The effect of Porphyromonas gingivalis on the gut microbiome of mice in relation to aging.

BACKGROUND AND OBJECTIVE: The translocation of oral bacteria, including Porphyromonas gingivalis, to the gut has been shown to alter gut microbiome. However, the effect of P. gingivalis on gut microbiome in relation to aging has not been demonstrated. We hypothesize that P. gingivalis has more detrimental effect on gut environment with increased age. The objective of this study is to investigate the effect of P. gingivalis on gut environment using aged mice. MATERIALS AND METHODS: C57BL/6J mice aged 4 weeks (young) or 76 weeks (old) were divided into four groups: control-young, control-old, P. gingivalis-administered young, and P. gingivalis-administered old. P. gingivalis was orally administered thrice weekly for 5 weeks. At 30 days after the last P. gingivalis administration, 16S rRNA sequencing was performed to study the gut microbiome. The mRNA and protein expression of intestinal junctional barrier molecules and the levels of the inflammatory cytokines IL-1ß and TNF-α in the serum were evaluated. RESULTS: Significant differences in the gut microbiomes between the groups, in terms of taxonomic abundance, bacterial diversity, and predicted metagenome function, were observed. A significant reduction in the alpha diversity and in the abundance of beneficial bacteria, such as Akkermansia and Clostridiaceae, in the P. gingivalis-administered old mice was observed. The mRNA and protein levels of Claudin-1 and Claudin-2 in the intestine were significantly elevated, while E-cadherin was significantly downregulated in the P. gingivalis-administered old mice, as were the serum levels of IL-1ß and TNF-α. CONCLUSION: The effect of P. gingivalis on the gut environment is more pronounced in old mice than in young mice.

Inattentive Driving Detection Using Body-Worn Sensors: Feasibility Study.

This study aims to build a system for detecting a driver's internal state using body-worn sensors. Our system is intended to detect inattentive driving that occurs during long-term driving on a monotonous road, such as a high-way road. The inattentive state of a driver in this study is an absent-minded state caused by a decrease in driver vigilance levels due to fatigue or drowsiness. However, it is difficult to clearly define these inattentive states because it is difficult for the driver to recognize when they fall into an absent-minded state. To address this problem and achieve our goal, we have proposed a detection algorithm for inattentive driving that not only uses a heart rate sensor, but also uses body-worn inertial sensors, which have the potential to detect driver behavior more accurately and at a much lower cost. The proposed method combines three detection models: body movement, drowsiness, and inattention detection, based on an anomaly detection algorithm. Furthermore, we have verified the accuracy of the algorithm with the experimental data for five participants that were measured in long-term and monotonous driving scenarios by using a driving simulator. The results indicate that our approach can detect both the inattentive and drowsiness states of drivers using signals from both the heart rate sensor and accelerometers placed on wrists.

Trends and Future Prospects of the Drowsiness Detection and Estimation Technology.

Drowsiness is among the important factors that cause traffic accidents; therefore, a monitoring system is necessary to detect the state of a driver's drowsiness. Driver monitoring systems usually detect three types of information: biometric information, vehicle behavior, and driver's graphic information. This review summarizes the research and development trends of drowsiness detection systems based on various methods. Drowsiness detection methods based on the three types of information are discussed. A prospect for arousal level detection and estimation technology for autonomous driving is also presented. In the case of autonomous driving levels 4 and 5, where the driver is not the primary driving agent, the technology will not be used to detect and estimate wakefulness for accident prevention; rather, it can be used to ensure that the driver has enough sleep to arrive comfortably at the destination.

A Review of Heartbeat Detection Systems for Automotive Applications.

Many accidents are caused by sudden changes in the physical conditions of professional drivers. Therefore, it is quite important that the driver monitoring system must not restrict or interfere with the driver's action. Applications that can measure a driver's heartbeat without restricting the driver's action are currently under development. In this review, examples of heartbeat-monitoring systems are discussed. In particular, methods for measuring the heartbeat through sensing devices of a wearable-type, such as wristwatch-type, ring-type, and shirt-type devices, as well as through devices of a nonwearable type, such as steering-type, seat-type, and other types of devices, are discussed. The emergence of wearable devices such as the Apple Watch is considered a turning point in the application of driver-monitoring systems. The problems associated with current smartwatch- and smartphone-based systems are discussed, as are the barriers to their practical use in vehicles. We conclude that, for the time being, detection methods using in-vehicle devices and in-vehicle cameras are expected to remain dominant, while devices that can detect health conditions and abnormalities simply by driving as usual are expected to emerge as future applications.

Aberrant expression of DUSP4 is a specific phenomenon in betel quid-related oral cancer.

Oral cancer due to betel quid chewing habit is very common in South Asian countries. We attempted to detect the presence of a novel gene in epithelial cells stimulated with arecoline, a main component of betel quid. Human gingival epithelial progenitors were cultured and treated with a 3-day alternating regimen with/without 50 µg/ml arecoline for 1 month. DNA microarray and methylation arrays were analyzed to identify the candidate genes. Immunohistochemical staining was performed in the tissue samples. Genome-wide analyses, quantitative reverse transcription PCR and quantitative methylation-specific PCR revealed DUSP4 as the most significant and promising gene. The methylation levels of DUSP4 were significantly higher in the betel quid-related oral squamous cell carcinoma (OSCC) than those in the non-related OSCC and controls (Mann-Whitney U test, p < 0.05). The number of DUSP4 immunopositive cells in betel quid-related OSCC was significantly higher than those from the non-chewing patients and the controls (p < 0.05). Hypermethylation of DUSP4 may be considered as a specific event in betel quid-related oral cancer.

Recent Research and Developing Trends of Wearable Sensors for Detecting Blood Pressure.

Blood pressure is considered an index to measure a person's health or state. The IEEE published a standard for wearable cuffless blood pressure measuring devices, which was certified as IEEE1708 on 26 August 2014, and, according to this standard, the development of wearable devices based on blood pressure is expected in the future. Considering this, blood pressure should be detectable all the time and everywhere, and this can help improve health consciousness. In this review, we introduce the recent development of wearable blood pressure measuring devices and research trends, and present the future prospects for blood pressure measuring devices.

Immunohistochemical evaluation of Klotho and DNA methyltransferase 3a in oral squamous cell carcinomas.

Carcinoma follows a course of multiple changes that are affected by several important factors, with epigenetic silencing of the promoter gene being one of them. A series of studies have suggested that epigenetic changes in the anti-aging gene Klotho may be one of the emerging areas of concern in the study of carcinogenesis. We hypothesized that epigenetic silencing of Klotho due to hypermethylation of DNMT3a may be one of the causes of carcinoma in the oral and maxillofacial region. In this study, we analyzed the immunohistochemical expressions of Klotho and DNMT3a in tissues obtained from oral dysplasia and oral squamous cell carcinoma. Our results showed increased immune expression of DNMT3a, and decreased expression of Klotho in cells of the cancer tissues when compared with those in the dysplasia and healthy control samples. Chi-square tests complemented by adjusted residual analysis revealed significantly higher number of Klotho-positive and DNMT3a-negative cases in healthy controls, Klotho-negative and DNMT3a-negative cases in ODL, and Klotho-negative and DNMT3a-positive cases in OSCC when compared with the other types among the three groups (X 2 = 46.66, p < 0.001). Thus, downregulation of Klotho may be associated with the overexpression of DNMT3a in cancer tissues.

Video tracking analysis of behavioral patterns during estrus in goats.

Here, we report a new method for measuring behavioral patterns during estrus in goats based on video tracking analysis. Data were collected from cycling goats, which were in estrus (n = 8) or not in estrus (n = 8). An observation pen (2.5 m × 2.5 m) was set up in the corner of the female paddock with one side adjacent to a male paddock. The positions and movements of goats were tracked every 0.5 sec for 10 min by using a video tracking software, and the trajectory data were used for the analysis. There were no significant differences in the durations of standing and walking or the total length of movement. However, the number of approaches to a male and the duration of staying near the male were higher in goats in estrus than in goats not in estrus. The proposed evaluation method may be suitable for detailed monitoring of behavioral changes during estrus in goats.

Dioscorea japonica extract down-regulates prostaglandin E2 synthetic pathway and induces apoptosis in lung cancer cells.

Prostaglandin E2 plays a role in an array of pathophysiological responses, including inflammation, carcinogenesis and so on. Prostaglandin E2 is synthesized from arachidonic acid by the enzymes cyclooxygenase and prostaglandin E synthase. In some pathological conditions, the isozymes cyclooxygenase-2 and microsomal prostaglandin E synthase-1 are transiently induced, leading to prostaglandin E2 overproduction. The present study showed that Dioscorea japonica extract suppresses mRNA expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 in human non-small-cell lung carcinoma A549 cells in a dose-dependent manner. The suppressive effects of Dioscorea japonica extract on the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 were confirmed by Western blotting, cyclooxygenase activity and prostaglandin E2 production. Dioscorea japonica extract induced the translocation of nuclear factor-κB from the nucleus to the cytosol and inhibited the activity of the cyclooxygenase-2 promoter. Furthermore Dioscorea japonica extract suppressed the expression of the anti-apoptotic factor B-cell chronic lymphocytic leukemia/lymphoma 2 and enhanced apoptotic terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive intensity in A549 cells. These results suggest that Dioscorea japonica extract suppresses the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, with the regulation of the transcriptional activity of cyclooxygenase-2, and induces apoptosis in cancer cells. Thus, Dioscorea japonica may contribute to the prevention of prostaglandin E2-mediated pathophysiological responses such as carcinogenesis and inflammation.

Oral infection with Porphyromonas gingivalis augmented gingival epithelial barrier molecules alteration with aging.

OBJECTIVE: Disruption of the gingival epithelial barrier is often mediated by aging or the pathogen Porphyromonas gingivalis. This study examined the combined effects of aging and P. gingivalis exposure on gingival epithelial barrier molecules. METHODS: In vitro experiments involved treating young- and senescence-induced primary human gingival epithelial progenitor cells (HGEPp) with P. gingivalis lipopolysaccharide (LPS). Transepithelial electrical resistance (TER) and paracellular permeability were measured. In vivo, male C57BL/6J mice aged 10 (young) and 80 (old) weeks were divided into four groups: young, old, young with P. gingivalis (Pg-Young) inoculation, and old with P. gingivalis (Pg-Old) inoculation. P. gingivalis was inoculated orally thrice a week for 5 weeks. The mice were sacrificed 30 days after the last inoculation, and samples were collected for further procedures. The junctional molecules (Claudin-1, Claudin-2, E-cadherin, and Connexin) were analyzed for mRNA expression using qRT-PCR and protein production using western blotting and immunohistochemistry. The alveolar bone loss and inflammatory cytokine levels in gingival tissues were also assessed. RESULTS: LPS-treated senescent cells exhibited a pronounced reduction in TER, increased permeability to albumin protein, significant upregulation of Claudin-1 and Claudin-2, and significant downregulation of E-cadherin and Connexin. Furthermore, the Pg-Old group showed identical results with aging in addition to an increase in alveolar bone loss, significantly higher than that in the other groups. CONCLUSION: In conclusion, the host susceptibility to periodontal pathogens increases with age through changes in the gingival epithelial barrier molecules.

VAMP4 is required to maintain the ribbon structure of the Golgi apparatus.

The Golgi apparatus forms a twisted ribbon-like network in the juxtanuclear region of vertebrate cells. Vesicle-associated membrane protein 4 (VAMP4), a v-SNARE protein expressed exclusively in the vertebrate trans-Golgi network (TGN), plays a role in retrograde trafficking from the early endosome to the TGN, although its precise function within the Golgi apparatus remains unclear. To determine whether VAMP4 plays a functional role in maintaining the structure of the Golgi apparatus, we depleted VAMP4 gene expression using RNA interference technology. Depletion of VAMP4 from HeLa cells led to fragmentation of the Golgi ribbon. These fragments were not uniformly distributed throughout the cytoplasm, but remained in the juxtanuclear area. Electron microscopy and immunohistochemistry showed that in the absence of VAMP4, the length of the Golgi stack was shortened, but Golgi stacking was normal. Anterograde trafficking was not impaired in VAMP4-depleted cells, which contained intact microtubule arrays. Depletion of the cognate SNARE partners of VAMP4, syntaxin 6, syntaxin 16, and Vti1a also disrupted the Golgi ribbon structure. Our findings suggested that the maintenance of Golgi ribbon structure requires normal retrograde trafficking from the early endosome to the TGN, which is likely to be mediated by the formation of VAMP4-containing SNARE complexes.

SNARE proteins are not excessive for the formation of post-Golgi SNARE complexes in HeLa cells.

To evaluate the role of SNARE proteins in the constitutive exocytosis, we knocked down syntaxin 3, 4, 5, 6, 7, and VAMP3, 5, 7, 8 with their siRNAs, and determined the cell-to-medium ratio of CLuc, a secreted luciferase of Cypridina noctiluca. Although the protein level of SNAREs in HeLa cells was markedly reduced by the siRNA treatment, the cell/medium ratio was scarcely increased by any siRNAs except for syntaxin 5. The accumulation of GFP-tagged human growth hormone was also visible only by the knockdown of syntaxin 5. To examine whether the residual amount of SNAREs are sufficient for maintaining normal constitutive exocytosis, we estimated the effect of siRNAs on the level of post-Golgi SNARE complexes containing syntaxin 4, SNAP23, and VAMP3 or VAMP8. The amount of SNARE complexes was robustly decreased by siRNAs and was well correlated with the residual amount of SNAREs in the lysates, suggesting that SNAREs are unnecessarily excessive for the formation of post-Golgi SNARE complexes in HeLa cells.

The profiling and analysis of gene expression in human periodontal ligament tissue and fibroblasts.

OBJECTIVES: The periodontal ligament (PDL) is an important component of periodontium to support dental structure in the alveolar socket. Regeneration of PDL tissue is an effective treatment option for periodontal disease and the profiling of genes involved in this process will be informative. Therefore, our study aims to accurately delineate the profiling of gene expression for PDL tissue regeneration. MATERIALS AND METHODS: We isolated PDL tissues and PDL fibroblasts (PDLFs) from premolar teeth, which were extracted from healthy periodontal status patients undergoing orthodontic treatment. Messenger RNA (mRNA) expression in PDL tissue and PDLFs were analyzed using Cap analysis gene expression, which is a second-generation sequencing technique to create profiling. We also determined the protein expression using Western blot. RESULTS: Collagens (type I, III, and VI), noncollagenous proteins (periostin and osteonectin), and proteoglycans (asporin, lumican, decorin, and osteomodulin) were highly expressed in PDL tissue. Integrin, ß1 was also expressed in PDL tissue. On comparison of gene expression between PDL tissue and PDLFs, four PDL marker genes, osteopontin, asporin, periostin, and osteonectin, were decreased in PDLFs. The genes for gene regulation were also highly expressed. CONCLUSIONS: Our study demonstrated the overall profiling of mRNA expression in PDL tissue and analyzed the important genes which may be useful for providing specific information for the reconstruction of PDL. We also identified the difference in gene expression between PDL tissue and PDLFs which might provide insights towards PDL regeneration.

A polymerase chain reaction-based method for constructing a linear vector with site-specific DNA methylation.

DNA methylation is an important epigenetic modification that leads to a wide variety of biological functions, including transcription, growth and development, and diseases associated with altered gene expression such as cancers. However, tools to insert site-specific methylation into DNA for analyzing epigenetic functions are limited. Here we describe a novel polymerase chain reaction (PCR)-based approach to provide site-specific DNA methylation at any site, including CpG or CpNpG islands. This method is simple and versatile, and it consists of four steps to construct the DNA methylation vector: (I) design and synthesis of methylated primers, (II) PCR amplification, (III) isolation of single-stranded DNA, and (IV) annealing and ligation of isolated single-stranded DNAs. First we produced and validated a linear green fluorescence protein (GFP) vector by this method. Next we applied this method to introduce methyl groups into the promoter of the cyclooxygenase-2 (COX-2) gene and found that site-specific DNA methylation at the CRE element significantly altered COX-2 gene expression. These results demonstrate that this PCR-based approach is useful for the analysis of biological functions that depend on DNA methylation.

Nicotine induces upregulated expression of beta defensin-2 via the p38MAPK pathway in the HaCaT human keratinocyte cell line.

Human beta-defensins (hBDs), a group of antimicrobial peptides, are involved in the protective barrier of the oral epithelium. Nicotine induces periodontal and oral epithelial diseases. The purpose of the present study was to investigate the effect of nicotine on the expression pattern of hBD-2 in keratinocytes. HaCaT cells, a keratinocyte cell line, were incubated with 8, 15, 30, or 80 µM nicotine for 24 h. Expression of hBD-2 was observed by RT-PCR, qRTPCR, and ELISA assay. The cells were treated with inhibitors for intracellular pathways (p38MAP kinase, NF-κB, JNK, MAPK-ERK) and with nicotinic acetylcholine receptor (nAChR) inhibitors in a series of experiments. Data were analyzed using Student's t test. qRT-PCR revealed that the expression level of hBD-2 mRNA was significantly higher at 30 and 80 µM nicotine than the control without nicotine (P < 0.05). The 80 µM cell extraction contained significantly higher hBD-2 peptide levels than the control (P < 0.05). The p38MAP kinase inhibitor abolished the upregulated expression of hBD-2 by nicotine. Both nAChR inhibitors also abolished the upregulation of hBD-2 by nicotine. The present study demonstrated that nicotine causes upregulated expression of hBD-2 via the p38MAP kinase pathway in keratinocytes.

Possibility of Autonomous Estimation of Shiba Goat's Estrus and Non-Estrus Behavior by Machine Learning Methods.

Mammalian behavior is typically monitored by observation. However, direct observation requires a substantial amount of effort and time, if the number of mammals to be observed is sufficiently large or if the observation is conducted for a prolonged period. In this study, machine learning methods as hidden Markov models (HMMs), random forests, support vector machines (SVMs), and neural networks, were applied to detect and estimate whether a goat is in estrus based on the goat's behavior; thus, the adequacy of the method was verified. Goat's tracking data was obtained using a video tracking system and used to estimate whether they, which are in "estrus" or "non-estrus", were in either states: "approaching the male", or "standing near the male". Totally, the PC of random forest seems to be the highest. However, The percentage concordance (PC) value besides the goats whose data were used for training data sets is relatively low. It is suggested that random forest tend to over-fit to training data. Besides random forest, the PC of HMMs and SVMs is high. However, considering the calculation time and HMM's advantage in that it is a time series model, HMM is better method. The PC of neural network is totally low, however, if the more goat's data were acquired, neural network would be an adequate method for estimation.

Role of VAMP8/endobrevin in constitutive exocytotic pathway in HeLa cells.

To evaluate the role of VAMP8/endobrevin in constitutive exocytosis, we have examined the exocytotic pathways of VAMP8 and human growth hormone, both GFP-tagged, by total internal reflection fluorescence microscopy (TIRF-M). Human GH-GFP and VAMP8-GFP were similarly expressed in small round vesicles and elongated tubular vesicles in HeLa cells, and were mostly exocytosed at the peripheral area of the cells. VAMP8-GFP gave 2 types of exocytotic images: a burst type and a non-burst type. The burst type showed a sharp transient increase in the peak fluorescence intensity and a much slower decrease in the average intensity in the active windows, where exocytosis took place, as observed in the "full-fusion" type of exocytosis. The non-burst type showed a relatively long-lasting fusion to the plasma membrane with little transfer of VAMP8-GFP to the plasma membrane, as observed in the so-called "kiss-and-run" type of exocytosis. Endogenous VAMP8 and hGH-GFP were colocalized on the same vesicles at least in part. However, the constitutive exocytosis of hGH-GFP and CLuc, a secreted luciferase from Cypridina noctiluca, was normal, even when siRNAs for VAMP8 and VAMP3 robustly decreased their proteins. These results suggest that VAMP8 is not essential for constitutive exocytosis, although it can be involved in the exocytosis.

High frequency of hypermethylation of p14, p15 and p16 in oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

BACKGROUND: Oral squamous cell carcinoma and the most common oral pre-malignancies appear to be related to the habit of betel-quid chewing in Sri Lanka. Although hypermethylation of the tumour suppressor genes in oral cancer have been well documented, little information has been available concerning hypermethylation in oral pre-cancerous lesions. In the present study, we investigated the hypermethylation of p14, p15 and p16 in pre-cancerous lesions including epithelial dysplasia and submucous fibrosis. METHODS: All samples were obtained from patients with a betel-quid chewing habit in Sri Lanka. Sixty-four patients were clinically diagnosed with leukoplakia, and histopathologically diagnosed with mild or severe dysplasia. Ten patients were diagnosed with submucous fibrosis without epithelial dysplasia. CpG island hypermethylation was assessed by a methylation-specific PCR method. Immunohistochemical staining was performed using anti-p53 antibodies. RESULTS: A high frequency of hypermethylation of p14, p15 and p16 was detected in the pre-cancerous lesions, although no hypermethylation was found in normal epithelium. The frequency of hypermethylation was higher than that of positive staining for p53 mutation except in the case of p16 in mild dysplasia. No significant correlation was observed between p53-positive reactions and hypermethylation in any lesions. The hypermethylation was highly detectable even in p53-negative lesions, suggesting that hypermethylation of p14, p15 and p16 occur regardless of whether the lesions have p53 mutations or not. CONCLUSIONS: The present study indicates that hypermethylation may be involved in the pathogenesis of oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

Profiling of differentially expressed genes in porcine epithelial cells derived from periodontal ligament and gingiva by DNA microarray.

OBJECTIVE: It is unknown which genes are differentially expressed in cultured epithelial cells derived from the epithelial rests of Malassez (ERM) in periodontal ligament and oral gingival epithelium (OGE). This study analysed the different gene expression of OGE and ERM cells using a DNA microarray technique. DESIGN: Epithelial cells from ERM and OGE were isolated from porcine periodontal ligament and oral gingival epithelium. Each RNA sample extracted from the cells was reverse transcribed into cDNA and labelled with either cytidine 5-dUTP (Cy5) or cytidine 3-dUTP (Cy3). These labelled cDNA probes were then mixed and simultaneously hybridised to the Pig 13K microarray plate bearing 13,295 different genes (Operon, AL). Cellular enzyme-linked immunosorbent assay (CELISA) was performed to confirm the expression at protein level. RESULTS: There were nine genes common to the triplicate microarrays in ERM cells and one in OGE cells. Four of the nine genes including tissue factor (TF), FAT cadherin (FAT) and two unknown genes were expressed at levels more than threefold higher in ERM cells than in OGE cells. The protein levels of both TF and FAT in ERM cells were significantly higher than those in OGE. CONCLUSION: TF and FAT may act as markers to distinguish ERM cells from OGE cells in vitro.

SNAP-23 is not essential for constitutive exocytosis in HeLa cells.

We applied the small interfering RNA (siRNA) technique and over-expression of a dominant-negative mutant to evaluate the role of SNAP-23, a non-neuronal isoform of SNAP-25, in constitutive exocytosis from HeLa cells. Although the protein level of SNAP-23 was reduced to less than 10% of the control value by siRNA directed against SNAP-23, exocytosis of SEAP (secreted alkaline phosphatase) was normal. Double knockdown of SNAP-23 and syntaxin-4 also failed to inhibit the secretion. Furthermore, over-expression of deltaC8-SNAP-23, a dominant-negative mutant of SNAP-23, did not abrogate SEAP secretion. These results suggest that SNAP-23 is not essential for constitutive exocytosis of SEAP.