To investigate the glucose profile of women with and without gestational diabetes mellitus (GDM) by simultaneously analyzing several factors of continuous glucose monitoring (CGM) data. METHODS: CGM was conducted for 2 weeks in the second trimester of pregnant women whose random blood glucose level was ≥100 mg/dl. A 75-g oral glucose tolerance test was performed around day 7, and the index of hyperglycemia, relative hypoglycemia, and indices of glucose variability were extracted from CGM data. Unsupervised hierarchical clustering was performed to categorize glucose profiles of the participants. RESULTS: CGM data were obtained from 29 women. Glucose profiles were categorized into three clusters: low glucose levels with less glucose variability group (L group, n = 7); moderate glucose levels with moderate-to-high glucose variability group (M group, n = 18); and high glucose levels with high glucose variability group (H group, n = 4). The waveforms of the glucose profiles were very different among the three groups. Women with GDM tended to be more frequent in the H group than in the M and L groups (75.0%, 16.7%, and 14.3%, respectively; p = 0.053). Maternal age was significantly higher and the proportion of multiparous women was significantly larger in the H group compared to L group (p = 0.002 and 0.015, respectively). CONCLUSIONS: A comprehensive analysis of CGM data could help us extract a subgroup of women with characteristics of GDM.

Transforming growth factor-ß signal regulates gut bending in the sea urchin embryo.

During gastrulation, one of the most important morphogenetic events in sea urchin embryogenesis, the gut bends toward the ventral side to form an open mouth. Although the involvement of transforming growth factor-ß (TGF-ß) signals in the cell-fate specification of the ectoderm and endoderm along the dorsal-ventral axis has been well reported, it remains unclear what controls the morphogenetic behavior of gut bending. Here, using two sea urchin species, Hemicentrotus pulcherrimus and Temnopleurus reevesii, we show that TGF-ß signals are required for gut bending toward the ventral side. To search for the common morphogenetic cue in these two species, we initially confirmed the expression patterns of the dorsal-ventral regulatory TGF-ß members, nodal, lefty, bmp2/4, and chordin, in T. reevesii because these factors are appropriate candidates to investigate the cue that starts gut bending, although genetic information about the body axes is entirely lacking in this species. Based on their expression patterns and a functional analysis of Nodal, the dorsal-ventral axis formation of T. reevesii is likely regulated by these TGF-ß members, as in other sea urchins. When the Alk4/5/7 signal was inhibited by its specific inhibitor, SB431542, before the late gastrula stage of T. reevesii, the gut was extended straight toward the anterior tip region, although the ectodermal dorsal-ventral polarity was normal. By contrast, H. pulcherrimus gut bending was sensitive to SB431542 until the prism stage. These data clearly indicate that gut bending is commonly dependent on a TGF-ß signal in sea urchins, but the timing of the response varies in different species.

P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas.

UNLABELLED: Peas carrying the cyv1 recessive resistance gene are resistant to clover yellow vein virus (ClYVV) isolates No.30 (Cl-No.30) and 90-1 (Cl-90-1) but can be infected by a derivative of Cl-90-1 (Cl-90-1 Br2). The main determinant for the breaking of cyv1 resistance by Cl-90-1 Br2 is P3N-PIPO produced from the P3 gene via transcriptional slippage, and the higher level of P3N-PIPO produced by Cl-90-1 Br2 than by Cl-No.30 contributes to the breaking of resistance. Here we show that P3N-PIPO is also a major virulence determinant in susceptible peas that possess another resistance gene, Cyn1, which does not inhibit systemic infection with ClYVV but causes hypersensitive reaction-like lethal systemic cell death. We previously assumed that the susceptible pea cultivar PI 226564 has a weak allele of Cyn1 Cl-No.30 did not induce cell death, but Cl-90-1 Br2 killed the plants. Our results suggest that P3N-PIPO is recognized by Cyn1 and induces cell death. Unexpectedly, heterologously strongly expressed P3N-PIPO of Cl-No.30 appears to be recognized by Cyn1 in PI 226564. The level of P3N-PIPO accumulation from the P3 gene of Cl-No.30 was significantly lower than that of Cl-90-1 Br2 in a Nicotiana benthamiana transient assay. Therefore, Cyn1-mediated cell death also appears to be determined by the level of P3N-PIPO. The more efficiently a ClYVV isolate broke cyv1 resistance, the more it induced cell death systemically (resulting in a loss of the environment for virus accumulation) in susceptible peas carrying Cyn1, suggesting that antagonistic pleiotropy of P3N-PIPO controls the resistance breaking of ClYVV. IMPORTANCE: Control of plant viral disease has relied on the use of resistant cultivars; however, emerging mutant viruses have broken many types of resistance. Recently, we revealed that Cl-90-1 Br2 breaks the recessive resistance conferred by cyv1, mainly by accumulating a higher level of P3N-PIPO than that of the nonbreaking isolate Cl-No.30. Here we show that a susceptible pea line recognized the increased amount of P3N-PIPO produced by Cl-90-1 Br2 and activated the salicylic acid-mediated defense pathway, inducing lethal systemic cell death. We found a gradation of virulence among ClYVV isolates in a cyv1-carrying pea line and two susceptible pea lines. This study suggests a trade-off between breaking of recessive resistance (cyv1) and host viability; the latter is presumably regulated by the dominant Cyn1 gene, which may impose evolutionary constraints upon P3N-PIPO for overcoming resistance. We propose a working model of the host strategy to sustain the durability of resistance and control fast-evolving viruses.

Mechanism for Increased Expression of UGT2B in the Liver of Mice with Neuropathic Pain.

Approximately 30% of patients with cancer pain experience concurrent neuropathic pain. Since these patients are not sufficiently responsive to morphine, the development of an effective method of pain relief is urgently needed. Decreased function of the µ opioid receptor, which binds to the active metabolite of morphine M-6-G in the brain, has been proposed as a mechanism for morphine resistance. Previously, we pharmacokinetically examined morphine resistance in mice with neuropathic pain, and demonstrated that the brain morphine concentration was decreased, expression level of P-glycoprotein (P-gp) in the small intestine was increased, and expression level and activity of uridine diphosphate glucuronosyltransferase (UGT)2B in the liver were increased. In order to clarify the mechanism of the increased expression of UGT2B, we examined the phase of neuropathic pain during which UGT2B expression in the liver begins to increase, and whether this increased expression is nuclear receptor-mediated. The results of this study revealed that the increased expression of UGT2B in the liver occurred during the maintenance phase of neuropathic pain, suggesting that it may be caused by transcriptional regulation which was not accompanied by increased nuclear import of pregnane X receptor (PXR).

Preoperative planning for implant placement with consideration of pelvic tilt in total hip arthroplasty: postoperative efficacy evaluation.

BACKGROUND: In total hip arthroplasty (THA), tilting of the pelvis alters the cup placement angles. Thus, the cup angles need to be planned with consideration of the effects of pelvic tilt. In the present study, we evaluated the efficacy of preoperative planning for implant placement with consideration of pelvic tilt in THA, and the accuracy of a CT-based computer navigation for implant positioning. METHODS: We examined 75 hips of 75 patients who underwent THA and were followed-up for one year postoperatively. The patients were divided into three groups (anterior, intermediate, posterior tilt) according to their preoperative pelvic tilt. Preoperative planning for implant placement was made with consideration of pelvic tilt and a CT-based navigation was used to execute the preoperative planning. Cup inclination, cup anteversion, and combined anteversion (CA) in supine and standing positions were examined 1 year after THA. The accuracy of the computer navigation was also examined. RESULTS: Mean CA was 35.0 ± 5.8° in supine position and 39.3 ± 5.7° in standing position. CA did not differ among the three subgroups (anterior, intermediate, posterior tilt) in either supine or standing position, indicating implant placements to be equally effective. The desired CA (37.3°) was midway between those in supine and standing positions for each subgroup. Respective mean absolute errors between preoperative planning and postoperative CT measurement was 5.3 ± 5.2° for CA. CONCLUSION: We obtained favorable THA results with preoperative planning with consideration of pelvic tilt by demonstrating supine and standing CA to be unaffected by preoperative pelvic tilt one year postoperatively. Mean absolute error of CA between preoperative planning and postoperative measurement was 5° with use of the CT-based navigation.

Postural and Chronological Change in Pelvic Tilt Five Years After Total Hip Arthroplasty in Patients With Developmental Dysplasia of the Hip: A Three-Dimensional Analysis.

The pelvis generally tilts to the posterior with movement from the supine to standing position, and with time after total hip arthroplasty (THA). This study aimed to investigate changes in pelvic tilt from the preoperative supine position to the standing position at 5 years after THA (pelvic change, PC). We measured pelvic tilt using a 2D-3D matching technique in 77 unilaterally affected patients who underwent primary THA. PC in 8% of all patients was ≤-20°, and the greatest PC was -25°. In these patients, posterior pelvic tilt continued up to 5 years after THA. These patients were older, and their lumbo-lordotic angle was small. For such cases, cup orientation should be planned to account for continuous posterior change in pelvic tilt after THA.

Highly fluorescent flavins: rational molecular design for quenching protection based on repulsive and attractive control of molecular alignment.

Unprecedented intense fluorescent emission was observed for a variety of flavin compounds bearing a perpendicular cyclic imide moiety at the C7 position of an isoalloxazine platform. A series of alloxan-substituted flavins was prepared selectively by reduction of the corresponding N-aryl-2-nitro-5-alkoxyanilines with zinc dust and subsequent reaction with alloxan monohydrate in the presence of boric acid. Analogues bearing oxazolidine-2,4-dione functionality were obtained on methylation of the alloxan-substituted flavins with methyl iodide and subsequent rearrangement in the presence of an inorganic base. The flavin compounds exhibit intense white-green fluorescent emission in the solution state under UV excitation at 298 K, with emission efficiencies Φ298 K greater than 0.55 in CH3 CN, which are higher than the values for all reported flavin compounds under similar conditions. The highest Φ298 K value of 0.70 was obtained in CH3 CN for isoalloxazine bearing C7-alloxan and N10-2,6-diisopropylphenyl groups. The temperature dependence of the emission intensities indicates that the pronounced emission properties at 298 K are attributable to the highly heat resistant properties towards emission decay with increasing temperature. Mechanistic studies, including X-ray diffraction analysis, revealed that the good emission properties and high heat resistance of the alloxan-substituted flavins are due to a synergetic effect of the associative nature of the C7-alloxan unit and the repulsive nature of the perpendicular bulky substituents at the C7 and N10 positions.

Highly efficient activation of organosilanes with η2-aldehyde nickel complexes: key for catalytic syntheses of aryl-, vinyl-, and alkynyl-benzoxasiloles.

An η(2)-aldehyde nickel complex was utilized as an effective activator for an organosilane in order to generate a hypervalent silicate reactant for the first time. This method was successfully applied to the highly efficient syntheses of 3-aryl-, vinyl-, and alkynyl-2,1-benzoxasiloles from benzaldehydes with aryl-, vinyl-, and alkynylsilyl groups at the ortho position. Initial mechanistic studies revealed that an intermolecular aryl transfer process was involved in the reaction mechanism. The formation of an η(2)-aldehyde complex was directly confirmed by NMR.

Effects of aromatherapy massage on face-down posture-related pain after vitrectomy: a randomized controlled trial.

Postoperative face-down posturing (FDP) is recommended to optimize the effects of intraocular gas tamponade after vitrectomy. However, patients undergoing FDP usually experience physical and psychological burdens. This 3-armed, randomized, single-center trial investigated the effects of aromatherapy on FDP-related physical pain. Sixty-three patients under FDP were randomly allocated to one of three treatment groups: aromatherapy massage with essential oil (AT), oil massage without essential oil (OT), and a control group. The AT and OT groups received 10 minutes of massage by ward nurses trained by an aromatherapist, while the control group received usual care. Outcomes were assessed as short-term (pre- to post-intervention) and long-term (first to third postoperative day) changes in physical pain in five body regions using face-scale. The AT and OT groups both revealed similar short-term pain reductions after intervention, compared with the control group. Regarding long-term effects, neither group experienced significant effects until the second day. Significantly more pain reduction compared with usual care occurred on the third day, mainly in the AT group, though there were few significant differences between the AT and OT groups. In conclusion, this study suggests that simple oil massage is an effective strategy for immediate pain reduction in patients undergoing FDP, while aromatherapy may have a long-term effect on pain reduction.

Body Part Pain Affects Subjective and Objective Handball Performance in Japanese Male National Athletes-Results of Short-Term Practical Monitoring of Athletes' Conditions.

This short-term survey examined the effect of body part pain on subjective and objective handball performance in Japanese male national handball athletes. Fourteen athletes participated in this study. Assessments of pain in 10 body parts and subjective performance (concentration and satisfaction with body movement) were performed using a visual analog scale from 0 to 10 over four consecutive training days. Monitoring of heart rate and body acceleration during training was also performed to quantify the objective performance. Path analysis and linear mixed modeling were employed to assess the relationship between body pain scores and subjective/objective handball performance. Over the four days of the study period, the body part in which most athletes reported pain was the dominant shoulder (6 of 14 athletes), followed by the dominant knee, the dominant elbow, the dominant ankle joint, and the non-dominant ankle joint (3 of 14 athletes). The path analysis revealed that pain in the dominant elbow negatively correlated with concentration (standardized path coefficient = -0.644, p = 0.00), which was associated with satisfaction with body movement (standardized path coefficient = 0.704, p = 0.00). No significant effect of body pain on objective performance (heart rate and body acceleration) was found among the athletes in this study. The results suggested that the elite athletes were practicing with pain. Even if pain does not physically affect athletes' objective performance, pain in the upper extremities, associated with the primary handball movement of throwing, may reduce the quality of practice by lowering athletes' subjective performance.

Epithelial granuloma occurring on the staple-stump after segmentectomy for ovarian cancer lung metastasis.

When a mass occurs at the staple line following lung resection, it can be difficult to distinguish between local cancer recurrence and granuloma. We present a case of a staple-line granuloma with 18F-fluorodeoxyglucose-positron emission tomography uptake and elevated serum carbohydrate antigen 19-9 (CA19-9) in a patient with ovarian cancer lung metastasis. After granuloma resection, serum CA19-9 levels normalized, and CA19-9 positive cells were identified in the resected tumor. Therefore, serum CA19-9 elevation does not rule out a staple-line granuloma. Whereas granulomas on computed tomography (CT) scans tend to show smooth shadows along the staple line unilaterally, detailed CT evaluation may help diagnostic differentiation. Differentiation based on imaging and tumor markers has limitations. However, core needle biopsy has the risk of misdiagnosis and tumor cell dissemination, therefore surgical resection should be considered when comprehensive findings indicate a potential recurrence.

18­α­glycyrrhetinic acid induces apoptosis in gingival fibroblasts exposed to phenytoin.

Phenytoin (PHT)-induced gingival overgrowth is caused by the increased proliferation and reduced apoptosis of gingival fibroblasts in inflammatory gingiva. Licorice has long been used as a component of therapeutic preparations. It inhibits cell proliferation, induces cell apoptosis and has anti-inflammatory effects. 18-α-glycyrrhetinic acid (18α-GA), the active compound in licorice, promotes apoptosis in various types of cells. The present study determined whether 18α-GA affects apoptosis in gingival fibroblasts exposed to PHT. The present study aimed to establish a basis for the therapeutic application of 18α-GA to treat the gingival overgrowth induced by PHT. Human gingival fibroblasts from healthy donors were cultured to semi-confluence and then stimulated in serum-free DMEM containing PHT with or without 18α-GA for subsequent experiments. Apoptotic cells were detected by ELISA. Analysis of the distribution of cell cycle phases and the apoptotic cell population was performed by flow cytometry. The expression levels of mRNAs and proteins of apoptotic regulators were measured using reverse transcription-quantitative PCR and western blotting, respectively. Caspase (CASP) activities were assessed by an ELISA. Treatment with 18α-GA markedly increased the number of apoptotic cells, reduced BCL2 mRNA expression, increased CASP2 and receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1) domain containing adaptor with death domain, Fas (TNFRSF6)-associated via death domain, RIPK1, tumor necrosis factor receptor superfamily; member 1A, TNF receptor-associated factor 2, CASP2, CASP3 and CASP9 mRNA expression, and also upregulated the protein expression levels and activities of caspase-2, caspase-3 and caspase-9. These results demonstrated that 18α-GA induced apoptosis through the activation of the Fas and TNF pathways in the death receptor signaling pathway in gingival fibroblasts treated with PHT. 18α-GA exhibited therapeutic potential for the treatment of PHT-induced gingival overgrowth.