Role of ferroptosis in periodontitis: An animal study in rats.

OBJECTIVE: This study aimed to investigate (1) the temporal pattern of ferroptosis, an iron-dependent cell death, in ligation-induced rat periodontitis and (2) the effect of ferrostatin-1, a ferroptosis inhibitor, on the model. BACKGROUND: Ferroptosis may contribute to various diseases. However, the role of ferroptosis in periodontitis is still fully understood. METHODS: In the first experiment, 25 rats with ligation-induced periodontitis were sacrificed on days 0, 1, 2, 7, and 10. Gingivae were obtained to determine tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and ferroptotic biomarkers, including solute carrier family 3 member 2 (SLC3A2) and solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (Gpx4), via immunoblotting. Using microcomputed tomography (µCT) and histology, the periodontal soft and hard tissue lesions, including dental alveolar bone crest level, bony characteristics of the surrounding alveolus, periodontal tissue inflammation, and periodontal tissue losses, were evaluated. In study two, 16 rats with induced periodontitis were grouped according to ferrostatin-1 treatment. The rats were intraperitoneally injected with solvent or ferrostatin-1 (1.5 mg/kg/day) 1 day before ligation and sacrificed on days 7 and 10. Gingival protein changes and periodontal tissue damage were also examined. RESULTS: In study one, SLC3A2/SLC7A11 and Gpx4 decreased since day 1; however, TNF-α/IL-1ß increased on days 7 and 10. Moreover, the µCT/histology revealed resorptive bony characteristics, inflamed gingival tissue, and periodontal attachment loss. In study two, ferrostatin-1-injected rats exhibited significantly increased SLC3A2/SLC7A11 and Gpx4 but decreased TNF-α/IL-1ß than vehicle rats. They also revealed lessened bone resorption, tissue inflammation, and attachment loss. CONCLUSION: This study highlights the role of ferroptosis, via the system Xc/Gpx4 pathway, in experimental periodontitis and may serve as a regulatory strategy.

Chrysophanol Suppresses Cell Growth via mTOR/PPAR-α Regulation and ROS Accumulation in Cultured Human Tongue Squamous Carcinoma SAS Cells.

Oral cancer, a type of head and neck cancer, can pose a significant risk of death unless diagnosed and treated early. Alternative treatments are urgently needed owing to the high mortality rate, limitations of conventional treatments, and many complications. The anthraquinone compound chrysophanol acts as a tumor suppressor on some types of cancer cells. To date, it has not been clarified how chrysophanol affects human tongue squamous carcinoma. This study was aimed to examine the effects of chrysophanol on oral cancer treatment. The results show that chrysophanol caused cell death, reduced the expression of the mammalian target of rapamycin (mTOR)/peroxisome proliferator-activated receptor-alpha (PPAR-α), and increased reactive oxygen species (ROS) production. We also used two ion chelators, deferoxamine (DFO) and liproxstatin-1 (Lipro), to further determine whether chrysophanol inhibits cell growth and regulates mTOR/PPAR-α expression and ROS production, both of which are involved in iron homeostasis. The results show that DFO and Lipro reversed the increase in cell death, downregulation of mTOR/PPAR-α, and decrease in ROS accumulation. In conclusion, chrysophanol inhibits the growth of oral squamous cell carcinoma cells by modulating mTOR/PPAR-α and by causing ROS accumulation.

Fucoidan Prevents RANKL-Stimulated Osteoclastogenesis and LPS-Induced Inflammatory Bone Loss via Regulation of Akt/GSK3ß/PTEN/NFATc1 Signaling Pathway and Calcineurin Activity.

Excessive osteoclast differentiation and/or function plays a pivotal role in the pathogenesis of bone diseases such as osteoporosis and rheumatoid arthritis. Here, we examined whether fucoidan, a sulfated polysaccharide present in brown algae, attenuates receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis in vitro and lipopolysaccharide (LPS)-induced bone resorption in vivo, and investigated the molecular mechanisms involved. Our results indicated that fucoidan significantly inhibited osteoclast differentiation in RANKL-stimulated macrophages and the bone resorbing activity of osteoclasts. The effects of fucoidan may be mediated by regulation of Akt/GSK3ß/PTEN signaling and suppression of the increase in intracellular Ca2+ level and calcineurin activity, thereby inhibiting the translocation of nuclear factor-activated T cells c1 (NFATc1) into the nucleus. However, fucoidan-mediated NFATc1 inactivation was greatly reversed by kenpaullone, a GSK3ß inhibitor. In addition, using microcomputer tomography (micro-CT) scanning and bone histomorphometry, we found that fucoidan treatment markedly prevented LPS-induced bone erosion in mice. Collectively, we demonstrated that fucoidan was capable of inhibiting osteoclast differentiation and inflammatory bone loss, which may be modulated by regulation of Akt/GSK3ß/PTEN/NFATc1 and Ca2+/calcineurin signaling cascades. These findings suggest that fucoidan may be a potential agent for the treatment of osteoclast-related bone diseases.

Butyrate increases methylglyoxal production through regulation of the JAK2/Stat3/Nrf2/Glo1 pathway in castration­resistant prostate cancer cells.

Cancer cells are characterized by increased glycolysis, known as the Warburg effect, which leads to increased production of cytotoxic methylglyoxal (MGO) and apoptotic cell death. Cancer cells often activate the protective nuclear factor erythroid 2­related factor2 (Nrf2)/glyoxalase1 (Glo1) system to detoxify MGO. The effects of sodium butyrate (NaB), a product of gut microbiota, on Nrf2/Glos/MGO pathway and the underlying mechanisms in prostate cancer (PCa) cells were investigated in the present study. Treatment with NaB induced the cell death and reduced the proliferation of PCa cells (DU145 and LNCap). Moreover, the protein kinase RNA-like endoplasmic reticulum kinase/Nrf2/Glo1 pathway was greatly inhibited by NaB, thereby accumulating MGO-derived adduct hydroimidazolone (MG-H1). In response to a high amount of MGO, the expression of Nrf2 and Glo1 was attenuated, coinciding with an increased cellular death. NaB also markedly inhibited the Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (Stat3) pathway. Conversely, co­treatment with Colivelin, a Stat3 activator, significantly reversed the effects of NaB on Glo1 expression, MG-H1 production, and the cell migration and viability. As expected, overexpression of Stat3 or Glo1 reduced NaB­induced cell death. The activation of calcium/calmodulin dependent protein kinase II gamma and reactive oxygen species production also contributed to the anticancer effect of NaB. The present study, for the first time, demonstrated that NaB greatly increases MGO production through suppression of the JAK2/Stat3/Nrf2/Glo1 pathway in DU145 cells, a cell line mimicking castration­resistant PCa (CRPC), suggesting that NaB may be a potential agent for PCa therapy.

Comparison of Different Hepatocyte Nuclear Factor 4α Clones for Invasive Mucinous Adenocarcinoma of the Lung.

Invasive mucinous adenocarcinoma (IMA) is a rare variant of adenocarcinoma that comprises mucinous epithelial cells. The expression of hepatocyte nuclear factor 4α (HNF4α) has been previously reported as a marker for IMA, but controversy remains regarding whether HNF4α is a reliable marker for lung IMAs. In the present study, we compared HNF4α expression levels between IMA and nonmucinous adenocarcinoma (NMA) cases using 2 different HNF4α clones. We used 2 HNF4α antibody clones, H1 and H1415, to examine HNF4α expression in 36 IMA and 40 NMA cases, which comprised the control group. HNF4α immunostaining intensity (range, 0 to 3) and percentage of intensity (range, 0% to 100%) were evaluated by 3 pathologists and ImageJ software, and average H-scores were calculated for each case. Interobserver agreement was assessed using intraclass correlation coefficient. Receiver-operating characteristic curve was used to analyze sensitivity and specificity of the clones. The mean H-score was higher in the IMA group than in the NMA group for both the H1415 (141.3 vs. 9.3) and H1 (67.3 vs. 3.4) clones. The intraclass correlation coefficient for agreement among the 4 observers was good (0.806 and 0.711). The H1415 clone exhibited comparable sensitivity (83.3% vs. 83.3%) with higher specificity (97.5% vs. 92.5%) compared with the H1 clone when using cutoff values of 36.2 (H1415) and 9.5 (H1), respectively. Our analyses suggest that HNF4α should be considered as a reliable marker for primary IMA of the lung. The H1415 clone should be preferred for use in clinical practice.

Cyanidin-3-O-glucoside downregulates ligation-activated endoplasmic reticulum stress and alleviates induced periodontal destruction in rats.

OBJECTIVE: Cyanidin-3-O-glucoside (C3G), a dietary anthocyanin, possesses various biological properties, including alleviating endoplasmic reticulum (ER) stress. This study examined the effect of C3G on periodontitis via ER stress in rats. DESIGN: Periodontitis was induced by placing silk sutures around maxillary second molars. C3G (0, 3, or 9 mg/kg) was fed on the day before ligation (10 rats/group). Further, 10 non-ligation control rats received deionized water. On day 8, gingivae were obtained to determine CCAAT/enhancer-binding protein homologous protein (CHOP), c-Jun N-terminal kinase (JNK), phospho-JNK (p-JNK), and nuclear factor-κB (NF-κB) by immunoblotting. Periodontal destruction was evaluated using micro-computed tomography (µCT) and histology. RESULTS: Gingival expression of CHOP, p-JNK/JNK, and NF-κB significantly increased in ligation rats (0 mg/kg C3G) than that in controls. However, protein expression in ligation groups presented a negative association with C3G concentration. By µCT, the distance of cemento-enamel junction to bone significantly increased in ligation groups; however, distances showed a negative association with C3G concentration. In the region of interest, bone volume and trabecular thickness and number significantly decreased in ligation groups but they were positively associated with C3G concentration. In terms of trabecular separation, opposite results were found. Histologically, infiltrated connective tissue (ICT) and periodontal destructions increased in ligation groups; however, they were negatively associated with C3G concentration. Moreover, ICT area is positively correlated with µCT- and histologically measured destructions and protein expression of CHOP, p-JNK/JNK, or NF-κB. CONCLUSION: C3G promotes favorable modulation of ER stress and alleviates destruction of periodontitis, which may imply a new strategy.

Direct determination of ECD in ECD Kit: a solid sample quantitation method for active pharmaceutical ingredient in drug product.

Technetium-99m ethyl cysteinate dimer (Tc-99m-ECD) is an essential imaging agent used in evaluating the regional cerebral blood flow in patients with cerebrovascular diseases. Determination of active pharmaceutical ingredient, that is, L-Cysteine, N, N'-1,2-ethanediylbis-, diethyl ester, dihydrochloride (ECD) in ECD Kit is a relevant requirement for the pharmaceutical quality control in processes of mass fabrication. We here presented a direct solid sample determination method of ECD in ECD Kit without sample dissolution to avoid the rapid degradation of ECD. An elemental analyzer equipped with a nondispersive infrared detector and a calibration curve of coal standard was used for the quantitation of sulfur in ECD Kit. No significant matrix effect was found. The peak area of coal standard against the amount of sulfur was linear over the range of 0.03-0.10 mg, with a correlation coefficient (r) of 0.9993. Method validation parameters were achieved to demonstrate the potential of this method.

Tensile strength, growth factor content and proliferation activities for two platelet concentrates of platelet-rich fibrin and concentrated growth factor.

BACKGROUND/PURPOSE: Platelet-rich fibrin (PRF) can be obtained by centrifuging fresh blood in the absence of anticoagulants. Varying the centrifugation speeds may produce tougher and richer concentrated growth factors (CGF). This study examines tensile strength, growth factor content, and the potential of CGF and PRF in promoting periodontal cell proliferation. MATERIALS AND METHODS: Blood (40 mL/subject) was collected from 44 healthy subjects. PRF and CGF were prepared by centrifuging at 3000 rpm and switching speeds ranging within 3000 rpm, respectively. Fibrin strip was prepared and its tensile strength was measured. Transforming growth factor beta 1 (TGF-ß1), platelet-derived growth factor BB (PDGF-BB), and epidermal growth factor (EGF) in the residual serum and fibrin clots were determined by enzyme-linked immunosorbent assay, and their effects on the proliferation of hFOB1.19 osteoblasts and human gingival fibroblasts were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. RESULTS: Compared with PRF, tensile strength of CGF was significantly higher. Concentrations and amounts of PDGF-BB and EGF in CGF were significantly higher than those in PRF. Osteoblast number was significantly higher in the cultures with fetal bovine serum (FBS, 10%) and with PRF or CGF fibrin clots (5%, 10%, and 50%) compared to that without FBS. Moreover, osteoblast number in CGF, regardless of the preparation of 10% and 50%, was significantly greater than that in PRF. Similar findings were also observed for gingival fibroblasts among the various subjects. CONCLUSION: Varying centrifugation speeds can modify the tensile strength and biological activities of platelet fibrin clots.

Successful Identification of Nasopharyngeal Carcinoma in Nasopharyngeal Biopsies Using Deep Learning.

Pathologic diagnosis of nasopharyngeal carcinoma (NPC) can be challenging since most cases are nonkeratinizing carcinoma with little differentiation and many admixed lymphocytes. Our aim was to evaluate the possibility to identify NPC in nasopharyngeal biopsies using deep learning. A total of 726 nasopharyngeal biopsies were included. Among them, 100 cases were randomly selected as the testing set, 20 cases as the validation set, and all other 606 cases as the training set. All three datasets had equal numbers of NPC cases and benign cases. Manual annotation was performed. Cropped square image patches of 256 × 256 pixels were used for patch-level training, validation, and testing. The final patch-level algorithm effectively identified NPC patches, with an area under the receiver operator characteristic curve (AUC) of 0.9900. Using gradient-weighted class activation mapping, we demonstrated that the identification of NPC patches was based on morphologic features of tumor cells. At the second stage, whole-slide images were sequentially cropped into patches, inferred with the patch-level algorithm, and reconstructed into images with a smaller size for training, validation, and testing. Finally, the AUC was 0.9848 for slide-level identification of NPC. Our result shows for the first time that deep learning algorithms can identify NPC.

Immediate hyperbaric oxygen after tooth extraction ameliorates bisphosphonate-related osteonecrotic lesion in rats.

BACKGROUND: This study aims to assess whether hyperbaric oxygen (HBO) applied immediately after tooth extraction could ameliorate medication-related osteonecrosis of the jaw in rats. METHODS: To evaluate whether osteonecrosis could be successfully induced, healing of extraction maxillary molars was examined in 40 female Sprague Dawley rats received zoledronic acid (7.5 µg/kg) plus dexamethasone (1 mg/kg). Rats were divided into four groups, receiving zero, two, four, or seven injection(s) for 7 days, respectively. Effect of HBO, pressurized to 2.5 atmospheres absolute (ATA) at rate of 0.15 ATA/min with 100% oxygen for 90 minutes, applied immediately after tooth extraction, on the development of osteonecrosis was evaluated. Lesions among groups were compared by size of ulceration, exact area (mm2 ) or relative area (%), and by histology. RESULTS: Unhealed ridge was observed in all nine rats in four and seven injection groups, but none of 10 rats in the control (non-injection) group. Immediate HBO significantly reduced the lesions in rats that received four injections, regardless of the distribution and the total/relative areas of lesions (P <0.01). Histological findings showed the lesions were uncovered epithelium and severe tissue inflammation. CONCLUSION: This is the first in vivo study demonstrating the HBO applied immediately after tooth extraction effectively decreases the development of medication-related osteonecrosis.

Immunochemical features in the classification of human alcohol dehydrogenase family.

Human alcohol dehydrogenase (ADH) constitutes a complex family with diversified functions. Rabbit antihuman class I, II, III, and IV ADH antisera were prepared and used as probes to compare cross-reactivity with the isozymes across classes by semiquantitative Western blotting and quantitative enzyme-linked immunosorbent assay (ELISA). The interclass cross-reactivities with the noncognate isozymes by ELISA, generally approximately 0-35%, appeared considerably lower than those of the intraclass cross-reactivities except with the class IV isozyme. The anti-ADH1B1, ADH1C1, and ADH3 antisera, but not the anti-ADH2, exhibited approximately 80% cross-reactivity with ADH4. The intraclass cross-reactivities among class I isozymes ADH1A, ADH1B1, and ADH1C1 with anti-ADH1B1 or anti-ADH1C1 antisera were approximately 90%. Immunohistochemistry detecting with class-specific antibodies for ADH1-4 isolated from the corresponding antisera demonstrated that ADH4 was the predominant isoform expressed in the basal and suprabasal layer of human esophagus mucosa, whereas it was virtually devoid in the adjacent squamous cell carcinoma. Thus, the setup is more valuable for scanning ADH expression at protein level in different tissues and under different conditions, and maybe not as a tool for classification.

Functional comparison after reconstruction with a radial forearm free flap or a pectoralis major flap for cancer of the tongue.

OBJECTIVE: Numerous patients in Taiwan with tongue carcinoma require tongue reconstruction. We compared the abilities of 2 methods of tongue reconstruction to reserve tongue function. STUDY DESIGN AND SETTING: Sixty patients underwent resection of the tumors and reconstruction with a pectoralis major flap or a radial forearm flap. The Chinese articulation test was used to evaluate the place and manner of error production, and a questionnaire on dietary habits was used to evaluate deglutition 6 months to 10 years after reconstruction. RESULTS: Patients with the free flap had more intelligible speech. The questionnaire study showed no significant difference between the 2 groups in swallowing rating. Motility caused by flap pliability increased speech intelligibility more than it did on swallowing function. CONCLUSION: Our experience in a few selected patients shows that the functional outcome of tongue surgery is related to the reconstruction methods used (for speech) and to the extent of tongue resection (for swallowing).

Association of early-onset dementia with activities of daily living (ADL) in middle-aged adults with intellectual disabilities: the caregiver's perspective.

Few studies have investigated in detail which factors influence activities of daily living (ADL) in adults with intellectual disabilities (ID) comorbid with/without dementia conditions. The objective of the present study was to describe the relation between early onset dementia conditions and progressive loss of ADL capabilities and to examine the influence of dementia conditions and other possible factors toward ADL scores in adults with ID. This study was part of the "Healthy Aging Initiatives for Persons with an Intellectual Disability in Taiwan: A Social Ecological Approach" project. We analyzed data from 459 adults aged 45 years or older with an ID regarding their early onset symptoms of dementia and their ADL profile based on the perspective of the primary caregivers. Results show that a significant negative correlation was found between dementia score and ADL score in a Pearson's correlation test (r=-0.28, p<0.001). The multiple linear regression model reported that factors of male gender (ß=4.187, p<0.05), marital status (ß=4.79, p<0.05), education level (primary: ß=5.544, p<0.05; junior high or more: ß=8.147, p<0.01), Down's syndrome (ß=-9.290, p<0.05), severe or profound disability level (ß=-6.725, p<0.05; ß=-15.773, p<0.001), comorbid condition (ß=-4.853, p<0.05) and dementia conditions (ß=-9.245, p<0.001) were variables that were able to significantly predict the ADL score (R(2)=0.241) after controlling for age. Disability level and comorbidity can explain 10% of the ADL score variation, whereas dementia conditions can only explain 3% of the ADL score variation in the study. The present study highlights that future studies should scrutinize in detail the reasons for the low explanatory power of dementia for ADL, particularly in examining the appropriateness of the measurement scales for dementia and ADL in aging adults with ID.

Caregivers' reported functional limitations in activities of daily living among middle-aged adults with intellectual disabilities.

This study was conducted to describe the functioning of Activities of Daily Living (ADL) and to examine socio-economic effects on ADL functioning among adults with intellectual disabilities (ID) aged 45 years and older (N=480) in Taiwan. The Barthel Index (BI) was used to determine a baseline level of ADL functioning in the study participants. There are five categories of functional impairment using the following cut-off values in Taiwan: total dependence (BI score 0-20), severe (BI score 21-60), moderate (BI score 61-90), mild (BI score 91-99), and total independence (BI score 100) (Taiwan Department of Health, 2012). The results revealed that 2.3% of adults with ID were in total dependence, 11.9% were in severe dependence, 27.9% were in moderate dependence, 8.1% had a mild dependence, and 49.8% were totally independent. In the multiple linear regression model of the ADL score, we determined that educational level, comorbid Down's syndrome, and disability level are the variables able to significantly predict ADL score (R(2)=0.190) after controlling for the factors of age, marital status, and other comorbidity conditions. Those ID adults with a lower education level (primary vs. literate, ß=4.780, p=0.031; intermediate vs. literate, ß=6.642, p=0.030), with comorbid Down's syndrome (ß=-7.135, p=0.063), and with a more severe disability condition (severe vs. mild, ß=-7.650, p=0.007; profound vs. mild, ß=-19.169, p<0.001) had significantly lower ADL scores. The present study highlights the need to support mobility in older adults with ID as much as possible to optimize independence in this group.

Medial sural artery perforator flap for tongue and floor of mouth reconstruction.

BACKGROUND: The radial forearm flap is frequently considered the first choice for tongue reconstruction, but the disadvantages of donor site morbidity are well known. The search for another thin skin flap as an alternative has led to the application of the medial sural artery perforator flap. METHODS: We used 12 medial sural artery perforator flaps to reconstruct tongue and floor of mouth following cancer ablation. We paid attention to the major perforator (vein >or= 1 mm) as the vascular relay. RESULTS: Most flaps were raised with a single perforator. The size of the skin paddle varied from 9 cm x 5 cm to 14 cm x 12 cm. The mean thickness of the flap was 5.2 mm. We reexplored 1 patient for venous insufficiency and could not salvage the flap. CONCLUSIONS: The thin medial sural artery perforator flap permits high accuracy of tongue restoration and reduces the morbidity at the donor site.