Both excitatory and inhibitory neurons transiently form clusters at the outermost region of the developing mammalian cerebral neocortex.

During development of the mammalian cerebral neocortex, postmitotic excitatory neurons migrate toward the outermost region of the neocortex. We previously reported that this outermost region is composed of densely packed relatively immature neurons; we named this region, which is observed during the late stage of mouse neocortical development, the "primitive cortical zone (PCZ)." Here, we report that postmigratory immature neurons spend about 1-1.5 days in the PCZ. An electron microscopic analysis showed that the neurons in the PCZ tend to be in direct contact with each other, mostly in a radial direction, forming "primitive neuronal clusters" with a height of 3-7 cells and a width of 1-2 cells. A time-course analysis of fluorescently labeled neurons revealed that the neurons took their positions within the primitive clusters in an inside-out manner. The neurons initially participated in the superficial part of the clusters, gradually shifted their relative positions downward, and then left the clusters at the bottom of this structure. GABAergic inhibitory interneurons were also found within the primitive clusters in the developing mouse neocortex, suggesting that some clusters are composed of both excitatory neurons and inhibitory interneurons. Similar clusters were also observed in the outermost region of embryonic day (E) 78 cynomolgus monkey occipital cortex and 23 gestational week (GW) human neocortices. In the primate neocortices, including human, the presumptive primitive clusters seemed to expand in the radial direction more than that observed in mice, which might contribute to the functional integrity of the primate neocortex.

Validation of the digital PCR system in tyrosine kinase inhibitor-resistant EGFR mutant non-small-cell lung cancer.

The aim of this study was to compare the accuracy of the QuantStudio 3D Digital polymerase chain reaction (dPCR) system and a PCR-based next generation sequencing (NGS) system for detecting a secondary mutation in the epidermal growth factor receptor (EGFR) gene T790M in non-small cell lung cancer (NSCLC) patients previously diagnosed with EGFR-activating mutations. Twenty-five patients with NSCLC previously treated with EGFR-TKIs were examined. The patients were treated daily with either erlotinib or gefitinib. New biopsies, followed by DNA sequencing on an Ion Torrent systems using the Ion Torrent AmpliSeq Cancer Hotspot Panel and dPCR were performed. A comparison of NGS, sensitive PCR, and dPCR revealed that the sensitivities of NGS and dPCR were similar in this study. As well, T790M was detected in as low as about 5% of mutant allelic frequencies, which represented 5% of the total reads on site mapped reads in NGS and greater than 5% of the dPCR reads, which represented mutant and wild type copies. The strategy in which NGS sequencing is followed by revealed acquired mutation with dPCR may be a reasonable one. We demonstrated the utility of combining NGS and dPCR as a tool for monitoring T790M. NGS and dPCR with formalin-fixed paraffin-embedded (FFPE) specimens might become a standard genomic test for exploring acquired resistance to targeted molecular medicines.

Programmed death-ligand 1 expression and T790M status in EGFR-mutant non-small cell lung cancer.

BACKGROUND: Differential biology and prognosis between T790M+ and T790M- populations imply immunological differences also. METHODS: We retrospectively analyzed programmed death-ligand 1 (PD-L1) expression and T790M status in rebiopsied samples of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). PD-L1 immunohistochemistry was performed using the SP142 antibody for tumour cell (TC) and tumour-infiltrating immune cell (IC) and the 28-8 antibody for TC. PD-L1+ was defined as TC or IC ≥1%. RESULTS: We investigated 67 available rebiopsied histologic samples in 47 patients. Using the SP142, prevalence of PD-L1 any+, moderate+, and strong+ in T790M+ vs. T790M- samples were 31% vs. 61%, 8% vs. 15%, and 0% vs. 2%, respectively, representing PD-L1+ prevalence of T790M+ samples was significantly lower than that of T790M- (p=0.0149). Prevalence of any TC+/IC+ in T790M+ vs. T790M- samples were TC: 31% vs. 51% (p=0.0997) and IC: 8% vs. 27% (p=0.0536), respectively. Using the 28-8, median percentage of PD-L1+ in T790M+ samples was 1.9 (range, 0-27.2), whereas T790M- was 4.1 (range, 0-89.8) (p=0.0801). Prevalence of PD-L1+ ≥1%, ≥5%, and ≥10% in T790M+ vs. T790M- samples were 77% vs. 83% (p=0.5476), 31% vs. 49% (p=0.1419), and 12% vs. 27% (p=0.1213), respectively. In 9 of 11 patients receiving multiple rebiopsies, T790M and/or PD-L1 expression revealed temporal dynamism. Survival curves according to PD-L1 expression/T790M status suggested better prognosis in PD-L1-/T790M+ population. CONCLUSIONS: T790M+ status was correlated to lower PD-L1 expression. PD-L1 expression might have a prognostic value and interaction with T790M mutation in EGFR-mutant NSCLC.

Single nucleotide variant sequencing errors in whole exome sequencing using the Ion Proton System.

Errors in sequencing are a major obstacle in the interpretation of next-generation sequencing (NGS) results. In the present study, sequencing errors identified from analysis of single nucleotide variants (SNVs) identified during exome sequencing of human germline DNA were studied using the Thermo Fisher Ion Proton System. Two consanguineous cases were selected for sequencing using the AmpliSeq Exome capture kit, and SNVs found in both cases were validated using Sanger sequencing. A total of 98 SNVs detected by NGS were randomly selected for further analysis. Nine of the analyzed SNVs were shown to be false positives when confirmed by Sanger sequencing. All but one SNV were considered to be homopolymer regions, mainly through the insertion or deletion of nucleotides. The remaining error was considered to be related to the primer. The present results revealed that the majority of the SNV sequencing errors originated from homopolymer insertion/deletion errors, which are commonly observed when using the Ion Torrent system.

Association of impaired neuronal migration with cognitive deficits in extremely preterm infants.

Many extremely preterm infants (born before 28 gestational weeks [GWs]) develop cognitive impairment in later life, although the underlying pathogenesis is not yet completely understood. Our examinations of the developing human neocortex confirmed that neuronal migration continues beyond 23 GWs, the gestational week at which extremely preterm infants have live births. We observed larger numbers of ectopic neurons in the white matter of the neocortex in human extremely preterm infants with brain injury and hypothesized that altered neuronal migration may be associated with cognitive impairment in later life. To confirm whether preterm brain injury affects neuronal migration, we produced brain damage in mouse embryos by occluding the maternal uterine arteries. The mice showed delayed neuronal migration, ectopic neurons in the white matter, altered neuronal alignment, and abnormal corticocortical axonal wiring. Similar to human extremely preterm infants with brain injury, the surviving mice exhibited cognitive deficits. Activation of the affected medial prefrontal cortices of the surviving mice improved working memory deficits, indicating that decreased neuronal activity caused the cognitive deficits. These findings suggest that altered neuronal migration altered by brain injury might contribute to the subsequent development of cognitive impairment in extremely preterm infants.

PD-L1 Expression in Patients with Non-small Cell Lung Cancer.

AIM: The aim of this study was to evaluate whether irradiation induces the expression of tumor programed cell death ligand 1 (PD-L1) in patients with non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Seventeen patients with NSCLC who received chemoradiotherapy and underwent tumor resection and six patients whose pre-treatment biopsy specimens were available, were analyzed by immunohistochemistry for PD-L1 expression between September 2011 and June 2016 at the Institute of Biomedical Research and Innovation Hospital. RESULTS: Among six patients for which pre-irradiation biopsy samples were available, the H-score for PD-L1 was reduced after irradiation following staining with two different antibody clones (SP28-8 and SP142). A PD-L1 H-score >5 with SP28-8 antibody (hazard ratio=6.46; 95% confidence interval=1.209-34.53; p=0.029) was a significant negative factor for duration of progression-free survival after curative operation or chemoradiation. CONCLUSION: We showed that tumor PD-L1 expression decreased in patients with NSCLC who received chemoradiotherapy and radiation resistance might be due to pre-treatment PD-L1 expression.

Intentions and Factors Regarding Selection of Workplace by Postgraduate Dental Trainees after Clinical Training.

The purpose of the present study was to investigate the reasons governing selection of geographical location of workplace by postgraduate dental trainees. We believe that such data would aid in the development of measures aimed at reducing regional disparities in the number of dentists. A total of 10 dental colleges and faculties in Japan were invited to participate in the study. Anonymous, self-completed questionnaire surveys were given to 739 dentists at 13 clinical training facilities following training in 2012 and 2013; 713 responses were analyzed. The items surveyed included the local population in the area in which they wished to work and whether they wanted to work in rural areas or remote islands. The most common response to the question regarding population was "medium-size cities" with a population of 100,000 to 300,000 people (67.3%). The largest number of respondents stated that they hoped to work in their hometown or a neighboring area (49.9%). A large percentage wished to work in medium-size cities, which may reflect the population of their hometowns. The current system allows dentists a free choice in becoming practitioners or hospital dentists on completion of training. This may explain why so many expressed a preference to work in their hometown or a neighboring area here. These results suggest that policies relying on increasing the supply of dentists as the sole solution to rural shortages are flawed.

Programmed death-ligand 1 expression according to epidermal growth factor receptor mutation status in pretreated non-small cell lung cancer.

BACKGROUND: Current clinical trials have suggested poorer efficacies of anti-programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) immunotherapies for non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations, implying lower PD-L1 expression in EGFR-mutant NSCLC than in EGFR-wild type. METHODS: We retrospectively analyzed correlation between PD-L1 expression and EGFR status in clinical samples of pretreated NSCLC. PD-L1 immunohistochemistry was performed using the 28-8 anti-PD-L1 antibody for tumor cell membrane staining. H-score was adopted to evaluate both percentage and intensity. We investigated H-scores ≥1, ≥5, and ≥10 as PD-L1+ cut-offs. H-score ≥10 was defined as strong PD-L1+. RESULTS: We investigated 96 available histologic samples in 77 pretreated patients with NSCLC. Median H-score in EGFR-mutant samples (n=65) was 3 (range, 0-150), whereas EGFR-wild-type (n=31) was 8 (range, 0-134) (p=0.0075). Using H-scores ≥1, ≥5, and ≥10 cut-offs, incidence of PD-L1+ in EGFR-mutant vs. EGFR-wild-type samples were: 85% (55/65) vs. 94% (29/31) (p=0.2159); 42% (27/65) vs. 74% (23/31) (p=0.0027); and 22% (14/65) vs. 48% (15/31) (p=0.0074), respectively. Patient-oriented (n=77) univariate analysis for strong PD-L1+ found age of sample (p=0.0226) and EGFR mutation status (p=0.0490) as significant factors. Multivariate analysis identified EGFR mutation status as the only significant factor (p=0.0121, odds ratio 2.99) for strong PD-L1+. H-scores of PD-L1 expression varied in all 11 cases receiving multiple rebiopsies, and categories of positivity migrated in 10 (91%) of 11 patients. CONCLUSIONS: PD-L1 expression was significantly lower in EGFR-mutant NSCLC samples than in EGFR wild-type samples. Its expression could be dynamic and affected by age of sample.

Salusin-ß as a powerful endogenous antidipsogenic neuropeptide.

Salusin-ß is an endogenous parasympathomimetic peptide, predominantly localized to the hypothalamus and posterior pituitary. Subcutaneously administered salusin-ß (50 nmol/mouse) significantly increased water intake but did not affect locomotor activity or food intake. The salusin-ß-induced increase in water intake was completely abrogated by pretreatment with muscarinic antagonist, atropine sulphate. In contrast, intracerebroventricular injection of salusin-ß, at lower doses (10-100 fmol/mouse) caused a long-lasting decrease in water intake and locomotor activity throughout the entire dark phase of the diurnal cycle. Pre-injection of intracerebroventricular anti-salusin-ß IgG completely abrogated the central salusin-ß mediated suppression of water intake and locomotor activity. These results demonstrate contrasting actions of salusin-ß in the control of water intake via the central and peripheral systems and highlight it as a potent endogenous antidipsogenic neuropeptide.

Adult Oral Health Programs in Japanese Municipalities: Factors Associated with Self-Rated Effectiveness.

Health Japan 21 plan establishes specific targets for aspects of health including oral health for 2010, in an effort to increase health expectancy. Despite this, there has been insufficient improvement in oral health status in adults. The objective of this cross-sectional study was to determine the factors associated with effective oral health programs for adults in Japanese municipalities. Questionnaires were mailed to all 1,472 municipalities in Japan and responses were obtained from 862 municipalities (response rate: 58.6%). After excluding 71 municipalities with "unknown" answer, no answer, or lack of relevant information, we analyzed the data from the remaining 791 municipalities with or without oral health programs for adults self-reported as effective within three years. Multilevel Poisson regression models were used to examine the associations of effective programs with oral health personnel, contact with related agencies, the establishment of Health Japan 21 goals, financial status, the density of dentists and population density at the municipality level, and having oral health personnel at the prefecture level. Three hundred and fifty-four municipalities reported having effective programs. In the fully adjusted model, having dental hygienists in the municipal office (P < 0.05) and a high number of contacts with related agencies (P < 0.05) were significantly associated with having effective programs. These results suggest that having dental hygienists and contact with related agencies such as residents, local dental associations, companies, community general support centers, or medical, nursing or welfare facilities are promoting factors for effective adult oral health programs in Japanese municipalities.

Next-generation sequencing of tyrosine kinase inhibitor-resistant non-small-cell lung cancers in patients harboring epidermal growth factor-activating mutations.

BACKGROUND: The aim of this study was to detect the epidermal growth factor receptor (EGFR)-activating mutations and other oncogene alterations in patients with non-small-cell lung cancers (NSCLC) who experienced a treatment failure in response to EGFR-tyrosine kinase inhibitors (TKIs) with a next generation sequencer. METHODS: Fifteen patients with advanced NSCLC previously treated with EGFR-TKIs were examined between August 2005 and October 2014. For each case, new biopsies were performed, followed by DNA sequencing on an Ion Torrent Personal Genome Machine (PGM) system using the Ion AmpliSeq Cancer Hotspot Panel version 2. RESULTS: All 15 patients were diagnosed with NSCLC harboring EGFR-activating mutations (seven cases of exon 19 deletion, seven cases of L858R in exon 21, and one case of L861Q in exon 21). Of the 15 cases, acquired T790M resistance mutations were detected in 9 (60.0%) patients. In addition, other mutations were identified outside of EGFR, including 13 cases (86.7%) exhibiting TP53 P72R mutations, 5 cases (33.3%) of KDR Q472H, and 2 cases (13.3%) of KIT M541L. CONCLUSIONS: Here, we showed that next-generation sequencing (NGS) is able to detect EGFR T790M mutations in cases not readily diagnosed by other conventional methods. Significant differences in the degree of EGFR T790M and other EGFR-activating mutations may be indicative of the heterogeneity of disease phenotype evident within these patients. The co-existence of known oncogenic mutations within each of these patients may play a role in acquired EGFR-TKIs resistance, suggesting the need for alternative treatment strategies, with PCR-based NGS playing an important role in disease diagnosis.

Regulation of growth hormone secretion by (pro)renin receptor.

(Pro)renin receptor (PRR) has a single transmembrane domain that co-purifies with the vacuolar H(+)-ATPase (V-ATPase). In addition to its role in cellular acidification, V-ATPase has been implicated in membrane fusion and exocytosis via its Vo domain. Results from the present study show that PRR is expressed in pituitary adenoma cells and regulates growth hormone (GH) release via V-ATPase-induced cellular acidification. Positive PRR immunoreactivity was detected more often in surgically resected, growth hormone-producing adenomas (GHomas) than in nonfunctional pituitary adenomas. GHomas strongly expressing PRR showed excess GH secretion, as evidenced by distinctly high plasma GH and insulin-like growth factor-1 levels, as well as an elevated nadir GH in response to the oral glucose tolerance test. Suppression of PRR expression in rat GHoma-derived GH3 cells using PRR siRNA resulted in reduced GH secretion and significantly enhanced intracellular GH accumulation. GH3 treatment with bafilomycin A1, a V-ATPase inhibitor, also blocked GH release, indicating mediation via impaired cellular acidification of V-ATPase. PRR knockdown decreased Atp6l, a subunit of the Vo domain that destabilizes V-ATPase assembly, increased intracellular GH, and decreased GH release. To our knowledge, this is the first report demonstrating a pivotal role for PRR in a pituitary hormone release mechanism.

Validation of an Ion Torrent Sequencing Platform for the Detection of Gene Mutations in Biopsy Specimens from Patients with Non-Small-Cell Lung Cancer.

BACKGROUND: Treatment for patients with advanced non-small cell lung cancer (NSCLC) is often determined by the presence of biomarkers that predict the response to agents targeting specific molecular pathways. Demands for multiplex analysis of the genes involved in the pathogenesis of NSCLC are increasing. METHODS: We validated the Ion Torrent Personal Genome Machine (PGM) system using the Ion AmpliSeq Cancer Hotspot Panel and compared the results with those obtained using the gold standard methods, conventional PCR and Sanger sequencing. The cycleave PCR method was used to verify the results. RESULTS AND CONCLUSION: The Ion Torrent PGM resulted in a similar level of accuracy in identifying multiple genetic mutations in parallel, compared with conventional PCR and Sanger sequencing; however, the Ion Torrent PGM was superior to the other sequencing methods in terms of increased ease of use, even when taking into account the small amount of DNA that was obtained from formalin-fixed paraffin embedded (FFPE) biopsy specimens.

Ectopic ACTH syndrome caused by desmopressin-responsive thymic neuroendocrine tumor.

A 32-year-old Chinese woman with rapid weight gain and progressive edema was found to have typical Cushingoid features. Her endocrine data were consistent with a diagnosis of ACTH-dependent Cushing's syndrome. To differentiate ectopic ACTH syndrome (EAS) from Cushing's disease (CD), various dynamic endocrine and imaging tests were performed. Her ACTH response was negative to corticotropin-releasing hormone (CRH) and positive to desmopressin. Magnetic resonance imaging of the pituitary showed no mass lesion. Computed tomography scan of the chest revealed a large mass (21 × 15 mm) in the anterior mediastinum, where positron emission tomography showed accumulation of [(18)F] fluorodeoxyglucose. Selective venous sampling showed marked step-up in ACTH level in the internal thoracic vein but not in the cavernous sinus after CRH stimulation. These data are compatible with the diagnosis of EAS. The resected tumor was pathologically consistent with thymic neuroendocrine tumor (NET) positive for ACTH by immunohistochemistry and abundant V1b receptor gene expression by RT-PCR. Postoperatively, her circulating ACTH/cortisol levels became normalized, and responded to stimulation with CRH but not with desmopressin. Her Cushingoid appearance gradually disappeared, and she was free from recurrence 5 years after surgery. This is a rare case of desmopressin-responsive EAS caused by thymic NET with predominant V1b gene expression, which was successfully localized by imaging modalities combined with selective venous sampling.

A Case of Rathke's Cleft Cyst Associated with Transient Central Adrenal Insufficiency and Masked Diabetes Insipidus.

A 73-year-old woman admitted to our hospital because of headache, poor appetite, malaise, weight loss, and vomiting was found to have central adrenal insufficiency and thyrotoxicosis due to silent thyroiditis. Polyuria developed after replacement with glucocorticoid (masked diabetes insipidus), which was controlled with nasal administration of desmopressin. Magnetic resonance imaging of the brain showed a large cystic pituitary mass (18 × 18 × 12 mm) extending suprasellarly to the optic chiasm. Transsphenoidal surgery revealed that the pituitary tumor was Rathke's cleft cyst. Following surgery, replacement with neither glucocorticoid nor desmopressin was needed any more. Therefore, it is suggested that Rathke's cleft cyst is responsible for the masked diabetes insipidus and the central insufficiency. Furthermore, it is speculated that thyrotoxicosis with painless thyroiditis might induce changes from subclinical adrenal insufficiency to transiently overt insufficiency.

Association between the longest job and oral health: Japan Gerontological Evaluation Study project cross-sectional study.

BACKGROUND: Inequality in oral health is a major challenge. Oral diseases and their risk factors accumulate throughout life. The objective of this cross-sectional study was to examine the association of longest job with oral health status and oral health behavior among older Japanese. METHODS: Subjects were a total of 23,191 (11,310 males and 11,881 females) community-dwelling individuals aged 65 or over, living independently and able to perform daily activities from 30 municipalities across Japan. The outcome variables were oral health status (number of teeth, use of denture or bridge and subjective oral health status) and oral health behavior (dental visit for treatment and use of interdental brush or dental floss). The longest job was used as an explanatory variable. Age, educational attainment, equivalent income, and densities of dentists and population in municipalities were used as covariates. Two-level (first level: individual, second level: municipality) multilevel Poisson regression analyses were performed for each sex. RESULTS: Multilevel Poisson regression analyses showed that all variables of oral health status and oral health behavior were significantly associated with longest job after adjusting for all covariates except denture/bridge use and dental visit for females. People whose longest jobs were sales/service, skilled/labor, agriculture/forestry/fishery or others, or who had no occupation were more likely to have poor oral health status and oral health behavior compared to those whose longest jobs were professional/technical. CONCLUSIONS: The longest job may be one of the major determinants of oral health status and oral health behavior in Japanese older people.

Social determinants of denture/bridge use: Japan gerontological evaluation study project cross-sectional study in older Japanese.

BACKGROUND: Studies suggest that using a denture/bridge may prevent disability in older people. However, not all older people with few remaining teeth use a denture/bridge. This cross-sectional study aimed to examine the social determinants which promote denture/bridge use among older Japanese. METHODS: A total of 54,388 (25,630 males and 28,758 females) community-dwelling individuals aged 65 or over, living independently, able to perform daily activities, and with 19 or fewer teeth. The dependent variable was denture/bridge use. Socio-demographics, number of teeth, present illness, social participation, social support, and social networks were used as individual-level independent variables. Data for social capital were aggregated and used as local district (n = 561 for males, n = 562 for females) -level independent variables. Number of dentists working in hospitals/clinics per population and population density were used as municipality (n = 28) -level independent variables. Three-level multilevel Poisson regression analysis was performed for each sex. RESULTS: High equivalent income, low number of teeth, present illness, and living in a municipality with high population density were significantly associated with denture/bridge use in both sexes in the fully adjusted models (p < 0.05). Denture/bridge use was significantly associated with high educational attainment in males and participating in social groups in females in the fully adjusted model (p < 0.05). No significant associations were observed between denture/bridge use and social capital. CONCLUSIONS: Denture/bridge use was significantly associated with high economic status and present illness in both sexes, high educational attainment in males, and participation in social groups in females among community-dwelling older Japanese after adjusting for possible confounders.

Dental health behavior of parents of children using non-fluoride toothpaste: a cross-sectional study.

BACKGROUND: One of the dental health goals of Health Japan 21, in which the Japanese government clarified its health policy, was to ensure the use of fluoride toothpaste in 90% or more of schoolchildren. This goal was not achieved. The aim of this cross-sectional questionnaire study was to evaluate the characteristics of parents whose children use non-fluoride toothpaste. METHODS: In December 2010, questionnaire forms were sent to 18 elementary schools or school dentists. Students (6-12 years old) were asked to take the forms home for their parents to fill in, and to bring the completed questionnaire to school. The collected questionnaires were mailed from schools to the author's institution by the end of March 2011. The relationship between fluoride in toothpaste and reasons for choice of toothpaste, the child's toothbrushing habits, and attitude toward child caries prevention was examined in the 6,069 respondents who answered all the questions for the analyses and indicated that their children use toothpaste. RESULTS: Non-fluoride toothpaste users accounted for 5.1% of all toothpaste users. Among the children using non-fluoride toothpaste, significantly greater numbers gave 'anti-gingivitis', 'halitosis prevention' or 'tartar control' as reasons for choice of toothpaste; did not give 'has fluoride', 'is cheaper' or 'tastes good' as reasons for choice of toothpaste; or used toothpaste sometimes, or were in 4th - 6th grades. There was no significant relationship between use of non-fluoride toothpaste and measures taken for caries prevention in children. Multilevel (first level: individual, second level: school) logistic regression analysis indicated that use of non-fluoride toothpaste was significantly related to: giving 'anti-gingivitis' (odds ratio: 1.44) as a reason for choice of toothpaste; not giving 'has fluoride' (0.40), 'tastes good' (0.49) or 'is cheaper' (0.50) as the reason for choice of toothpaste; to toothbrushing less often (twice a day: 1.34, once a day or less: 1.46) and to using toothpaste less often (sometimes: 1.39). CONCLUSIONS: It is necessary to teach parents that dental caries is the dental health issue with the highest priority for children, and therefore fluoride toothpaste should be used.

Multimolecular salivary mucin complex is altered in saliva of cigarette smokers: detection of disulfide bridges by Raman spectroscopy.

Saliva contains mucins, which protect epithelial cells. We showed a smaller amount of salivary mucin, both MG1 and MG2, in the premenopausal female smokers than in their nonsmoking counterparts. Smokers' MG1, which contains almost 2% cysteine/half cystine in its amino acid residues, turned out to be chemically altered in the nonsmoker's saliva. The smaller acidic glycoprotein bands were detectable only in smoker's saliva in the range of 20-25 kDa and at 45 kDa, suggesting that degradation, at least in part, caused the reduction of MG1 mucin. This is in agreement with the previous finding that free radicals in cigarette smoke modify mucins in both sugar and protein moieties. Moreover, proteins such as amylase and albumin are bound to other proteins through disulfide bonds and are identifiable only after reduction with DTT. Confocal laser Raman microspectroscopy identified a disulfide stretch band of significantly stronger intensity per protein in the stimulated saliva of smokers alone. We conclude that the saliva of smokers, especially stimulated saliva, contains significantly more oxidized form of proteins with increased disulfide bridges, that reduces protection for oral epithelium. Raman microspectroscopy can be used for an easy detection of the damaged salivary proteins.

Dental status and incident falls among older Japanese: a prospective cohort study.

OBJECTIVE: To examine if self-reported number of teeth, denture use and chewing ability are associated with incident falls. DESIGN: Longitudinal cohort study (the Aichi Gerontological Evaluation Study). SETTING: 5 Japanese municipalities. PARTICIPANTS: 1763 community-dwelling individuals aged 65 years and older without experience of falls within the previous year at baseline. MAIN OUTCOME MEASURES: Self-reported history of multiple falls during the past year at the follow-up survey about 3 years later. Baseline data on the number of teeth present and/or denture use and chewing ability were collected using self-administered questionnaires. Logistic regression analyses controlled for sex, age, functional disability during follow-up period, depression, self-rated health and educational attainment. RESULTS: 86 (4.9%) subjects reported falls at the follow-up survey. Logistic regression models fully adjusted for all covariates showed that subjects having 19 or fewer teeth but not using dentures had a significantly increased risk for incident falls (OR 2.50, 95% CI 1.21 to 5.17, p=0.013) compared with those having 20 or more teeth. Among subjects with 19 or fewer teeth, their risk of falls was not significantly elevated so long as they wore dentures (OR 1.36, 95% CI 0.76 to 2.45, p=0.299). No significant association was observed between chewing ability and incident falls in the fully adjusted model. CONCLUSIONS: Having 19 or fewer teeth but not using dentures was associated with higher risk for the incident falls in older Japanese even after adjustment for multiple covariates. Dental care to prevent tooth loss and denture treatment for older people might prevent falls, although the authors cannot exclude the possibility that the association is due to residual confounding.