Autophagy Plays a Crucial Role in Ameloblast Differentiation.

Tooth enamel is generated by ameloblasts. Any failure in amelogenesis results in defects in the enamel, a condition known as amelogenesis imperfecta. Here, we report that mice with deficient autophagy in epithelial-derived tissues (K14-Cre;Atg7F/F and K14-Cre;Atg3F/F conditional knockout mice) exhibit amelogenesis imperfecta. Micro-computed tomography imaging confirmed that enamel density and thickness were significantly reduced in the teeth of these mice. At the molecular level, ameloblast differentiation was compromised through ectopic accumulation and activation of NRF2, a specific substrate of autophagy. Through bioinformatic analyses, we identified Bcl11b, Dlx3, Klk4, Ltbp3, Nectin1, and Pax9 as candidate genes related to amelogenesis imperfecta and the NRF2-mediated pathway. To investigate the effects of the ectopic NRF2 pathway activation caused by the autophagy deficiency, we analyzed target gene expression and NRF2 binding to the promoter region of candidate target genes and found suppressed gene expression of Bcl11b, Dlx3, Klk4, and Nectin1 but not of Ltbp3 and Pax9. Taken together, our findings indicate that autophagy plays a crucial role in ameloblast differentiation and that its failure results in amelogenesis imperfecta through ectopic NRF2 activation.

Spatiotemporal MicroRNA-Gene Expression Network Related to Orofacial Clefts.

Craniofacial structures change dynamically in morphology during development through the coordinated regulation of various cellular molecules. However, it remains unclear how these complex mechanisms are regulated in a spatiotemporal manner. Here we applied natural cubic splines to model gene and microRNA (miRNA) expression from embryonic day (E) 10.5 to E14.5 in the proximal and distal regions of the maxillary processes to identify spatiotemporal patterns of gene and miRNA expression, followed by constructing corresponding regulatory networks. Three major groups of differentially expressed genes (DEGs) were identified, including 3,927 temporal, 314 spatial, and 494 spatiotemporal DEGs. Unsupervised clustering further resolved these spatiotemporal DEGs into 8 clusters with distinct expression patterns. Interestingly, we found 2 clusters of differentially expressed miRNAs: 1 had 80 miRNAs monotonically decreasing and the other had 97 increasing across developmental stages. To evaluate the phenotypic relevance of these DEGs during craniofacial development, we integrated data from the CleftGeneDB database and constructed the regulatory networks of genes related to orofacial clefts. Our analysis revealed 2 hub miRNAs, mmu-miR-325-3p and mmu-miR-384-5p, that repressed cleft-related genes Adamts3, Runx2, Fgfr2, Acvr1, and Edn2, while their expression increased over time. On the contrary, 2 hub miRNAs, mmu-miR-218-5p and mmu-miR-338-5p, repressed cleft-related genes Pbx2, Ermp1, Snai1, Tbx2, and Bmi1, while their expression decreased over time. Our experiments indicated that these miRNA mimics significantly inhibited cell proliferation in mouse embryonic palatal mesenchymal (MEPM) cells and O9-1 cells through the regulation of genes associated with cleft palate and validated the role of our regulatory networks in orofacial clefts. To facilitate interactive exploration of these data, we developed a user-friendly web tool to visualize the gene and miRNA expression patterns across developmental stages, as well as the regulatory networks (https://fyan.shinyapps.io/facebase_shiny/). Taken together, our results provide a valuable resource that serves as a reference map for future research in craniofacial development.

Radiation doses of workers engaged in decontamination of the environment.

After the accident at Fukushima Daiichi nuclear power plant on 11 March 2011, radioactive materials were released into the atmosphere resulting in environmental contamination. Following the implementation of environmental decontamination efforts, the Radiation Dose Registration Centre of the Radiation Effects Association established the radiation dose registration system for decontamination and related workers to consolidate and prevent the loss of radiation records. This article presents statistics on the radiation doses of decontamination and related workers using official records. Since approximately 10 years have passed since the accident in Fukushima, the types of work conducted in the affected restricted areas have changed over time. Therefore, changes in radiation dose for each type of work and comparisons with nuclear workers are presented.

Polyethylene glycol-based linkers as hydrophilicity reservoir for antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are an established therapeutic entity in which potent cytotoxic drugs are conjugated to a monoclonal antibody. In parallel with the great emphasis put on novel site-specific bioconjugation technologies, future advancements in this field also rely on exploring novel linker-drug architectures that improve the efficacy and stability of ADCs. In this context, the use of hydrophilic linkers represents a valid strategy to mask or reduce the inherent hydrophobicity of the most used cytotoxic drugs and positively impact the physical stability and in vivo performance of ADCs. Here, we describe the use of linkers containing monodisperse poly(ethylene glycol) (PEG) moieties for the construction of highly-loaded lysine-conjugated ADCs. The studied ADCs differ in the positioning of PEG (linear or pendant), the bonding type with the antibody (amide or carbamate), and the drug-to-antibody ratio (DAR). These ADCs were first evaluated for their stability in solution under thermal stress, showing that both the drug-linker-polymer design and the nature of the antibody-linker bonding are of great importance for their physical and chemical stability. Amide-coupled ADCs bearing two pendant 12-unit poly(ethylene glycol) chains within the drug-linker structure were the best performing conjugates, distancing themselves from the ADCs obtained with a conventional linear 24-unit PEG oligomer or the linker of Kadcyla®. The pharmacokinetic profiles of amide-linked ADCs, with a linear or pendant configuration of the PEG, were tested in mice in comparison to Kadcyla®. Total antibody pharmacokinetics paralleled the trends in aggregation tendency, with slower clearance rates for the ADCs based on the pendant drug-linker format. The above-mentioned findings have provided important clues on the drug-linker design and revealed that the positioning and configuration of a PEG unit have to be carefully tuned to achieve ADCs with improved stability and pharmacokinetics.

Development of the Asia Pacific Consortium on Osteoporosis (APCO) Framework: clinical standards of care for the screening, diagnosis, and management of osteoporosis in the Asia-Pacific region.

Guidelines for doctors managing osteoporosis in the Asia-Pacific region vary widely. We compared 18 guidelines for similarities and differences in five key areas. We then used a structured consensus process to develop clinical standards of care for the diagnosis and management of osteoporosis and for improving the quality of care. PURPOSE: Minimum clinical standards for assessment and management of osteoporosis are needed in the Asia-Pacific (AP) region to inform clinical practice guidelines (CPGs) and to improve osteoporosis care. We present the framework of these clinical standards and describe its development. METHODS: We conducted a structured comparative analysis of existing CPGs in the AP region using a "5IQ" model (identification, investigation, information, intervention, integration, and quality). One-hundred data elements were extracted from each guideline. We then employed a four-round Delphi consensus process to structure the framework, identify key components of guidance, and develop clinical care standards. RESULTS: Eighteen guidelines were included. The 5IQ analysis demonstrated marked heterogeneity, notably in guidance on risk factors, the use of biochemical markers, self-care information for patients, indications for osteoporosis treatment, use of fracture risk assessment tools, and protocols for monitoring treatment. There was minimal guidance on long-term management plans or on strategies and systems for clinical quality improvement. Twenty-nine APCO members participated in the Delphi process, resulting in consensus on 16 clinical standards, with levels of attainment defined for those on identification and investigation of fragility fractures, vertebral fracture assessment, and inclusion of quality metrics in guidelines. CONCLUSION: The 5IQ analysis confirmed previous anecdotal observations of marked heterogeneity of osteoporosis clinical guidelines in the AP region. The Framework provides practical, clear, and feasible recommendations for osteoporosis care and can be adapted for use in other such vastly diverse regions. Implementation of the standards is expected to significantly lessen the global burden of osteoporosis.

Stimulatory effect of insulin on H+-ATPase in the proximal tubule via the Akt/mTORC2 pathway.

PURPOSE: Acid-base transport in renal proximal tubules (PTs) is mainly sodium-dependent and conducted in coordination by the apical Na+/H+ exchanger (NHE3), vacuolar H+-adenosine triphosphatase (V-ATPase), and the basolateral Na+/HCO3- cotransporter. V-ATPase on PTs is well-known to play an important role in proton excretion. Recently we reported a stimulatory effect of insulin on these transporters. However, it is unclear whether insulin is involved in acid-base balance in PTs. Thus, we assessed the role of insulin in acid-base balance in PTs. METHODS: V-ATPase activity was evaluated using freshly isolated PTs obtained from mice, and specific inhibitors were then used to assess the signaling pathways involved in the observed effects. RESULTS: V-ATPase activity in PTs was markedly enhanced by insulin, and its activation was completely inhibited by bafilomycin (a V-ATPase-specific inhibitor), Akt inhibitor VIII, and PP242 (an mTORC1/2 inhibitor), but not by rapamycin (an mTORC1 inhibitor). V-ATPase activity was stimulated by 1 nm insulin by approximately 20% above baseline, which was completely suppressed by Akt1/2 inhibitor VIII. PP242 completely suppressed the insulin-mediated V-ATPase stimulation in mouse PTs, whereas rapamycin failed to influence the effect of insulin. Insulin-induced Akt phosphorylation in the mouse renal cortex was completely suppressed by Akt1/2 inhibitor VIII and PP242, but not by rapamycin. CONCLUSION: Our results indicate that stimulation of V-ATPase activity by insulin in PTs is mediated via the Akt2/mTORC2 pathway. These results reveal the mechanism underlying the complex signaling in PT acid-base balance, providing treatment targets for renal disease.

Prophylactic effect of rikkunshito, an herbal medicine, for chemotherapy-induced nausea in thoracic cancer patients receiving carboplatin-based chemotherapy.

Rikkunshito has been shown to improve upper gastrointestinal symptoms and anorexia. The aim of this study was to evaluate whether rikkunshito improves chemotherapy-induced nausea in thoracic cancer patients receiving carboplatin (CBDCA)-based chemotherapy. A retrospective before-and-after comparison study was conducted in patients with thoracic cancer receiving the first cycle of CBDCA-based chemotherapy. Among 61 eligible patients, 34 received standard antiemetic therapy with a combination of 5-hydroxytryptamine-3 receptor antagonist and dexamethasone from September 2012 and June 2013 (standard group), while the other 27 received the standard antiemetic therapy plus oral rikkunshito from July 2013 and December 2014 (rikkunshito group). The rates of no nausea showed no significant difference between the standard and rikkunshito group (Overall phase: 64.7 % for standard group vs 74.1 % for rikkunshito group, p = 0.579). Subgroup analysis indicated that, in female patients, the rates of no nausea in rikkunshito groups was significantly higher than in standard group (overall phase: 44.4 % vs 100 %, p = 0.034). Rikkunshito did not demonstrate an additional prophylactic effect on standard antiemetic therapy for nausea in patients with thoracic cancer receiving CBDCA-based chemotherapy, but showed a prophylactic effect of nausea in female patients.

Telomere length determined by the fluorescence in situ hybridisation distinguishes malignant and benign cells in cytological specimens.

BACKGROUND: Telomeres are tandem repeats of TTAGGG at the end of eukaryotic chromosomes that play a key role in preventing chromosomal instability. The aim of the present study is to determine telomere length using fluorescence in situ hybridisation (FISH) on cytological specimens. METHODS: Aspiration samples (n = 41) were smeared on glass slides and used for FISH. RESULTS: Telomere signal intensity was significantly lower in positive cases (cases with malignancy, n = 25) as compared to negative cases (cases without malignancy, n = 16), and the same was observed for centromere intensity. The difference in DAPI intensity was not statistically significant. The ratio of telomere to centromere intensity did not show a significant difference between positive and negative cases. There was no statistical difference in the signal intensities of aspiration samples from ascites or pleural effusion (n = 23) and endoscopic ultrasound-guided FNA samples from the pancreas (n = 18). CONCLUSIONS: The present study revealed that telomere length can be used as an indicator to distinguish malignant and benign cells in cytological specimens. This novel approach may help improve diagnosis for cancer patients.

18F-fluorodeoxyglucose specimen-positron emission mammography delineates tumour extension in breast-conserving surgery: Preliminary results.

OBJECTIVES: We aimed to determine whether high-resolution specimen-positron emission mammography (PEM) using fluorodeoxyglucose (18F-FDG) can reveal extension of breast cancer in breast-conserving surgery (BCS), and assess the safety of radiation exposure to medical staff. METHODS: Sixteen patients underwent positron emission tomography, and then BCS with intraoperative frozen section analysis on the same day. Resected specimens with remaining 18F-FDG accumulation were scanned by high-resolution PEM. At least 1 day after surgery, tumour extension was evaluated by three independent experienced readers and by binarized images from the specimen-PEM data. Intraoperative exposure of medical staff to 18F-FDG was measured. RESULTS: Specimen-PEM evaluations of binarized images and the three investigators detected all (100 %, 12/12) invasive lesions and 94.4 % (17/18) of in situ lesions using both methods. The positive predictive value of the accumulated lesions was 74.4 % (29/39) for the binarized images and 82.9 % (29/35) for the three investigators. Analysis of intraoperative frozen sections detected 100 % (2/2) of the margin-positive cases, also detected by both specimen-PEM evaluation methods with no false-positive margin cases. The mean exposure of the medical staff to 18F was 18 µSv. CONCLUSIONS: Specimen-PEM detected invasive and in situ lesions with high accuracy and allowable radiation exposure. KEY POINTS: • Specimen-PEM detected invasive and in situ lesions with high accuracy. • Specimen-PEM predicted complete resection with the same accuracy as frozen section analysis. • Breast-conserving surgery after fluorodeoxyglucose injection was performed with low medical staff exposure.

Development of water vapor transmission rate measuring device using a quadrupole mass spectrometer and standard gas barrier films down to the 10-6 g m-2 day-1 level.

Water vapor transmission rate (WVTR) measuring devices with a quadrupole mass spectrometer (QMS) have an advantage in measuring low WVTRs because measurements are taken under an extremely low background of water vapor by realizing ultrahigh vacuum conditions. Here, the reliability of the QMS measurements was improved by including a porous plug with known molecular conductance in the device to generate a reference molar flux for in situ QMS calibration. Then, standard gas barrier (SGB) films made from a clay-polyimide nanocomposite film were also developed and used to validate the measurement. The measurement results for the SGB films were on the extrapolated calibration curve obtained with the porous plug down to WVTR at the 10-6 g m-2 day-1 level within the estimated measurement uncertainty.

MOB1-YAP1/TAZ-NKX2.1 axis controls bronchioalveolar cell differentiation, adhesion and tumour formation.

Mps One Binder Kinase Activator (MOB)1A/1B are core components of the Hippo pathway. These proteins, which coactivate LArge Tumour Suppressor homologue kinases, are also tumour suppressors. To investigate MOB1A/B's roles in normal physiology and lung cancer, we generated doxycycline (Dox)-inducible, bronchioalveolar epithelium-specific, null mutations of MOB1A/B in mice (SPC-rtTA/(tetO)7-Cre/Mob1aflox/flox/Mob1b-/-; termed luMob1DKO mice). Most mutants (70%) receiving Dox in utero (luMob1DKO (E6.5-18.5) mice) died of hypoxia within 1 h post-birth. Their alveolar epithelial cells showed increased proliferation, impaired YAP1/TAZ-dependent differentiation and decreased surfactant protein production, all features characteristic of human respiratory distress syndrome. Intriguingly, mutant mice that received Dox postnatally (luMob1DKO (P21-41) mice) did not develop spontaneous lung adenocarcinomas, and urethane treatment-induced lung tumour formation was decreased (rather than increased). Lungs of luMob1DKO (P21-41) mice exhibited increased detachment of bronchiolar epithelial cells and decreased numbers of the bronchioalveolar stem cells thought to initiate lung adenocarcinomas. YAP1/TAZ-NKX2.1-dependent expression of collagen XVII, a key hemidesmosome component, was also reduced. Thus, a MOB1-YAP1/TAZ-NKX2.1 axis is essential for normal lung homeostasis and expression of the collagen XVII protein necessary for alveolar stem cell maintenance in the lung niche.

Cytological features of solid variants of papillary thyroid carcinoma: a fine needle aspiration cytology study of 18 cases.

OBJECTIVE: Solid variants of papillary thyroid carcinoma (SV-PTC) are rare, and there have been few reports describing the cytological findings of such variants. METHODS: The cytological features of cellular specimens aspirated from 18 histologically confirmed SV-PTC cases were evaluated, retrospectively. RESULTS: Solid and small papillary clusters were observed in 14 (77.8%) and 13 (72.2%) cases, respectively. The incidences of large papillary clusters (11.1%) and sheet-like arrangements (11.1%) were low. Nuclear features were consistent with conventional PTC. The background was clean, and there were no colloid materials, foamy histiocytes, multinucleated giant cells, psammoma bodies, or necrotic materials. CONCLUSIONS: Solid clusters and small papillary clusters in conjunction with a clean background are diagnostic clues that indicate SV-PTC cytologically. It is thought that small papillary clusters reflect the micropapillary growth pattern seen within the lumen of middle-sized follicular structures. The presence of nuclear findings typical of conventional PTC and the absence of mitotic figures and necrotic materials are important for distinguishing SV-PTC from poorly differentiated carcinoma.

Determinant factors of postoperative recurrence of endometriosis: difference between endometrioma and pain.

OBJECTIVE: Although the postoperative use of hormonal treatment for endometriosis is recommended in the European Society of Human Reproduction and Embryology guidelines to prevent the recurrence of endometriosis-associated dysmenorrhoea, hormonal treatment may not be necessary for all patients who undergo surgical treatment for endometriosis. The aim of this study was to clarify the determinant factors that predict the recurrence of endometriosis after surgery in order to develop personalized hormonal treatment recommendations. Factors associated with the recurrence of endometrioma and pain were investigated independently to identify the likelihood of recurrence in each individual patient. STUDY DESIGN: Between 2008 and 2013, 352 patients underwent surgery and were diagnosed with endometriosis based on pathological findings at the study hospital. Among these patients, 191 experienced a recurrence of endometrioma in the absence of pre- or postoperative hormonal treatment. Various clinical factors such as pre-operative pain, intra-operative findings and postoperative improvement of pain were compared between patients who experienced recurrence after surgery and those who did not. RESULTS: The cumulative 5-year recurrence rate of endometrioma was 28.7% among the 191 patients who did not undergo pre- or postoperative hormonal treatment. Significant differences were detected in maximum tumour diameter, revised American Society for Reproductive Medicine score (r-ASRM score), operative time and operative blood loss between patients in the recurrent endometrioma group and the non-recurrent endometrioma group; only the r-ASRM score was significantly correlated with recurrence of endometrioma in the multivariate analysis. The cumulative 5-year rate of persistent/recurrent pain was 33.4%. There were significant differences in the postoperative improvement of pain between the persistent/recurrent pain group and the non-recurrent pain group according to the univariate and multivariate analyses. CONCLUSION: This study suggests that the risk factors for recurrence of endometrioma differ from the risk factors for recurrence of pain. The use of postoperative hormonal treatment should be considered based on the dominant risk factors and needs of each patient.

Bilberry extract supplementation for preventing eye fatigue in video display terminal workers.

OBJECTIVES: To examine the effect of a dietary supplement containing bilberry extract (BE) on eye fatigue induced by acute video display terminal (VDT) loads. DESIGN AND SETTING: A prospective, randomized, double-blind, placebo-controlled study was performed from August 2012 to February 2013 in the Medical Corporation Jico-kai Yagi Hospital, and the Shinyokohama Shinoharaguchi Orthopedic Surgery and Dermatology Clinic, in Japan. PARTICIPANTS: Two hundred eighty-one office workers aged 20-40 years that used VDTs were screened by critical flicker fusion (CFF) and near point accommodation (NPA). INTERVENTION: The participants were randomized to either a BE (480 mg/day) or placebo (vehicle) group, and took allocated capsule, daily, for 8 weeks. MEASUREMENTS: The CFF, NPA, contrast visual acuity, functional visual acuity, keratoconjunctival epithelial damage, and fluorescein tear film break-up time were examined, and 18 subjective symptoms of eye fatigue were evaluated by questionnaire. Adverse events were reported via medical interviews. Data were collected both before and after VDT load at baseline, and 4, and 8 weeks after daily supplementation with either BE or placebo. RESULTS: Of 281 participants screened, 88 having relatively lower levels of CFF and NPA were enrolled in the study. Of these, 37 control and 43 BE group subjects completed the study. The VDT load-induced reduction in CFF was alleviated after 8 weeks of BE supplementation (95% confidence interval, 0.10-1.60; p=0.023), in contrast to placebo supplementation, while NPA variation was not. Of the subjective symptoms of eye fatigue, VDT load-induced ocular fatigue sensation, ocular pain, eye heaviness, uncomfortable sensation, and foreign body sensation were mitigated more in the BE group than in the control group, at week 8 (p<0.05). There were no severe adverse events in either group. CONCLUSIONS: BE supplementation improved some of the objective and subjective parameters of eye fatigue induced by VDT loads.

The AURKA/TPX2 axis drives colon tumorigenesis cooperatively with MYC.

BACKGROUND: The MYC oncogene has long been established as a central driver in many types of human cancers including colorectal cancer. However, the realization of MYC-targeting therapies remains elusive; as a result, synthetic lethal therapeutic approaches are alternatively being explored. A synthetic lethal therapeutic approach aims to kill MYC-driven tumors by targeting a certain co-regulator on the MYC pathway. PATIENTS AND METHODS: We analyzed copy number and expression profiles from 130 colorectal cancer tumors together with publicly available datasets to identify co-regulators on the MYC pathway. Candidates were functionally tested by in vitro assays using colorectal cancer and normal fibroblast cell lines. Additionally, survival analyses were carried out on another 159 colorectal cancer patients and public datasets. RESULTS: Our in silico screening identified two MYC co-regulator candidates, AURKA and TPX2, which are interacting mitotic regulators located on chromosome 20q. We found the two candidates showed frequent co-amplification with the MYC locus while expression levels of MYC and the two genes were positively correlated with those of MYC downstream target genes across multiple cancer types. In vitro, the aberrant expression of MYC, AURKA and TPX2 resulted in more aggressive anchorage-independent growth in normal fibroblast cells. Furthermore, knockdown of AURKA or TPX2, or treatment with an AURKA-specific inhibitor effectively suppressed the proliferation of MYC-expressing colorectal cancer cells. Additionally, combined high expression of MYC, AURKA and TPX2 proved to be a poor prognostic indicator of colorectal cancer patient survival. CONCLUSIONS: Through bioinformatic analyses and experiments, we proposed TPX2 and AURKA as novel co-regulators on the MYC pathway. Inhibiting the AURKA/TPX2 axis would be a novel synthetic lethal therapeutic approach for MYC-driven cancers.