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Supplementary Figures 1-2 from PEA-15 Induces Autophagy in Human Ovarian Cancer Cells and Is Associated with Prolonged Overall Survival

AIM We identified chemical components that exhibited antitumor activity against oral squamous cell carcinoma (OSCC) cells and examined their effective concentrations and additive and/or synergistic effects in combinational usage on the proliferation, apoptosis and cell cycle of OSCC cells. MATERIALS AND METHODS Using high-performance liquid chromatography, nuclear magnetic resonance spectroscopy and electrospray ionization-mass spectrometry, we identified the main chemical components of the methanol extracts from Paeonia lutea. We investigated the pharmaceutical effects of those components on the proliferation, apoptosis, and cell cycle of an OSCC cell line, SAS, using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and caspase assays, as well as flow cytometry cell cycle analysis. We also examined the effects of those components on the mitogen-activated protein kinase signal transduction pathway by western blotting. Finally, the effects on normal human epidermal keratinocyte cells were also examined in similar experiments. RESULTS Three chemicals have been identified in P. lutea leaves using high performance liquid chromatography: gallic acid methyl ester (GAME), pentagalloyl glucose (PGG) and paeoniflorin (PF). Both GAME and PGG significantly suppressed cell proliferation, and their combined effects were synergistic, while the effect of PF was minimal. However, those chemicals did not induce apoptosis. Cell cycle and western blotting analysis showed that the suppressive effects on cell proliferation resulted from G2 arrest and the suppression of phosphorylation of Akt/PKB. No effect was identified on normal human epidermal keratinocyte cells. CONCLUSION These results indicate that GAME and PGG are the main chemical components of P. lutea leaves that have potential anti-cancer therapeutic effects.

Cells generally control the concentration of mRNA via transcriptional and posttranscriptional regulation, so the separate contributions of synthesis and degradation (decay) cannot be discriminated by the quantification of mRNA. To elucidate the contribution of posttranscriptional regulation, all experimental procedures for the analysis of the total transcript level, transcriptional induction, degradation of the target mRNA, and inhibition of mRNA translation are performed either individually or in combination. From our experience, measurement of the steady-state levels of mRNA using quantitative real-time polymerase chain reaction is an essential first step in quantifying the ccn2 gene expression. Subsequently, the effect of transcription rates should be assessed by reporter assays of the ccn2 promoter and nuclear run-on assays. The stability of ccn2 mRNAs is then evaluated in the presence of a metabolic inhibitor actinomycin D, followed by mRNA degradation assays in vitro. Finally, repression of ccn2 mRNA translation can be estimated by comparing the expression of mRNA and protein changes. We herein report the strategic methods used in a series of analyses to elucidate the possible involvement of the posttranscriptional regulatory mechanism of the ccn2 gene and show how this approach can, in theory, be used to elucidate the posttranscriptional regulation of other genes belonging to the CCN family.

This study aimed to examine the effects of a change in medication from suvorexant to lemborexant among patients with insomnia.Patients with chronic insomnia who had persistent insomnia for 3 months or longer and who had been taking suvorexant for 3 months or longer were selected. The participants were divided into two groups: the 'modified' group and the 'non-modified' group. Four sub-types of insomnia (i.e., difficulty initiating sleep, difficulty maintaining sleep, early-morning awakening, and non-restorative sleep) were investigated. Logistic regression was used to investigate improvements in both the groups after 12 weeks.Among the 77 participants, 43 and 34 patients were in the modified drug group and the non-modified drug group, respectively. Comparing sleep disorders between the two groups, we found significant improvement after 12 weeks in the modified drug group in terms of difficulty initiating sleep, compared with the non-modified drug group (odds ratio = 0.036, P = 0.008, 95% CI = 0.003-0.415). However, no significant differences were found between the two groups in terms of difficulty maintaining sleep, early-morning awakening, and non-restorative sleep.Sleep disorders can be treated by alleviating difficulties in initiating sleep by changing from suvorexant to lemborexant. In addition, it was confirmed that the drug change caused no serious side effects and that it was highly safe and tolerated.

Background There is uncertainty surrounding the causal relationship between serum uric acid and hypertension. The aim was to investigate the association between serum uric acid and prevalence of hypertension in a general population of Japanese. Methods This was a population-based cross-sectional study using health check-up data of the residents of the Iki City, Nagasaki Prefecture, Japan. A total of 7,484 participants aged 30 years or older were included in this study. Serum uric acid was classified into four groups: group 1 (< 357 µmol/L (< 6 mg/dL)), group 2 (357 - 415 µmol/L (6 - 6.9 mg/dL)), group 3 (416 - 475 µmol/L (7 - 7.9 mg/dL)) and group 4 (≥ 476 µmol/L (≥ 8 mg/dL)). Hypertension was defined as blood pressure (BP) levels of ≥ 140/90 mm Hg or use of BP lowering medications. Results Hypertension was observed among 3,467 participants (prevalence 46.3%). The prevalence of hypertension increased with elevation of serum uric acid levels: 42.8% in group 1, 55.0% in group 2, 57.6% in group 3 and 59.8% in group 4 (P < 0.001 for trend). This association was significant even after adjustment for other risk factors including age, sex, current smoking, current alcohol intake, obesity, diabetes, dyslipidemia, estimated glomerular filtration rate and proteinuria: odds ratios (95% confidence intervals) were 1.50 (1.28 - 1.77) for group 2, 1.58 (1.25 - 1.99) for group 3 and 1.89 (1.36 - 2.64) for group 4 compared with the reference group of group 1 (P < 0.001 for trend). Conclusions Serum uric acid was clearly associated with prevalence of hypertension in a general population of Japanese.

The novel conceptual disease model, the oral–gut axis, which represents the immunomodulatory mutual relationship between oral and gut microbial compartments, has been attracting attention in relation to systemic health issues. We investigated whether this unique crosstalk influences the systemic condition of patients with COVID-19 infections who received extracorporeal membrane oxygenation (ECMO) in the intensive care unit (ICU) during April and December 2020. In this case-control study, patients were divided into two groups according to their survival (total entry size, n = 21; survivors, n = 13; non-survivors, n = 8). Patients were evaluated using the oral assessment guide from Fukuoka University (OAG-F) and the Bristol Stool Form Scale (BSFS) to examine the oral and fecal conditions. A blood-based inflammatory factor, the neutrophil-to-lymphocyte ratio (NLR), was used as an indicator of systemic immunity. The high total OAG-F scores were associated with both elevated BSFS and NLR values, and a mutually positive correlation between BSFS and NLR was observed. This indicated an interplay between oral deterioration, gut dysbiosis, and the impairment of immunity. Furthermore, oral deterioration was more frequently observed in non-survivors on day 14 of ICU admission. In addition, on days 7 and 21 of ICU admission, impaired immunity, reflected by an elevated NLR, was observed in non-survivors. However, the distribution of the gut microbiome—reflected by increased BSFS values—with the time it was examined was not directly observed in non-survivors. Taken together, these findings suggested that oral–gut health may be specifically associated with mortality in COVID-19 patients receiving ECMO in the ICU.

The oral health of coronavirus disease 2019 (COVID-19) patients in the intensive care unit (ICU) is an important issue in treatment of respiratory failure. We retrospectively investigated the oral health history of severe COVID-19 patients who received extracorporeal membrane oxygenation (ECMO) from April 2020 to December 2020 using the oral assessment guide from Fukuoka University (OAG-F). Nineteen consecutive patients (median age: 62 years) were divided into two groups according to survival (survivors, n = 12; non-survivors, n = 7). A univariate analysis revealed no significant differences between the groups in sex, age, body mass index (BMI), or the number of remaining teeth, whereas the ECMO assistance of non-survivors (median: 34 days) was prolonged in comparison to survivors (median: 8 days; p < 0.05). Among the factors of OAG-F, significant differences were observed between the groups in the conditions of the saliva, mucous membrane, and gingiva. The total scores in non-survivors (median: 19) were significantly higher in comparison to survivors (Median: 15.5), suggesting that the frequency of oral health deterioration was higher in non-survivors (p < 0.05). Taken together, these findings suggest that poor oral health is associated with mortality in COVID-19 patients receiving ECMO in the ICU.

We aimed to investigate the association between serum uric acid (SUA) level and development of hypertension as well as the interaction effect of chronic kidney disease (CKD) on this relationship in the general Japanese population. We included 7895 participants aged ≥30 years from the ISSA-CKD study, a population-based retrospective cohort study that used annual health check-up data of residents from Iki Island, Japan. After the exclusion of 1881 with l < 1-year follow-up, 2812 with hypertension at baseline, and 165 with missing information on SUA, a total of 3037 participants were enrolled in this analysis. Participants were divided into four groups according to the quartiles of SUA level at baseline, and multivariable-adjusted hazard ratios for new-onset hypertension were calculated. Stratified analyses were performed for each subgroup (defined by sex, age, alcohol intake, and CKD) to assess the interaction effects. During a mean follow-up period of 4.4 years, 943 participants developed hypertension. The first quartile group was set as the reference group, and the multivariable-adjusted hazard ratios (95% confidence interval) for new-onset hypertension were 1.11 (0.90-1.36) in the second quartile, 1.25 (1.02-1.54) in the third quartile, and 1.35 (1.07-1.70) in the fourth quartile compared with those in the reference group (p = .007 for trend). The stratified analyses showed that the association between SUA and hypertension was significantly stronger in participants with CKD than in those without CKD (p = .035 for interaction). SUA level is an independent risk factor for new-onset hypertension. This tendency was significantly stronger in participants with CKD.

Cellular communication network factor 2 (CCN2, also known as CTGF), is a modular and matricellular protein and a well-known angiogenic factor in physiological and pathological angiogenesis. However, its roles in lymphangiogenesis and intracellular signaling in lymphatic endothelial cells (LECs) remain unclear. Here, we investigated CCN2 signaling in LECs and its effects on lymphangiogenesis. In primary cultured LECs, gene expressions of lymphatic endothelial markers lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), Podoplanin and prospero homeobox 1 (Prox1) and lymphangiogenic factors vascular endothelial cell growth factor c (Vegfc), vascular endothelial cell growth factor d (Vegfd) and fms-related tyrosine kinase 4 (Flt4, also known as Vegfr3) were upregulated by CCN2. Subsequently, we found that CCN2 induced phospho-ERK and that was decreased by suppression of integrin αv. CCN2 slightly decreased the growth of LECs due to enhancement of the interaction of ERK and dual specific protein phosphatase 6 (DUSP6), and knockdown of DUSP6 increased CCN2-induced phospho-ERK levels. In in vivo Matrigel plug assays, the number of Podoplanin-positive vessels was increased by exogenous CCN2, and phospho-ERK-positive LEC and DUSP6-positive LEC were detected in CCN2 plugs. These results suggest that CCN2-related lymphangiogenesis is regulated by DUSP6, which enables negative modulation of ERK-signaling.

Objective Rectal sedation has been in use for many years in paediatric dentistry. The purpose of this study was to retrospectively investigate the incidence of rectal sedation with midazolam plus ketamine for oral minor surgery among paediatric patients in our department. Methods The incidence of rectal sedation for paediatric outpatients under the age 6 years requiring oral surgery of our department between January 2007 and December 2016 was retrospectively analysed. Rectal sedation was given to 10 paediatric patients (8 boys and 2 girls) and consisted of a 0.1 mg/kg dose of midazolam and a 5 mg/kg dose of ketamine. Results Every case was a traumatic injury, the most common region was the tongue, and the most frequent type of wound was a laceration. With respect to sedative effects, approximately 30 min after rectal administration of the two drugs, good anxiolysis and cooperation were obtained, and few adverse effects were seen in all 10 cases although there were some cases that were difficult to treat. Conclusions Rectal administration of midazolam plus ketamine provides a safe and reliable method for paediatric outpatients requiring an oral surgery procedure.

Lower third molar extraction is the most common surgical treatment among routine dental and oral surgical procedures. while the surgical procedures for lower third molar extraction are well established, the difficulty of tooth extraction and the frequency of postoperative complications differ depending on the patient’s background. To establish a management protocol for the lower third molars, the prevalence of and risk factors for postoperative complications after lower third molar extraction were investigated in a large number of Japanese patients in a multicenter prospective study. During 6 consecutive months in 2020, 1826 lower third molar extractions were performed at the 20 participating institutions. The medical records of the patients were reviewed, and relevant data were extracted. The prevalence of and risk factors for postoperative complications were analyzed. The prevalence of postoperative complications after lower third molar extraction was 10.0%. Multivariate analysis indicated that age (≤32 vs >32, odds ratio [OR]: 1.428, 95% confidence interval [95% CI]: 1.040–1.962, P < .05), the radiographic anatomical relationship between the tooth roots and mandibular canal (overlapping of the roots and canal vs no close anatomical relationship between the roots and the superior border of the canal, OR: 2.078, 95% CI: 1.333–3.238, P < .01; overlapping of the roots and canal vs roots impinging on the superior border of the canal, OR: 1.599, 95% CI: 1.050–2.435, P < .05), and impaction depth according to the Pell and Gregory classification (position C vs position A, OR: 3.7622, 95% CI: 2.079–6.310, P < .001; position C vs position B, OR: 2.574, 95% CI: 1.574–4.210, P < .001) are significant independent risk factors for postoperative complications after lower third molar extraction. These results suggested that higher age and a deeply impacted tooth might be significant independent risk factors for postoperative complications after lower third molar extraction.

Background: We investigated whether eating speed was associated with the incidence of diabetes in a Japanese general population. Methods: A total of 4853 Japanese individuals without diabetes at baseline were analyzed. Self-reported eating speed was categorized as slow, medium, and fast on the basis of questionnaire responses. The study outcome was the incidence of diabetes. Results: After an average follow-up period of 5.1 years, 234 individuals developed diabetes. The incidence of diabetes per 1000 person-years was 4.9 in the slow eating speed group, 8.8 in the medium eating speed group, and 12.5 in the fast eating speed group, respectively (*** p &lt, 0.001 for trend). The HRs were 1.69 (95%CI 0.94–3.06) for the medium eating speed and 2.08 (95%CI 1.13–3.84) for the fast eating speed, compared to the slow eating speed (* p = 0.014 for trend) after adjustment for age, gender, smoking status, drinking, exercise, obesity, hypertension, and dyslipidemia. Conclusion: Faster eating speed increased a risk for the incidence of diabetes in a general Japanese population.

Objectives This study aimed to clarify the relationship between the white blood cell (WBC) count and hypertension in the general Japanese population. Methods We conducted a population-based retrospective cohort study using annual health check-up data of residents of Iki City, Nagasaki Prefecture, Japan. A total of 2935 participants without hypertension at baseline were included in the present analysis. WBC counts were classified as tertile 1 ( Result During an average follow-up of 4.5 years, 908 participants developed hypertension. The incidence (per 100 person-years) of hypertension increased with an elevation in the WBC count (6.3 in tertile 1, 7.0 in tertile 2, and 7.4 in tertile 3). This association was significant, even after adjustment for other risk factors, including age, sex, current smoking habits, current alcohol intake, exercise habits, obesity, elevated blood pressure, diabetes mellitus, and dyslipidemia. The hazard ratios were 1.07 for tertile 2 (95% CI 0.90–1.26) and 1.27 for tertile 3 (95% CI 1.06–1.51) compared with the reference group of tertile 1 (p = 0.009). Conclusion The WBC count was associated with future development of hypertension in the general Japanese population.

In this study, we investigated the regional differences in the composition of farm bulk milk produced at three different dairy areas in Hokkaido, Japan. A field survey was conducted at Central, Tokachi, and North areas of Hokkaido, three or four times a year. At each farm, an interview questionnaire for farm basal data was conducted, and 500 ml of bulk tank milk sample was obtained. Fatty acid composition, and vitamin and carotenoid concentrations in the milk samples were determined. In Central and Tokachi areas, corn silage was used as the main forage. In North area, fresh herbage was the dominant feed in the summer season, and grass hay was the main feed in the winter season. Discriminant analysis revealed that the composition of milk samples differed among the areas and seasons. Milk from Central and Tokachi areas contained a higher ratio of linoleic acid compared with that from North area, but there were only slight differences in the composition of milk between Central and Tokachi areas. The concentrations of carotenoids and alpha-tocopherol were higher in samples from North area and the ratios of trans-vaccenic acid and conjugated linoleic acid were higher in the summer season than in the indoor season.

Six ruminal-cannulated nonlactating Holstein Friesian cows (mean body weight:660 ± 42.9 kg) were used to investigate the effect of soybean meal (SBM) supplementation on voluntary rice straw (RS) intake, feed particle size reduction, and passage kinetics in the rumen. They were allocated to two dietary treatments: RS alone or RS supplemented with SBM. Voluntary dry matter intake of RS and total tract fiber digestibility was increased by SBM supplementation (p < 0.05). Supplementation with SBM decreased rumination time per dietary dry matter (DM) and neutral detergent fiber (NDFom) intake (p < 0.01). Particle size distribution in the rumen and total ruminal NDFom digesta weights were not affected by SBM supplementation. However, the disappearance rates of total digesta and large and small particles from the rumen were increased by SBM supplementation (p < 0.01). Moreover, SBM supplementation increased the rate of size reduction in ruminal particles (p < 0.05). In situ disappearance of DM and NDFom of RS in the rumen was greater in SBM-supplemented cows than in nonsupplemented cows (p < 0.05). This study clearly showed that increased ruminal RS particle size reduction, passage, and fermentation due to SBM supplementation accelerated the RS particle clearance from the rumen and resulted in increased voluntary RS intake of dairy cows.

In this paper, the authors proposed the “resident participation-type slope measurement and monitoring system” to detect the premonitory symptoms of sediment disasters in the mountainous area (Takasu Town, Fukui City, Japan). The key concepts of resident participation-type slope measurement/monitoring are (1) visualization, (2) daily routine, and (3) voluntary/proactive involvement. The residents will not only monitor the danger slopes and notice unusual events, but also try to establish a system to grasp the slope deformation/displacement quantitatively. For this purpose, some different types of sensors based on the on-site visualization (OSV) concept were introduced to the dangerous slopes and retaining walls selected by the residents and geotechnical experts. The OSV sensors are low cost and visually excellent measuring device with simple measurement principle. Further improvements of measurement and recording methodology are required to increase the participation of residents to the slope measurement and monitoring.

The aim of this study was to determine the relationship between eating before bed and the development of hypertension in a general Japanese population. We conducted a population-based retrospective cohort study using annual health check-up data collected from the residents of Iki City, Nagasaki Prefecture, Japan. In total, 2930 participants without hypertension at baseline (mean age 57.0 years, male 42.8%) were included in the present analysis. Eating before bed was defined as eating within 2 h of bedtime. The outcome of this study was incident hypertension (blood pressure ≥140/90 mmHg or initiation of blood pressure-lowering medications). Multivariable-adjusted hazard ratios and 95% confidence intervals (CIs) were estimated using Cox proportional hazards models. During an average follow-up of 4.5 years, 909 participants developed hypertension. The incidence (per 1000 person-years) of hypertension in the group of individuals who ate before bed was 82.8, whereas that in the group of individuals who did not eat before bed was 65.8. The association was significant even after adjusting for other risk factors, including age, sex, current smoking status, current alcohol intake, regular exercise, obesity, elevated blood pressure, diabetes mellitus, and dyslipidemia, with a hazard ratio of 1.23 (95% CI: 1.05-1.44) for the group of individuals who ate before bed compared with the group of individuals who did not eat before bed (P = 0.01 for trend). Eating before bed was correlated with a future risk of developing hypertension in the general Japanese population.

Submental tracheal intubation is an alternative technique for use in patients with airway difficulties in whom oro- or naso-tracheal intubation is contraindicated. Here, a planned submental tracheal intubation was used during orthognathic surgery in an acromegalic patient who had previously undergone trans-sphenoidal surgery (TSS) for removal of a pituitary adenoma, which involves removal of the sellar bone covering the anterior surface of the cavernous sinus. This helped prevent serious complications, such as intracranial penetration during nasotracheal intubation. Submental tracheal intubation can reduce the morbidity associated with repeated attempts at tracheal intubation in acromegalic patients who have undergone cranial base surgery and in whom orthognathic surgery is planned.

A number of studies report that epithelial to mesenchymal transition (EMT) supports the generation and maintenance of cancer stem cells (CSCs), which show tumor seeding ability and drug resistance; however, the molecular mechanisms underlying induction of EMT-associated tumor malignancy remain unclear. The present study reports that oral cancer cells switch from expressing the CD44 variant form (CD44v) to expressing the standard form (CD44s) during acquisition of cisplatin-resistance, which resulted in EMT induction. CD44s induced an EMT phenotype in cisplatin resistant cells by up-regulating ZEB1, a transcriptional repressor of E-cadherin. More importantly, CD44s up-regulated ZEB1 by suppressing microRNA-200c, which is a non-coding RNA that directly represses the ZEB1 gene. These results demonstrate the importance of the association between platinum resistance and CD44s during EMT induction in oral cancer cells.

In this paper, we present the applications of our newly developed, highly sensitive fluorescent detection method referred to as scanning single-molecule counting (SSMC). We found that the target RNA added to the total RNA was detected with high sensitivity at 384 aM by combining a magnetic bead-based assay and SSMC (MB-BA + SSMC). Gene expression analysis without reverse transcription or amplification confirmed that the pattern of gene expression was identical to that of real-time polymerase chain reaction (PCR). MB-BA + SSMC was also applied to formalin-fixed paraffin-embedded (FFPE) samples. RNA fragmentation and crosslinking owing to FFPE processing slightly affected gene expression. Conversely, FFPE samples showed an increase in gene Ct values and a decrease in the number of detectable genes when analyzed using real-time PCR. Overall, our results suggested that SSMC is a powerful tool for target RNA detection and amplification-free gene expression analysis.

Pemphigus vulgaris (PV) is a chronic autoimmune bullous disease characterized by the formation of suprabasal cleavage and acantholysis. As this disease almost always affects the oral mucosa, conventional cytological smears of oral lesions can be used for the initial diagnosis of PV. We report two cases of PV that were initially diagnosed based on cytological smears of an oral sample. As atypical squamous cells were present even in the liquid-based cytological (LBC) smears of the oral lesion in these two cases, this ultimately led to the misinterpretation of squamous cell carcinoma. These findings demonstrate that cytological mimicry of oral PV can occur in malignant cases when there is an absence of appropriate clinical information.