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Objectives: Ketone bodies are endogenous metabolites produced during fasting or a ketogenic diet that have pleiotropic effects on aging pathways. Ketone esters (KEs) are compounds that induce ketosis without dietary changes, but KEs have not been studied in an older adult population. The primary objective of this trial was to assess the tolerability and safety of KE ingestion in a cohort of older adults. Design: Randomized, placebo-controlled, double-blinded, parallel-arm trial (NCT05585762). Setting: General community, Northern California, USA. Participants: Community-dwelling older adults, independent in activities of daily living, with no unstable acute medical conditions (n = 30; M = 15, F = 15; age = 76 y, range 65–90 y) were randomized and n = 23 (M = 14, F = 9) completed the protocol. Intervention: Participants were randomly allocated to consume either KE (25 g bis-octanoyl (R)-1,3-butanediol) or a taste, appearance, and calorie-matched placebo (PLA) containing canola oil daily for 12 weeks. Measurements: Tolerability was assessed using a composite score from a daily log for 2-weeks, and then via a bi-weekly phone interview. Safety was assessed by vital signs and lab tests at screening and weeks 0, 4 and 12, along with tabulation of adverse events. Results: There was no difference in the prespecified primary outcome of proportion of participants reporting moderate or severe nausea, headache, or dizziness on more than one day in a two-week reporting period (KE n = 2 (14.3% [90% CI = 2.6–38.5]); PLA n = 1 (7.1% [90% CI = 0.4–29.7]). Dropouts numbered four in the PLA group and two in the KE group. A greater number of symptoms were reported in both groups during the first two weeks; symptoms were reported less frequently between 2 and 12 weeks. There were no clinically relevant changes in safety labs or vital signs in either group. Conclusions: This KE was safe and well-tolerated in this study of healthy older adults. These results provide an initial foundation for use of KEs in clinical research with older adults.

Recently, it has been demonstrated that dysbiosis, an alteration in commensal microflora composition, is intimately involved in the onset of a variety of diseases. It is becoming increasingly evident that the composition of commensal microflora in the oral cavity is closely connected to oral diseases, such as periodontal disease, and systemic diseases, such as inflammatory bowel disease. Next-generation sequencing techniques are used as a method to examine changes in bacterial flora, but additional analytical methods to assess bacterial flora are needed to understand bacterial activity in more detail. In addition, the oral environment is unique because of the role of secretory antibodies contained in saliva in the formation of bacterial flora. The present study aimed to develop a new method for evaluating the compositional change of microbiota using flow cytometry (FCM) with specific antibodies against the bacterial surface antigen, as well as salivary antibodies. Using specific antibodies against Streptococcus mutans, a causative agent of dental caries, and human IgA, bacterial samples from human saliva were analyzed via FCM. The results showed that different profiles could be obtained depending on the oral hygiene status of the subjects. These results suggest that changes in the amount and type of antibodies that bind to oral bacteria may be an indicator for evaluating abnormalities in the oral flora. Therefore, the protocol established in this report could be applied as an evaluation method for alterations in the oral microbiota.

Background: Antimicrobial resistance to colistin, a widely used feed additive for farm animals across the world, has raised public health concern in recent years. Since July 2018, its use as feed additive has been banned in Japan to reduce the spread of plasmid-based mobilized colistin resistance (mcr) genes and the subsequent development of colistin-resistant bacteria. Evaluating the effects of these measures is required. Methods: We evaluated the effects of colistin use, as a feed additive, on colistin resistance in pigs (n = 5) from birth to finishing in the farm. Moreover, to evaluate changes in colistin resistance and mcr gene prevalence in response to colistin withdrawal, E. coli samples derived from pig faeces sourced from the fields of three geographically distinct farms were characterized before and after the withdrawal of colistin as a feed additive. Results: Colistin-resistant Escherichia coli in pigs (n = 5) increased during the colistin administration period and decreased immediately after its end. In three fields, the colistin resistance rate and prevalence of mcr-1 decreased immediately and significantly after the ban. However, colistin-resistant and mcr-1-positive E. coli were still detected in all three farm fields 12 months after the ban on colistin use. Conclusion: Agricultural colistin use caused selective pressure that contributed to widespread mcr dissemination in Japan. Colistin resistance and the presence of mcr genes should be continuously monitored in food-producing animals.

Tetracyclines (TCs) are widely used for livestock, and the high prevalence of TC-resistant Escherichia coli in livestock has become a serious concern worldwide. In Japan, the National Action Plan on Antimicrobial Resistance in 2016 aimed to reduce the TC resistance rate in E. coli derived from livestock. Flavophospholipol (FPL), used as a feed additive, has an inhibitory effect on the spread of plasmid-mediated antimicrobial resistance. The number of TC-resistant E. coli was determined in pigs administered TCs and/or FPL to clarify the effect of FPL on reducing the number of TC-resistant E. coli in pigs. TC-resistant E. coli and their plasmids were then analyzed. The pigs were divided into four groups: control, doxycycline (DOXY; a TC), FPL, and a DOXY-FPL combination. Their feces were collected from the nursing period to the day before being transported to the slaughterhouse, followed by estimation of TC-resistant E. coli (colony-forming units [CFU]/g). The number of TC-resistant E. coli increased with the use of DOXY, suggesting that DOXY administration provides a selective pressure for TC-resistant E. coli. Supplementation with FPL as a feed additive significantly suppressed the increase in the number of TC-resistant E. coli, especially during the DOXY administration period. Transfer and growth inhibition analyses were performed for TC-resistant isolates. FPL inhibited the conjugational transfer and growth of a few TC-resistant E. coli isolates. These results suggest that FPL is effective against the spread of TC-resistant E. coli.

Objective: Adenoid cystic carcinoma (AdCC) is a rare, indolent salivary gland tumor that is reported to be driven by fusion genes. However, MYB/MYBL1-NFIB fusions have been detected in <60% of all AdCC cases and the oncogenic driver mutations in approximately 40% of AdCC remain unknown. Our aim was to identify novel gene fusions in AdCC., Study Design: We investigated 20 AdCC cases using a targeted RNA sequencing panel to identify gene fusions and performed quantitative real-time reverse transcription polymerase chain reaction to assess MYB, MYBL1, and NFIB expression levels., Results: A total of 36 fusion transcripts in 15 cases were detected and validated by Sanger sequencing. The MYB-NFIB and MYBL1-NFIB fusion genes were detected in 9 and 3 cases, respectively, in a mutually exclusive manner. Furthermore, novel gene fusions, namely, NFIB-EPB41L2, MAP7-NFIB, NFIB-MCMDC2, MYBL1-C8orf34, C8orf34-NFIB, and NFIB-CASC20, were identified. Among them, NFIB-EPB41L2 and NFIB-MCMDC2 are thought to activate MYB and MYBL1 expression, respectively, through the insertion of a genomic segment in proximity to MYB and MYBL1 genes, respectively., Conclusion: Six novel gene fusions other than MYB/MYBL1-NFIB were identified. The detection of novel fusion genes and investigation of the molecular mechanism will contribute to the development of novel molecular targeted therapies for this disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: The objective of this study was to investigate the prognostic effects of clinical and histologic findings in patients with mucoepidermoid carcinoma (MEC) of minor salivary glands., Study Design: This retrospective clinical review included 63 patients (30 males, mean age 52.8 years) with minor salivary gland MEC treated at our hospital from 1994 to 2019. Overall survival (OS) or disease-free survival was determined using the Kaplan-Meier limit method. Correlations between different factors and survival rates were assessed using chi-square tests., Results: The 10-year OS rate was 91.2%. Low- or intermediate-grade MEC had a good prognosis regardless of the surgical margin, whereas high-grade MEC had a poor 10-year OS rate (64.2%). Ten patients developed recurrence or metastasis after primary surgical resection, of whom 6 were diagnosed with a high-grade tumor. The most frequently affected site was the palate, whereas the mandibular gingiva was the most commonly affected site during recurrence. Of 4 patients who received chemotherapy and/or radiotherapy postsurgery, 2 had local recurrence and/or neck lymph node metastasis and 1 died from MEC., Conclusion: Patients with low- or intermediate-grade MEC exhibited satisfactory survival after surgery. In patients with high-grade tumors, it has been suggested that survival rates are poor and do not improve following adjuvant therapy., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: In patients with advanced oral squamous cell carcinoma (OSCC), surgical treatment is often administered in combination with radiotherapy-based postoperative adjuvant therapy. The aim of the present study was to determine the most appropriate dose by comparing patient outcomes between doses of 50- and 66-Gy for postoperative cervical irradiation., Materials and Methods: This retrospective study included patients who underwent postoperative cervical irradiation following neck dissection for OSCC with lymph nodes metastases, and their primary lesions were controlled. They were divided into the 50- and 66-Gy irradiation groups and were examined for sex, age, primary lesion site, tumor/node/metastasis stage, initial treatment for primary cancer, neck dissection procedure, number of metastatic lymph nodes, presence or absence of extranodal extension (ENE), concomitant anticancer agents, and therapeutic outcomes., Results: A total of 78 patients met the clinical criteria for study enrollment. The 50- and 66-Gy postoperative cervical irradiation groups included 40 and 38 patients, with neck dissection performed at 45 and 38 sites, respectively. Cervical control rate was 97.5% and 97.3% in the 50- and 66-Gy irradiation groups (p = 0.74). The cumulative disease-specific 5-year survival rate was 85.6% and 88.3%, respectively, with no significant difference (p = 0.64)., Conclusion: The findings of our study indicate that the irradiation dose of 50 Gy is appropriate for postoperative cervical irradiation in patients with OSCC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objectives: Retropharyngeal lymph node (RPLN) metastasis is extremely rare, and prognosis is significantly poor in oral cancer. We retrospectively examined the management of RPLN metastases in oral cancer., Materials and Methods: A total of 1247 patients with oral cancer were treated at our department from January 2002 and December 2016. Among these patients, 374 (30%) had histologically positive lymph node metastases. Of these, 15 patients (1.2%) were diagnosed with RPLN metastases. We evaluated the diagnostic period, size, recurrence pattern, laterality, treatment, and therapeutic outcomes. The Kaplan-Meier method was used to determine overall survival (OS) among the RPLN metastasis group, cervical lymph node (CLN) metastases group, and treatment methods group for RPLN metastases., Results: One patient had RPLN involvement at the initial treatment, and RPLN involvement in other patients was found subsequently. The mean duration in confirming RPLN metastases was 228 days (range, 50-867 days). Surgical therapy was performed in 5 patients, chemoradiotherapy in 7 patients, and best supported care (BSC) in 3 patients. The cumulative 5-year OS rate for the RPLN metastasis group (n = 15) was 38.1%, compared with the rate of 71.3% for the CLN group (n = 359). Regarding the therapeutic approach for RPLN metastases, OS rates were 80.0% (n = 5) in the surgical therapy group, 28.6% (n = 7) in the chemoradiotherapy group, and 0% (n = 3) in the BSC group., Conclusion: Early detection and surgical treatment of RPLN metastases are associated with increased survival rate in oral cancer., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objectives: Postoperative mandibular fracture (PMF) after marginal resection (MR) of the mandible remains an unresolved issue, and it has been reported that at least 10 mm of postoperative mandibular body height (PMBH) is required to prevent PMF. This study evaluated the clinical, physical, and structural risk factors for PMF in MR patients and determined appropriate preventive measures for PMF., Study Design: This retrospective study included 44 patients with lower gingival carcinoma who underwent MR. PMF occurred in four of these patients. Thirteen associated factors identified from medical records and radiographs were statistically analyzed., Results: Mandibular body height (MBH) preservation ratio originally evaluated as less than 0.3, more than 20 remaining teeth after surgery, and inferior alveolar canal (IAC) exposure were significant risk factors for PMF. Prostheses and number of remaining teeth were also correlated with PMF., Conclusions: The preserved mandibular bone should be reinforced in patients with an MBH preservation ratio of less than 0.3, more than 20 remaining teeth after surgery, and intraoperative IAC exposure. Patients with prostheses are at an increased risk of PMF compared with those without because of stable occlusion and a strong occlusal force. Our novel findings provide useful reference standards for PMF prevention in MR patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Chronic osteomyelitis of the jaw (COMJ) is one of the most intractable diseases among head and neck infections. Antimicrobial agents are routinely administered for COMJ without sufficient bacterial information, resulting in frequent treatment failures. To improve our knowledge of the bacterial aetiology of COMJ and to assist in the development of effective treatments, we performed a comprehensive analysis of the microbiome. Sixteen patients with four clinical types of COMJ (four with suppurative osteomyelitis, three with osteoradionecrosis of the jaw, four with primary chronic osteomyelitis, and five with bisphosphonate-related osteonecrosis of the jaw) were enrolled in this study. Bone samples were subjected to bacterial community comparisons by 16S rRNA gene pyrosequencing. As a result, we clarified that COMJ was caused by a far greater range of bacterial species (12 phyla and 163 genera) than previously reported. Moreover, the bacterial structures in COMJ changed dramatically with disease stage and the condition of the affected bone. Multiple correlation analyses revealed that sequestration and bone exposure could affect the community structure. On the basis of these factors, we reclassified COMJ into three clinical stages: I, inflamed or sclerotic bone without exposure; II, sequestrum without exposure; and III, exposed sequestrum. In stage II, the bacterial diversity was significantly lower, and the anaerobe genera Fusobacterium, Tannerella (formerly Bacteroides) and Porphyromonas were more abundant, than observed during other stages. Because these bacteria habitually reside in any clinical stage, they were considered to constitute the core microbiome of COMJ. Targeting these bacteria should lead to the development of effective preventive measures and cures., (© 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.)

Plinabulin (NPI-2358) is a novel microtubule-depolymerizing agent. In HeLa cells, plinabulin arrests the cell-cycle at M phase and subsequently induces mitotic catastrophe. To better understand the effects on this compound on the cell-cycle, we used the fluorescent ubiquitination-based cell cycle indicator (Fucci), which normally enables G1 and S/G2/M cells to emit red and green fluorescence, respectively. When HeLa-Fucci cells were treated with 50 nM plinabulin, cells began to fluoresce both green and red in an unusual pattern; most cells exhibited the new pattern after 24 h of treatment. X-irradiation efficiently induced G2 arrest in plinabulin-treated cells and significantly retarded the emergence of the unusual pattern, suggesting that entering M phase is essential for induction of the pattern. By simultaneously visualizing chromosomes with GFP-histone H2B, we established that the pattern emerges after nuclear envelope breakdown but before metaphase. Pedigree assay revealed a significant relationship between the unusual expression and mitotic catastrophe. Nocodazole, KPU-133 (a more potent derivative of plinabulin), and paclitaxel also exerted similar effects. From these data, we conclude that the unusual pattern may be associated with dysregulation of late M phase-specific E3 ligase activity and mitotic catastrophe following treatment with anti-microtubule agents., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To explore the mechanism of bone destruction associated with oral cancer, we identified factors that stimulate osteoclastic bone resorption in oral squamous cell carcinoma. Two clonal cell lines, HSC3-C13 and HSC3-C17, were isolated from the maternal oral cancer cell line, HSC3. The conditioned medium from HSC3-C13 cells showed the highest induction of Rankl expression in the mouse stromal cell lines ST2 and UAMS-32 as compared to that in maternal HSC3 cells and HSC3-C17 cells, which showed similar activity. The conditioned medium from HSC3-C13 cells significantly increased the number of osteoclasts in a co-culture with mouse bone marrow cells and UAMS-32 cells. Xenograft tumors generated from these clonal cell lines into the periosteal region of the parietal bone in athymic mice showed that HSC3-C13 cells caused extensive bone destruction and a significant increase in osteoclast numbers as compared to HSC3-C17 cells. Gene expression was compared between HSC3-C13 and HSC3-C17 cells by using microarray analysis, which showed that CXCL2 gene was highly expressed in HSC3-C13 cells as compared to HSC3-C17 cells. Immunohistochemical staining revealed the localization of CXCL2 in human oral squamous cell carcinomas. The increase in osteoclast numbers induced by the HSC3-C13-conditioned medium was dose-dependently inhibited by addition of anti-human CXCL2-neutralizing antibody in a co-culture system. Recombinant CXCL2 increased the expression of Rankl in UAMS-32 cells. These results indicate that CXCL2 is involved in bone destruction induced by oral cancer. This is the first report showing the role of CXCL2 in cancer-associated bone destruction., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Angiogenesis is an important phenomenon for the growth and metastasis of solid tumors. The present study examined the characterization of angiogenic factors produced by human oral squamous cell carcinoma (oral SCC) cell lines established from lymph node metastatic tumors and primary tumor in different patients. The conditioned medium of HSC3 with the strongest metastatic ability among the examined lines enhanced a tube-forming activity of bovine carotid artery endothelial (BAE) cells in collagen gel cultures. The treatment of HSC3 with anti-vascular endothelial growth factor (VEGF) antibody or anti-basic fibroblast growth factor (bFGF) antibody, either alone or in combination, attenuated the activity of urokinase-type plasminogen activator (uPA) in the endothelial cells stimulated by the conditioned medium of HSC3. In contrast, neither anti-interleukin-8 (IL-8) antibody nor anti-hepatocyte growth factor (HGF beta) antibody affected uPA activity in the endothelial cells. Among these HSC cell lines, HSC3 secreted VEGF with the highest (1.92 +/- 0.24 ng/10(6) cells/24 h) level and bFGF. The level of bFGF secreted by HSC3 was lower than that secreted by BAE cells. Other oral SCC cell lines secreted lower levels of VEGF and undetectable levels of bFGF. By reverse transcriptase-polymerase chain reaction analysis of mRNA the production of VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206 in these cell lines was able to be detected. Moreover, the conditioned medium of HSC3 enhanced the tyrosine phosphorylation and expression of kinase insert domain-containing receptor (KDR/flk-1) in the endothelial cells. These results suggest that oral SCC promotes angiogenesis via expression of VEGF and upregulation of their receptor KDR/flk-1 expression in endothelial cells.