N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that are widely expressed in the central nervous system and which play key roles in excitatory synaptic transmission. There is, however, no data on the involvement of NMDAR subunits in the apoptotic and neurotoxic effects of TCS. Our experiments are the first to show that TCS used at environmentally relevant concentrations evoked NMDA-dependent effects in neocortical neurons in primary cultures, as MK-801, an uncompetitive NMDA receptor antagonist, reduced the levels of TCS-induced ROS production as well as caspase-3 activity and LDH release. TCS caused a decrease in protein expression of all the studied NMDA receptor subunits  that were measured at 3, 6 and 24 h post-treatment. However, at 48 h of the experiment, the level of the GluN1 subunit returned to the control level, and the levels of the other subunits showed a tendency to increase. In TCS-treated neocortical cells, protein profiles of NMDAR subunits measured up to 24 h were similar to mRNA expression of GluN1 and GluN2A, but not to GluN2B mRNA. In this study, cells transiently transfected with GluN1, GluN2A or GluN2B siRNA exhibited reduced levels of LDH release, which suggests the involvement of all of the studied NMDAR subunits in the neurotoxic action of TCS. According to our data, GluN1 and GluN2A were mainly responsible for neuronal cell death as evidenced by neutral red uptake, whereas GluN2A was involved in TCS-induced caspase-3-dependent apoptosis. We suggest that TCS-evoked apoptosis and neurotoxicity could be related to transient degradation of NMDAR subunits in mouse neurons. Furthermore, recycling of NMDAR subunits in response to TCS is possible. Because transfections with specific siRNA did not completely abolish the effects of TCS as compared to cells transfected with negative siRNA in this study, other NMDAR-independent mechanisms of TCS action are also possible.Triclosan (TCS) is an antimicrobial agent that is used extensively in personal care and in sanitising products. A number of studies have shown the presence of TCS in different human tissues such as blood, adipose tissue, the liver, brain as well as in breast milk and urine. The online version of this article (10.1007/s12035-018-1083-z) contains supplementary material, which is available to authorized users. Triclosan phenol; CAS number: 3380-34-5) (TCS) is an antimicrobial agent that is commonly used worldwide. Due to both its anti-bacterial and anti-mould properties, it is added to a wide range of personal care and sanitising products as well as to household items or medical devices –5. The mN-Methyl-D-aspartate receptors (NMDARs) mediate excitatory neuronal transmission. However, despite their physiological functions, a large amount of evidence indicates their involvement in excitotoxicity [toxicity , 18. NMDtoxicity . Functiotoxicity , 20. Di-toxicity , 22.Reactive oxygen species (ROS) have a wide spectrum of functions in neuronal cells, where they can be generated as by-products of cellular metabolism, primarily in the mitochondria . ROS andHaliotis tuberculata) hemocytes, zebra mussel (Dreissena polymorpha) hemocytes, terrestrial snail (Achatina fulica), Daphnia magna or earthworms (Eisenia fetida) [To date, it has been proved that TCS induces the activity of enzymes involved in ROS metabolism in cells from different organisms such as green ormer ( fetida) –31. Howe fetida) , 16, 32.The aim of this study was to determine the involvement of NMDAR subunits in triclosan-evoked apoptosis and neurotoxicity in mouse neocortical neurons in primary cultures. Molecular analyses including mRNA and protein expression measurements as well as siRNA silencing were applied to support biochemical data related to ROS, neutral red uptake and caspase-3 and LDH activities.Actb (Mm00607939_s1), GluN1 (Mm00433800_m1), GluN2A (Mm00433802_m1) and GluN2B (Mm00433820_m1) were purchased from Life Technologies . Trypsin, charcoal/dextran-treated fetal bovine serum (FBS), L-glutamate, penicillin, streptomycin, N-methyl-D-aspartate acid (NMDA), staurosporine, triclosan (Irgasan), neutral red uptake assay, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), dizocilpine ((+)-MK801 maleate) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich . The lactate dehydrogenase-based cytotoxicity detection kit was purchased from Roche Applied Science . INTERFERin siRNA transfection reagent was purchased from Polyplus-transfection . NMDAζ1 siRNA (GluN1) (sc-36082), NMDAε1 siRNA (GluN2A) , NMDAε2 siRNA (GluN2B) (sc-36086), anti-NMDAζ1 antibody (sc-1467), anti-NMDAε1 antibody (sc-1468), anti-NMDAε2 antibody (sc-1469) and anti-GAPDH antibody (sc-25778) were purchased from Santa Cruz Biotechnology, Inc. . Phosphate-buffered saline (PBS) was purchased from BIOMED . Caspase-3 substrate was purchased from Calbiochem . TCS and other reagents were dissolved in DMSO. The final DMSO concentration in the culture medium was always equal to 0.01%.Neurobasal medium without phenol red, B27-AO supplement and TaqMan probes corresponding to specific genes encoding 2 and killed by cervical dislocation. The animal care protocols were in accordance with official governmental guidelines, and all efforts were made to minimise the number of animals used and their suffering. All procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by a Bioethics Commission (no. 46/2014) in compliance with Polish law. The brains were removed from the foetuses and the cortical tissues were dissected. The dissected tissue was minced into small pieces and then gently digested with trypsin. Then, the cells were centrifuged and the pellet was suspended in phenol red-free Neurobasal medium supplemented with 5% charcoal/dextran-treated FBS. The cells were plated onto poly-L-ornithine-coated (0.01 mg/mL) multi-well plates. After 2 days, the culture medium was exchanged with Neurobasal medium supplemented with B27-AO (2 μL/mL), glutamine (2 mM), 10 U/mL penicillin and 0.01 mg/mL streptomycin, without FBS which is recommended for primary neuronal cultures [5 cells/cm2 for the experiments. The cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and were cultivated for 7 days in vitro prior to the experiment. Then, the culture medium was changed prior to treating the cultures with the compound selected for this study.The experiments were performed on primary cultures of mouse neocortical neurons. These cultures were prepared from the foetuses of pregnant female Swiss mice as previously described in Brewer  and modicultures , 34. TheGluN1, GluN2A and GluN2B siRNA was used to inhibit expression of NMDA receptor subunits in mouse neocortical neurons by using a modification of a previously described method . The siRDue to the different aspects measured by cell viability assays, authors have chosen LDH release and neutral red uptake assays. The cytotoxicity detection kit is a colorimetric assay for the quantification of cell death and cell lysis based on the release of lactate dehydrogenase (LDH) from the cytosol of damaged cells into the surrounding medium . An incrCaspase-3 activity was used as a marker of cell apoptosis and was assessed according to Nicholson et al. . For mea2O) for 10 min at room temperature on a rotating platform. Absorbance was measured at a wavelength of 540 nm using a FilterMax F5 Multi-Mode microplate reader .The number of viable cells in experimental conditions was evaluated using the neutral red uptake test. This method is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. The neutral red uptake cytotoxicity assay is commonly used to study the viability of in vitro cultured primary cells as well as cell lines of diverse origin . After 22DCFDA) was used to detect intracellular reactive oxygen species (ROS). After diffusing into the cell, H2DCFDA is de-acetylated by cellular esterases into a non-fluorescent compound that is subsequently oxidised by ROS into 2′,7′-dichlorofluorescein (DCF) [2DCFDA in order to determine the ability of TCS to induce ROS production in neocortical neurons. We selected 3 and 6 h to study these processes as ROS production caused by NMDAR activation occurs in a short time period. The cells were plated on black-sided, clear-bottomed 96-well plates and exposed to vehicle (control group), 10 μM TCS and L-glutamate for an appropriate time period with or without MK-801 for the ROS measurement. The cells were incubated in H2DCFDA in serum-free and phenol red-free Neurobasal medium for 45 min prior to TCS treatment. Before measurement, the culture medium was replaced with fresh Neurobasal medium (FBS free) to remove extracellular residual DCF and H2DCFDA to reduce the fluorescence background. Fluorescence was measured after 3 and 6 h of incubating the cells with TCS and tool compounds (5% CO2 at 37 °C). DCF fluorescence was detected using a microplate reader (FilterMax F5) at maximum excitation and emission spectra of 485 and 535 nm, respectively. Cells treated with 0.3% hydrogen superoxide were used as a positive control (results not shown). The interaction between TCS and H2DCFDA was tested under cell-free conditions prior to the experiments (results not shown) based on concerns that were raised about the H2DCFDA assay as previously described by Szychowski et al. [The fluorogenic dye 2′,7′-dichlorodihydrofluorescein diacetate (Hin (DCF) . We appli et al. , 41.V/Vis; Thermo Fisher NanoDrop, USA). Two-step real-time RT-PCR was conducted with both the reverse transcription (RT) reaction and the quantitative polymerase chain reaction (qPCR) run using the CFX96 Real Time System . The RT reaction was performed at a final volume of 20 μL with 300 ng of RNA (as a cDNA template) using the cDNA reverse transcription kit according to the manufacturer’s protocol. Products from the RT reaction were amplified using the TaqMan Gene Expression Master Mix  kit with TaqMan probes as primers for specific genes encoding Actb, GluN1, GluN2A and GluN2B. Amplification was conducted in a total volume of 20 μL containing the 1x TaqMan Gene Expression Master Mix and 1 μL of the RT product, which was used as the PCR template. Standard qPCR procedures were performed as follows: 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The threshold value (Ct) for each sample was set during the exponential phase, and the delta Δ Ct method was used for data analysis. To study the gene expression levels, five candidate reference genes  were selected and validated. To evaluate the reference gene expression, RefFinder web-based comprehensive tool has been used [Actb as the most stable reference genes according to 3 and 6 h exposure to 10 μM TCS , 0.5% Na-deoxycholate, 0.5% Nonidet NP-40 and 0.5% SDS. Then, the lysates were sonicated and clarified by centrifugation at 15,000×d method  with bovn = 8) and measured in triplicate; thus, the total number of replicates was 24. The average of the triplicate samples was used for the statistical analyses. Statistical analysis was performed on the original results. Considering the different data from the measurement of fluorescence or absorbance, the results were presented as a percentage of the controls. The data were analysed via one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison procedure in STATISTICA 10 software .The data were presented as means ± SD of three independent experiments. Each treatment was repeated eight times . Co-treatment with 10 μM MK-801 and 10 μM TCS decreased TCS-induced ROS production as compared to the control (decrease by 12.52%). L-Glutamate used as a positive control increased ROS production by 87.58% in the neurons as compared to the controls. The presence of MK-801 decreased the production of ROS induced by L-glutamate, ROS production back to control levels . Cell co-treatment with 10 μM MK-801 and 10 μM TCS decreased TCS-induced LDH release to the control level Fig. a.Fig. 2EFollowing 24 h of exposure to 10 μM TCS, TCS increased LDH release in the neurons as compared to the controls (increases by 81.12%). Cell co-treatment with MK-801 and 10 μM TCS decreased TCS-induced LDH release by 23.03% . In cells co-treated with 10 μM MK-801 and 10 μM TCS, we observed no decreased TCS-induced caspase-3 activity . In cells co-treated with MK-801 and 10 μM TCS, we observed a decrease in TCS-induced caspase-3 activity by 74.73%   Fig. a.Fig. 4EGluN1 mRNA as compared to the vehicle control (decrease of 25.39%). However, in the same time period, 10 μM TCS increase in the expression GluN2B mRNA compared to the vehicle control (increase of 35.74%) . GluN2B protein expression started to decrease after 1 h (by 14.06% as compared to the control) and continued to decrease at 3, 6, 24 and 48 h  Fig. .Fig. 5ReFollowing 24 h of exposure to 10 μM concentrations of TCS, neurons transfected with negative siRNA showed a decrease in neutral red uptake as compared to the vehicle control (decrease by 36.72%). In neurons transfected with GluN2B siRNA, a similar decrease was observed in neutral red uptake as compared to the vehicle control (decrease by 51.75%). In neurons transfected with GluN1 or GluN2A siRNA, no changes were observed  and Western blot analyses were conducted to confirm involvement of the NMDA receptor in the apoptotic and neurotoxic action of TCS. Our study is the first to show that TCS caused a decrease in protein expression of all of the studied NMDA receptor subunits  that were measured at 3, 6 and 24 h post-treatment. However, at 48 h of the experiment, the level of the GluN1 subunit returned to the control level, and the levels of the other subunits, GluN2A and GluN2B, showed a tendency to increase. In TCS-treated neocortical cells, the protein profiles of NMDAR subunits measured up to 24 h were similar to the mRNA expression of GluN1 and GluN2A, but not GluN2B mRNA. We suggest that the differences between mRNA and the protein profiles of the NMDA subunits were due to subunit-specific regulation in response to TCS.GluN2B subunit in the brain tissue of rats that were prenatally exposed to TCDD [There has been no other study on TCS-evoked changes in mRNA and protein expression of NMDA receptor subunits in order to be able to compare them to our results. In the environment, TCS is converted to dioxins such as 2,8-DCDD, 2,3,7-TCDD, 1,2,8-TriCDD and 1,2,3,8-TCDD , 55. Bec to TCDD . As was  to TCDD . Enhance to TCDD .Specific siRNA-mediated silencing was applied to verify the involvement of individual NMDAR subunits in the apoptotic and neurotoxic action of TCS. According to our data, cells transiently transfected with GluN1, GluN2A or GluN2B siRNA exhibited reduced levels of LDH release, which suggests the involvement of all of the studied NMDAR subunits in the neurotoxic action of TCS. It has been documented that NMDAR subunits have various physiological functions in neuronal cells. GluN2A-containing synaptically located NMDARs have been postulated to activate pro-survival signalling pathways, while GluN2B-containing primarily extrasynaptic NMDARs trigger neuronal death signalling , 60. BecWe suggest that the TCS-evoked apoptosis and neurotoxicity that is accompanied by a decrease in protein expression of GluN1, GluN2A or GluN2B as observed from 3 to 24 h of the experiment could be related to transient degradation of NMDAR subunits in mouse neurons. Furthermore, recycling of NMDAR subunits in response to TCS is possible since the expression of GluN1 was normalised and the expression of GluN2A and GluN2B showed a tendency to increase at 48 h of exposure.Our experiments are the first to show that TCS used at environmentally relevant concentrations evoked NMDA-dependent apoptosis and neurotoxicity. Triclosan-evoked neurotoxicity involved all studied NMDAR subunits, with the particular role of GluN2A in caspase-3-dependent apoptosis. TCS also disrupted mRNA and protein expression of GluN1, GluN2A and GluN2B in mouse neurons in primary cultures. We postulate that TCS-induced apoptosis and neurotoxicity is related to transient degradation of NMDAR subunits; however, other NMDAR-independent mechanisms of TCS action are possible since the silencing of specific NMDAR subunits did not completely abolish the effects of TCS.Supplementary dataEvaluation of reference genes using RefFinder web-based tool. Computational algorithms  were used to evaluate and identify suitable reference genes. In our study, NormFinder, BestKeeper and delta Ct recommended β-actin as the most stable reference genes according to 3 h (panel A) and 6 h (panel B) exposure to 10 μM TCS. (XLSX 36 kb)


Prevention of deafness and hearing loss calls on hearing health-care stakeholders to develop and implement strategies for improved service provision, especially in low- and middle-income countries.Hearing loss affects close to 1.3 billion people and is a growing global health concern as the fourth leading contributor to years lived with disability.,While half of all hearing losses can be prevented, most of the remainder will benefit from significantly reduced functional impairment made possible through early detection and treatment.Clinical hearing assessment apps typically determine hearing sensitivity across tones of different pitch.,,,Examples of community-based screening programmes for children and adults in low- and middle-income countries have used inexpensive, clinically certified smartphone test solutions with calibrated headphones.Many consumer apps claim to provide accurate tests on Android and iOS operating systems,Speech-based consumer apps typically assess users’ ability to recognize speech in noise. Unlike pure tone audiometry, these tests do not require calibration, allowing use across various devices and headphones.The World Health Organization recently adopted this approach for the hearWHO app (iOS and Android) released on World Hearing Day 2019. This app has a new version of the digits-in-noise test and uses antiphasic digit stimuli that make the app sensitive to a range of hearing losses including sensorineural, conductive and asymmetrical losses.,Advantages of mobile-based digital hearing test solutions , include accessibility, affordability, advanced sensors and software-based quality control, alongside integrated cloud-based data management.,,A growing concern around health apps is the unstandardized approach to reporting and issues surrounding data security.Rapid global advancement in connectivity and technology is changing the landscape of affordable health-care access.


Hearing loss (HL) is the third most common chronic physical condition in the United States. Obesity has become an increasingly important public health concern, as the prevalence in children, adolescents and adults has increased over the past few decades. The objectives of this study is to investigate whether obesity is associated with audiometric notches indicative of noise-induced hearing loss (NIHL), speech frequency hearing loss (SFHL), and high frequency hearing loss (HFHL) in adolescent participants of the National Health and Nutrition Examination Survey 2007–2010. The prevalence of overall audiometric notches in the adolescent population was 16.0% with higher prevalence in females than males. The prevalence of SFHL and HFHL were higher in males than females . Obese adolescents had a higher adjusted OR to have audiometric notches  and HFHL . Continued preventative efforts towards reducing obesity might also help to reduce the risk for HL and NIHL. The most common preventable cause of HL is the damage to the auditory system caused by excessive noise exposure3. Although the onset of this type of HL is usually slow, it progresses as long as the exposure continues. One of the first signs of noise-induced hearing loss (NIHL) is an audiometric indication of a noise-induced hearing threshold shift (NITS), usually defined as an “audiometric notch” at 3, 4, or 6 kHz. Henderson and colleagues4 reported increased prevalence of audiometric notches in adolescents: 15.9% in NHANES 1998–1994 to 16.8% in NHANES 2005–2006. Furthermore, damage to hearing at a young age can lead to exacerbated hearing loss later in life2. Data from the National Health and Nutrition Examination Survey (NHANES) suggest that the prevalence of HL among United States adolescents (12–19 years old) increased from 1994 to 2006 for both high frequency HL (HFHL) and low frequency HL (LFHL). HFHL increased from 11.1% to 12.9% and LFHL increased from 5.2% to 6.5%4.Hearing loss (HL) is the third most common chronic physical condition in the United States5.Obesity has become an increasingly important public health concern, as the prevalence in children, adolescents and adults has increased over the past few decades both within the United States and worldwide. Among U.S. adolescents aged 12 to 19 years, obesity prevalence increased between 1988–1994 and 2013–2014, from 10.5%  to 20.6% 6. Moreover, obesity has been associated with unilateral low frequency HL, but not unilateral high frequency HL, in adolescent participants to NHANES 2005–20067. There are no studies about potential risk factors for audiometric notches in adolescents. Therefore, the objective of this study is to investigate whether obesity is associated with audiometric notches indicative of NIHL and hearing loss in adolescents in a nationally representative survey.Health problems during childhood and adulthood associated with obesity include an increased risk of diabetes, hypertension, nonalcoholic fatty liver disease, and depression8. Beginning in 1999, the survey has been conducted continuously and released in 2-year cycles. For our study, we merged the publicly available files for NHANES cycles 2007–2008 and 2009–2010 using the NCHS recommendations8. The survey employed a multistage stratified probability sample based on selected counties, blocks, households, and persons within households. During NHANES 2007–2010, 2,520 participants aged 12–19 years had complete audiometric data . We excluded participants with: 1) only a partial audio exam ; 2) otoscopic screening exam of the ear canals and eardrum for excessive or impacted ear cerumen (wax), physical abnormalities, or collapsing external ear canals that was not normal (n = 475), or ear compliance ≤0.2 mL (N = 42), or pressure lower than −150 dekapascals (daPa) (n = 18), and; 3) tinnitus based on the positive answer to the question “In the past 12 months, have you been bothered by ringing, roaring or buzzing in your ears or head that lasted for five minutes or more?” (n = 119). We, also, excluded participants that had missed information in the co-variates used in the analyses (n = 250). Therefore, the total sample size used in our analyses was of 1469 adolescent participants.NHANES is a cross-sectional, nationally representative survey of the non-institutionalized civilian population of the United States conducted by the National Center for Health Statistics at the Centers for Disease Control and Prevention (CDC)Trained examiners obtained audiometric measurements in a specifically designed and equipped Mobile Exam Center (MEC) sound-treated room using a standardized protocol.Air conduction thresholds were measured for each ear at 0.5, 1, 2, 3, 4, 6, and 8 kHz. The 1-kHz frequency was tested twice in each ear to measure the reliability of the participant’s responses and the average test response at 1-kHz frequency was used in the analyses. Pure-tone audiograms were not accepted if the difference between the 1-kHz test-retest thresholds was 10 dB or greater. For further details about the measurement techniques, see the National Center for Health Statistics websitehttps://wwwn.cdc.gov/nchs/nhanes/continuousnhanes/manuals.aspx?BeginYear=2007; https://wwwn.cdc.gov/nchs/nhanes/continuousnhanes/manuals.aspx?BeginYear=2009].[et al.9 when:one or more of the thresholds (the softest sound a person can hear) at 3, 4, or 6 kHz exceeds the pure-tone average (PTA) of the 0.5 and 1 kHz thresholds by 15 dB or more, andthe 8 kHz threshold is at least 5 dB lower (better) than the maximum threshold in the 3, 4, or 6 kHz range.In this study, we define the presence of a high-frequency audiometric notch as reported by Carroll 11 and the average of the three audiometric frequencies at 3, 4, and 6 kHz to define an high frequency (HF) PTA11. We used a PTA of 15 dB HL or greater in either ear as a cutoff threshold for both the SF and HF PTA to define speech frequency hearing loss (SFHL) and high frequency hearing loss (HFHL), respectively11.Similar to other studies using NHANES, we used the average of four audiometric frequencies at 0.5, 1, 2, and 4 kHz to define the speech frequency (SF) PTAIn our analyses, we used sociodemographic and hearing-related variables that have or are suspected to have an effect on hearing thresholds. These variables used for our analysis were age, sex, race/ethnicity, poverty income ratio (PIR), body weight status , serum cotinine, self-reported ear infection, and “exposure outside of a job to steady loud noise or music for 5 or more hours a week.”We categorized race/ethnicity as non-Hispanic white, non-Hispanic black, Mexican American, and other (other Hispanic and other race). PIR is a measure of socioeconomic status and represents the calculated ratio of household income to the poverty threshold after accounting for inflation and family size.2). However, since the relation between BMI and body weight in children depends on age and sex, obesity was defined as a BMI at or above the 95th percentile of the CDC sex-specific BMI-for-age growth charts from 200012. Overweight was defined as a BMI between the 85th and 95th percentiles. Underweight was defined as BMI less than the 5th percentile. Normal weight was defined as BMI between the 5th percentile to less than the 85th percentile.Body mass index (BMI) is calculated by dividing the weight by height squared  smokers, defined as participants who reported use of tobacco in the past 5 days or serum cotinine level ≥10 ng/mL.Serum cotinine has been used as a biomarker of exposure to both environmental and/or active tobacco smoke. In addition to serum cotinine, NHANES questionnaire on “recent tobacco use” report information about the self-report use of tobacco in past 5 days. For youth 12–19 years of age, the questions on tobacco use were self-administered using the Audio Computer-Assisted Self-Interview system. Therefore, tobacco exposure was categorized in: 1) no smokers, consisting of participants that did not report use of tobacco in the past five days and had serum cotinine below the limit of detection (LOD = 0.015 ng/ml); 2) second hand smokers exposure (SHS), defined as person who did not use tobacco in the past 5 days and had serum cotinine at or higher than the LOD and below 10 ng/mlThe household interview gathered information about self-reported ear infection and “exposure outside of a job to steady loud noise or music for 5 or more hours a week.” Generally, persons 16 years of age and older and emancipated minors were interviewed directly, whereas a responsible adult provided information for participants under 16 years of age.The NHANES physical activity questionnaire reports information on time of sedentary activity: “How much time do you usually spend sitting on a typical day?” The participants were asked ‘about sitting at work, at home, getting to and from places, or with friends, including time spent sitting at a desk, traveling in a car or bus, reading, playing cards, watching television, or using a computer. Excluding the time spent sleeping, how much time do you usually spend sitting on a typical day?’ Since the variable was right skewed, it was naturally log-transformed.8. SAS 9.3  and SAS-Callable SUDAAN 10  was used for all statistical analyses. We used logistic regression to calculate adjusted odds ratios (ORs) and 95% CIs for the audiometric notch and the hearing loss outcomes. Preliminary analyses did not found any significant interaction term between the predictors (p > 0.10). Statistical tests for linear trends were conducted by modeling ordinal variable using integer values. Two models, utilizing the various covariates were run: (1) model 1 included age, sex, race/ethnicity, obesity, PIR, ear infections, loud noise exposure and smoking; (2) and, as sensitivity analyses, model 2 included the covariates in model 1 plus hours of sedentary activity.All analyses were performed using the Mobile Exam Center weight as recommended by NCHS, and to account for the complex sampling design and non-response of NHANESTable 2 p value = 0.06). The weighted prevalence of audiometric notches in obese adolescents was higher compared to normal weight adolescents (24.8% vs 14.7%).Table In multivariate logistic regression analyses Table , obese aThe prevalence of SFHL was 7.3% in males and 5.4% in females Table . The wei2 p value = 0.02). The weighted prevalence of HFHL in obese adolescents was statistically significant higher compared to normal weight adolescents (17.9% vs 5.4%), , Cruickshanks et al.16 followed up 1925 participants (mean age 60.7 years at baseline) with a normal hearing baseline. They found that smoking and obesity were predicted risk factors for hearing loss . In a cross-sectional study comprising 61,052 Korean adults 30 years and older , Kim et al.17 reported that severely obese (BMI ≥ 30 kg/m2) persons had higher OR of hearing loss. Obesity was slightly associated with HL. Üçler et al.18 reported an association between obesity and hearing threshold in women aged 18–40 years. The associations were statistically significant at high frequencies of 4, 6, and 8 kHz, but not at lower frequencies.In the present study conducted using data from the NHANES 2007–2010 surveys we found that obesity was associated with increase odd of having audiometric notches and high frequency hearing loss, but not with speech frequency HL. These findings are notable because of the potential impact that hearing impairment at high frequency can have on the social behavior of children and adolescents. Children with HL are more likely to experience academic difficulties, have behavioral problems, and demonstrate lower performance in oral language, compared with their peers with normal hearinget al.19 reported that higher BMI and larger waist circumference were associated with increased risk of self-reported hearing loss. Conversely, Shargorodsky et al.20, in a study conducted in 26,917 adult men participants in the Health Professionals Follow-up Study, did not find any association between obesity with increased risk of self-reported hearing loss, but past smoking was independently associated with increased risk of hearing loss.In a prospective study conducted among 68421 women participants in the Nurses’ Health Study II from 1989 to 2009, Curham et al.21 hypothesized that plasma adiponectin mediates the effect of obesity on hearing loss. Adiponectin is a protein hormone that modulates several metabolic processes, among them glucose regulation and fatty acid oxidation22. Serum levels of adiponectin decrease with obesity23.The biological plausibility of a role of obesity as risk factor for hearing impairment maybe through the perturbation of the adiponectin hormone. Hwang in vivo studies Tanigawa et al.24 found that compared to the wild-type (WT) mice, adiponectin-knockout (APN-KO) mice had exacerbated hearing impairment, particularly in the high frequency range, with reduced cochlear blood flow and capillary density of the stria vascularis. The hearing impairment was prevented in APN-KO mice that were supplemented with adiponectin. Compared to the control group, Hwang et al.25 found that the auditory brainstem response threshold was significantly higher at high frequencies in mice with diet-induced obesity (DIO). Although susceptibility to damage from noise exposure is highly variable in the general population26, the potential reduction of cochlear blood flow in obese adolescents might make them more susceptible to the harmful effects of noise.However, the best evidence supporting a role of obesity with the development of hearing impairment comes from 27; however, we adjusted the models for several important confounding factors. A major study limitation is the measurement of audiometric thresholds at a single point in time, since the identified audiometric notches may only represent temporary threshold shifts. Concerns have been raised about potential false-positive rates of high frequency audiogram notches, such as 6 and 8 kHz, which might be due to high subject variability and calibration error for TDH-type headphones28.Our study has several limitations. The cross-sectional nature of this study limits the inferences that can be made based on the results. The associations reported in this study could be biased by uncontrolled factors such as genetic predisposition, otosclerosis, hypertension, diabetes, ototoxic medication, and exposure to ototoxic substances29, early basic hearing screenings such as whisper or finger rub tests may provide early diagnosis and opportunities for noise prevention counselling and access to hearing aids30. Continued preventative efforts towards reducing obesity, should also have a positive impact among adolescents at risk for NIHL and hearing loss.Being obese was associated with NIHL and hearing loss. Health care professionals should be aware of the increased risk of damage to hearing among their patients with obesity. Since hearing impairment often progresses insidiously for years before being self-perceived or diagnosed


Some previous studies reported hearing ability can be reduced by impaired masticatory ability, but there has been little evidence reported of an association between hearing loss and unilateral mastication. Therefore, this study aimed to investigate the relationship between unilateral mastication (UM), estimated from individual functional tooth units (FTUs), and hearing loss in a representative sample of Korean adults.The analyzed data were obtained from 1,773 adults aged 40–89 years who participated in Korean national survey. Hearing loss was defined as a pure-tone average of >25 dB at frequencies of 0.5, 1, 2, and 4 kHz in either ear. In each subject, UM was calculated as the difference in the sums of the FTU scores, which is an index of posterior tooth occlusion, on the two sides of the oral cavity. The scores were used to classify the UM into low, moderate, and high. The adjusted odds ratios (aORs) and their 95% confidence intervals (CIs) were calculated in multivariable logistic regression analyses.When controlling for sociodemographic factors, the aOR for hearing loss was 3.12  for high UM relative to low UM. This association remained in a fully-adjusted model containing factors related to noise exposure .Adults with high UM as measured using FTUs showed a higher occurrence of hearing loss than those with low UM. Furthermore, many older adults accept that hearing loss is inevitable. However, the severity of hearing loss among those of the same age varies with the individual sensitivity, and the risk of hearing loss is associated with being male, smoking, and having lower education, military service, industrial employment, and noise exposure.2–4 Mild hearing loss can also lead to symptoms of depression and social isolation due to its adverse effects on verbal communication.5,6 In addition, since presbycusis is rarely reversible, it is necessary to identify the hearing status in adults in order to avoid progressive hearing loss.7The World Health Organization (WHO) has estimated that almost 15% of adults worldwide have some degree of hearing loss.8 tooth loss,9,10 and impaired masticatory ability.11–13 Initially, an association between hearing loss and tooth loss was reported.9 Later, clinical evidence has shown that hearing can be restored through the removal of unilateral chewing habits via prosthodontic treatment.11,13 With respect to this connection, one experimental study found that one side of the maximum occlusal pressure affects the auditory-evoked magnetic fields.11 However, there has been little epidemiologic evidence reported of an association between tooth loss and hearing loss,9 and to our knowledge, no epidemiologic evidence of an association between hearing loss and UM.An association between dentate status and hearing loss has been reported. Previous studies suggest that hearing ability can be reduced by temporomandibular disorder (TMD),14 Therefore, the individual UM level can be estimated based on the difference in functional tooth units (FTUs). FTUs have been used as a masticatory ability index to evaluate the oral condition or dietary intake.15–18 FTUs was developed from the concept of occluding pairs (OPs) and defined as pairs of opposing posterior teeth—premolars and molars. A lack of FTUs is considered to be a key factor contributing to the loss of masticatory ability.16 The relationship between OPs and hearing loss had been examined previously,10 but, to our knowledge, no study has assessed the relationship between FTUs and auditory threshold. In addition, this approach on asymmetric FTUs can make it possible to compare changes in auditory thresholds according to the UM level. Therefore, the aim of this study was to determine the relationship between the UM level, estimated from individual FTUs, and hearing loss in a representative sample of Korean adults.One important aspect of UM is balancing of oral status. Preliminary research has found UM to be more common in individuals with an unequal distribution of residual teeth on both sides of the mouth than in those with an even distribution.The Korean National Health and Nutrition Examination Survey (KNHANES) is conducted to evaluate the health and nutritional status. It is a nationwide, multistage, and stratified survey of a representative sample of the South Korean population. This survey has been performed in a 3-year cycle since 1998, and it produces statistical information on health indicators requested by international organizations, such as the Organization for Economic Cooperation and Development (OECD) or WHO, that allows comparisons between countries. The data used in the present study constituted a subset of the data obtained in KNHANES 2010–2012, and it was necessary to determine periodontal status by assessing the masticatory ability using only a dental formula. We only used data from 2010 and 2012, since oral examination data for the community periodontal index (CPI) were not published in 2011. The initial sample size was 7,014 adults aged 40–89 years who completed oral and audiometric examinations.n = 2,300), since the advanced periodontal status can reportedly reduce the masticatory ability due to pain and teeth mobility.19,20 Second, we excluded an additional 2,037 participants who had artificial teeth on implant-supported, fixed (bridge pontics) and removable prostheses, since we obtained limited information only about the presence or absence of prostheses in each upper or lower jaw from national survey data. Additionally, 412 participants with decayed teeth (DT) were excluded, because it was difficult to confirm severity and pain/symptoms of DT in this study. Moreover, in the KNHANES dataset, information on the dental treatment needs (TN) of individual teeth is also available. Especially, TN codes 6–8 indicate needs for extraction-related severe caries, periodontitis, and so on. These conditions make it difficult to chew normally, so participants who had TN codes 6–8 were excluded (n = 272). Finally, those with external ear disease (n = 220) were excluded, leaving a total of 1,773 subjects for inclusion in the analysis.The key considerations when assessing the masticatory ability were factors related chewing impairment—periodontal disease and untreated caries. For the present analysis, we excluded participants with a CPI score of 4  of >25 dB, and the 4-PTA was calculated at frequencies of 0.5, 1, 2, and 4 kHz in either ear, which is consistent with the definition used by the WHO.The dentition status was evaluated using the WHO criteria by trained and calibrated dentists. Third molars were regarded as nonfunctional teeth, and the final FTUs were defined as pairs of opposing functioning teeth (FST), which were composed of filled and sound teeth. The complete dentition comprised 12 FTUs, with two opposing premolars defined as 1 FTU and two opposing molars defined as 2 FTUs. In each subject, UM was calculated as the difference in the sums of the FTU scores on the two sides of the oral cavity: |sum of right-side FTUs − sum of left-side FTUs|. Based on the scores, the UM was classified into low (0), moderate (1 or 2), and high (more than 2).Information on the study population including age, sex, household income, education level, BMI, waist circumference, noise exposure, smoking, and medical conditions was obtained from the KNHANES results. BMI was calculated by dividing the measured weight in kilograms by the square of the measured height in meters. Hypertension was defined as a systolic blood pressure of ≥140 mm Hg, a diastolic blood pressure of ≥90 mm Hg, or treatment with antihypertensive agents. Diabetes mellitus was defined as a fasting plasma glucose level of ≥126 mg/dL, treatment with oral hypoglycemic agents or insulin, or a diagnosis by a physician.All of the participants were requested to fill in self-reported questionnaires to obtain information on certain items such as household income, education level, exposure to noise, smoking, and medical conditions, such as tinnitus. Smoking habits were categorized into current smokers and others. Education level was divided into up to high school or beyond high school. The lowest quartile of household income was considered a low income. Exposure to occupational, firearm, and recreational noise may be an important confounding factor in the association of masticatory function with hearing loss, and so the participants were divided into exposed or unexposed to such noise. Occupational noise exposure was defined as performing tasks for more than 3 months at noisy locations where people have to talk loudly to make themselves heard. Noise from a firearm was defined as exposure to a very loud sound, such as gunfire noise. Recreational noise exposure was defined as being more than 5 hours per week outside the workplace in locations where people need to speak loudly in order to talk with one another or the experience of using an earphone device in a noisy location. Tinnitus was defined when the subject answered ‘yes’ to each question of experiencing symptoms during the past year. Depression was defined when the subject answered ‘yes’ to a question of experiencing anxiety/depression in the EuroQoL questionnaires, which measures quality of life.The analyses were conducted using SPSS  with a significance cutoff of 0.05. All results were analyzed using a complex sampling plans and sampling weights of the KNHANES to provide nationally representative prevalence estimates.t-test for continuous variables and the chi-squared test for categorical variables. To identify the distribution of FST on each side of the oral cavity according to UM level, the number of FST was divided into total teeth, posterior teeth, and premolar and molar teeth on the right or left side. In addition, the average subjective masticatory ability was compared among different UM levels. Generalized linear models were used to compare mean values of FST and subjective masticatory ability among UM groups. Using multiple logistic regression analyses with adjustment for the covariates, the risk of hearing loss was estimated according to the UM level and was reported as odds ratios (ORs) and 95% confidence intervals (CI). The adjusted odds ratios (aORs) and their 95% CIs were calculated in multivariable logistic regression analyses. Sequential models were used to control for potential confounders. The primary model (model A) was adjusted for age, sex, household income, education level, a number of teeth, and unilateral mastication. Model B further adjusted for all variables in the primary model plus the health-related factors of diabetes mellitus, hypertension, current smoking, waist circumference, BMI, depression, and tinnitus. The final model (model C) further adjusted for all variables in model B plus the noise-exposure-related factors of occupational noise, firearm noise, and recreational noise.Sociodemographic differences between participants with and those without hearing loss ascertained using the P < 0.001; Table n = 379, 64.8%) being significantly higher than that of women . The participants with hearing loss were older than the subjects with normal hearing (P < 0.001). The prevalence of hearing loss increased with age group. The subjects with hearing loss were more likely than were subjects with normal hearing to have a low income; low education level; obesity (BMI > 25); larger waist circumference (by about 3 cm); smoking behavior; exposure to occupational or firearm noise; chronic disease, such as diabetes or hypertension; and tinnitus. However, exposure to recreational noise and depression had no significant difference (P > 0.05). The rate of moderate and high UM was higher in subjects with hearing loss than in those with normal hearing.Overall, 678 of the included subjects (37.5%) had hearing loss P < 0.00; Table 1P < 0.05). Comparing the distribution of premolar and molar FST divided into right and left sides, subject with low UM had the highest number of premolar and molar FST on each side (P < 0.001). This pattern was consistent with the subjective masticatory ability using a 5-point Likert scale. Subject with low UM seemed to have significantly high ability in mastication (P < 0.001).Table P < 0.05, Bonferroni test, data not shown).The mean pure-tone threshold gradually increased with frequency in all groups Figure . The thrThe results obtained for the covariate-adjusted logistic regression model for hearing loss are presented in Table P = 0.017).Table To our knowledge, this is the first study to identify differences in hearing thresholds by UM level using large-scale epidemiologic data. In a representative sample of Korean adults aged 40 years and older who had taken part in KNHANES 2010–2012, the UM level was associated with reduced hearing loss, even after adjusting for confounding variables. Furthermore, we observed that the hearing threshold increased significantly with the UM level in this general population: the risk of hearing loss was about two-fold higher for high UM than for low UM.14 Based on their findings, the UM level in the present study was estimated from asymmetric FTUs, which were found to be the single best predictor of masticatory performance.15,22,23 In particular, the loss of posterior teeth can result in shifting of the adjacent teeth that causes the potential collapse of bite support that in turn leads to a reduced masticatory force.24 This was supported by the present study finding that the high UM group—as defined using the FTUs for the posterior teeth—had the most impaired subjective masticatory ability and the smallest number of posterior FST among the three groups categorized according to UM levels .9 In addition, it has been reported that the PTA is significantly higher in edentulous subjects.10,25 Consistent with these findings, the present study found that the risk of hearing loss was higher for high UM, for which the mean number of FST was lowest (25.08), compared to a mean number of FST of 27.38 for low UM . In another previous study, the mean difference in hearing loss between dentate and edentulous elderly was 5 dB (ranging from 4.9 to 8.6 dB), which was considered clinically significant.25 A direct comparison was not possible in the present study due to the absence of edentulous subjects, but the results of the present study were similar  and middle ear. The abnormal tensile force of the discomalleolar ligament (DML), which arises from the malleus among auditory ossicles in the middle ear and runs to the medial retrodiscal tissue of the TMJ, can cause subjective hearing loss in patients with temporomandibular disorder.26,27 Excessive tensile force from UM may stretch the DML so as to adversely affect the efficiency of the auditory ossicles in conducting sound from the eardrum to the cochlea. Second, it can be explained via a neurological pathway for somatic stimulation. The trigeminal nerve responsible for sensation on the face converges on the dorsal cochlear nucleus (DCN) with cranial nerve 7, 9, and 10 and cervical nerve 2 and 3. Nonspecific stimuli in the DCN are, therefore, able to induce auditory problems. Somatosensory information resulting from muscle spasms is not produced by compression of nerves or blood vessels in the ear, but rather from the convergence in the DCN of sensory signals from muscle spindles in the head and neck with sound signals from the cochlea.28 Prolonged asymmetric exercise can induce muscle pain,29 and so facial pain from excessive UM can cause nonspecific stimulation and hearing problems. This supports the hypothesis that facial sensitivity problems associated with chewing can affect hearing ability.The results of the present study are also consistent with previous clinical studies finding that a unilateral chewing preference can affect the hearing ability and can be recovered by bilateral mastication treatment.30 Asymmetric SDAs have a long side of the dental arches extending to the first molar, and so subjects with this feature compensate partly by chewing with the longest side. Therefore, our findings for high UM, which were based on asymmetry characterized by an FTUs score of more than 2, may reflect the effects on UM.This study postulated that the UM level, as measured by asymmetric FTUs, was associated with hearing loss. High UM clinically means that pairs of premolars and molars have been lost on one side of the mouth. It has been estimated that unilateral chewing occurs in 93% and 92% of cases of type-I and -II asymmetric shortened dental arches (SDAs), respectively.6,21 In addition to its broad generalizability, this is the first study to have considered the influence of unilateral mastication on hearing ability using the nationwide data set. A few previous studies have used FTUs,16,17 but no study has examined the influence of UM using a side-based asymmetry score of FTUs. Consequently, asymmetric FTUs can explain an association with hearing loss.The important strengths of this study include the use of data from a large-scale population and consideration of well-known risk factors for hearing loss. This study found that age was a strong confounder of risk factors. A logistic regression analysis revealed a strong association between elderly group (≥65 years) and hearing loss. The hearing loss was also significantly associated with being male; having a low income, low education, and tinnitus; and being subjected to occupational noise in the final model Table . These f31 Nevertheless, further analyses that includes these factors will need to account for our results.Some limitations of this cross-sectional study should be considered. First, our findings were based on cross-sectional data, so they cannot reveal cause and effect relationships. Second, this retrospective study was designed to analyze pre-existing data, and so we were unable to assess problems of the occlusal condition, subjective UM habits, and conductive hearing threshold. Moreover, we excluded the subject with untreated dental caries, artificial teeth , and CPI score 4 in order to estimate UM level. In case of CPI score of 4, it refers to advanced periodontal disease, which could cause impaired mastication due to biting pain, mobility of teeth, and decline in biting force during mastication. Although CPI index teeth do not include premolars, it can represent an individual’s periodontal status, even when compared to the entire recording.In conclusion, this study has shown that the UM, measured by asymmetric FTUs, is associated with hearing loss and confirmed that the individual FTUs can be used to assess the level of UM. These results indicate that adults with a higher probability of unilateral chewing experience greater impacts on impaired hearing ability than those without unilateral chewing based on assessing differences in FTUs.


Aim: We have recently demonstrated the presence of putative tumor stem cells (TSCs) in World Health Organization (WHO) grade I meningioma (MG) localized to the microvessels, which expresses components of the renin-angiotensin system (RAS). The RAS is known to be dysregulated and promotes tumorigenesis in many cancer types, including glioblastoma. Cathepsins B, D, and G are isoenzymes that catalyze the production of angiotensin peptides, hence providing bypass loops for the RAS. This study investigated the expression of cathepsins B, D, and G in WHO grade I MG in relation to the putative TSC population we have previously demonstrated.Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining with antibodies for cathepsins B, D, and G was performed on WHO grade I MG tissue samples from 10 patients. Three of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining to investigate co-expression of each of these cathepsins using combinations of smooth muscle actin (SMA) and embryonic stem cell marker OCT4. NanoString mRNA expression (n = 6) and Western blotting  analyses, and enzyme activity assays , were performed on snap-frozen WHO grade I MG tissue samples to confirm transcriptional activation, protein expression, and functional activity of these proteins, respectively.Results: DAB IHC staining demonstrated expression of cathepsins B, D, and G in all 10 MG samples. NanoString mRNA expression and WB analyses showed transcriptional activation and protein expression of all three cathepsins, although cathepsin G was expressed at low levels. EAAs demonstrated that cathepsin B and cathepsin D were functionally active. IF IHC staining illustrated localization of cathepsin B and cathepsin D to the endothelium and SMA+ pericyte layer of the microvessels, while cathepsin G was localized to cells scattered within the interstitium, away from the microvessels.Conclusion: Cathepsin B and cathepsin D, and to a lesser extent cathepsin G, are expressed in WHO grade I MG. Cathepsin B and cathepsin D are enzymatically active and are localized to the putative TSC population on the microvessels, whereas cathepsin G was localized to cells scattered within the interstitium, These results suggest the presence of bypass loops for the RAS, within WHO grade I MG. Meningioma (MG) is one of the most common primary neoplasms of the central nervous system with an age-adjusted incidence of 11.5 per 100,000 person-years . Risk faThe prevailing concept proposes a neuroectodermal origin of MG, specifically arachnoid cap cells . HoweverWhile the RAS is classically associated with regulation of blood pressure and blood volume, it also plays a role in cancer by inducing angiogenesis, promoting cellular proliferation, and inhibiting apoptosis –21. The The classical RAS cascade  is initiThe RAS can be activated through the classical cascade mechanisms initiated by (pro)renin and PRR or via bypass loops by a group of lysosomal proteases called cathepsins . Cathepsniche on the microvessels in GB has been reported recently  years included in our previous studies , 18, werThe diagnosis of WHO grade I MG was confirmed by an anatomical pathologist (HDB) using hematoxylin and eosin (H&E) stained 4 μm-thick formalin-fixed paraffin-embedded sections of MG samples from all 10 patients. 3,3-Diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining was performed, as previously described , 34, on To further characterize the expression of cathepsins B, D, and G, IF IHC staining was performed on three representative MG samples from the original cohort of 10 patients included in the DAB IHC staining. Dual IF IHC staining was performed for each of the cathepsins using identical primary antibodies and concentrations as for DAB IHC staining, in conjunction with SMA or OCT4. An appropriate fluorescent secondary antibody combination of Vectafluor Excel anti-rabbit 594  and Alexa Fluor anti-mouse 488  was used for detection.DAB IHC-stained slides were counterstained with hematoxylin and mounted in Surgipath Micromount mounting medium , whereas IF IHC-stained slides were mounted in Vectashield HardSet anti-fade mounting medium and counter-stained with 4′6-diamino-2-phenylinodole .Positive controls for DAB IHC staining included human placenta for cathepsin B, human breast tissue for cathepsin D and human splenic lymphoma for cathepsin G. Negative controls were performed on sections of WHO grade I MG using a matched isotype control for rabbit primary antibodies , to determine the specificity of the antibody. Negative controls for IF IHC staining were performed using a section of MG tissue with the combined use of primary isotype mouse  and rabbit  antibodies.The DAB IHC-stained slides were viewed and images were captured, using an Olympus BX53 light microscope fitted with an Olympus DP21 digital camera and processed with CellSens 2.0 software . The IF IHC-stained images were captured using the Olympus FV1200 biological confocal laser-scanning microscope and processed with CellSens Dimension 1.11 software using the 2D deconvolution algorithm (Olympus).Western blot (WB) analysis was performed on five snap-frozen WHO grade I MG samples from the original cohort of 10 patients included for DAB IHC staining. Total protein was extracted and precipitated from the tissue samples, as previously reported , followeTM F96 MicroWellTM black polystyrene plate  using the Varioskan Flash plate reader . Tonsil and denatured tonsil tissue lysates were used as appropriate positive and negative controls, respectively.Enzymatic activity of cathepsin B and cathepsin D were determined in snap-frozen WHO grade I MG tissue samples from three of the five samples used for WB analysis. Enzymatic activity assay (EAA) kits were utilized for cathepsin B  and cathepsin D . All steps of the procedure were performed according to the manufacturer's protocol, and as recently described . FluoresmRNA expression of cathepsins B, D, and G was determined by NanoString mRNA analysis in six snap-frozen WHO grade I MG samples of the original cohort of 10 patients used for DAB IHC staining. Total RNA was extracted separately from ~20 mg of each snap-frozen MG tissue and run through the NanoString nCounter™ Gene Expression Assay , as previously described , 36. Prot-tests using SPSS version 24 software.Data obtained from WB and NanoString mRNA analyses were subjected to paired The diagnosis of WHO grade I MG was confirmed by H&E staining for all 10 tissue samples . All DABThe expected positive staining patterns for cathepsin B , brown, To determine whether each cathepsin was expressed by the putative TSC population on the microvessels that express the ESC markers we have previously reported in WHO grade I MG , we co-sTo confirm the expression of both cathepsin B , red andp < 0.002) and cathepsin D (p <0.0001) compared to cathepsin G.WB of the snap-frozen WHO grade I MG samples demonstrated the presence of bands at the expected molecular weight of 25 kDa for cathepsin B , blue anIn light of the WB analysis, detecting only cathepsin B and cathepsin D, we limited our study to determine the functional activity of these two cathepsins. EAAs demonstrated enzymatic activity for cathepsin B and cathepsin D .t-tests, further confirmed that the mean levels of expression of cathepsin B (p <0.004) and cathepsin D (p <0.001) were significantly higher than that of cathepsin G, but only marginally different to each other (p <0.019).NanoString mRNA expression analysis demonstrated the presence of mRNA transcripts for cathepsins B, D, and G, normalized against the housekeeping gene GUSB, in all six MG samples . Statist+ cells may be of a mast cell phenotype, as previously reported  and Cancer Therapeutic (PCT/NZ2018/050006), and provisional patent application Novel Pharmaceutical Compositions for Cancer Therapy (US/62/711709). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycoplasma hyopneumoniae is an important respiratory pathogen that causes great economic losses to the pig industry worldwide. Although some putative virulence factors have been reported, pathogenesis remains poorly understood. Herein, we evaluated the relative abundance of proteins in virulent 168 (F107) and attenuated 168L (F380) M. hyopneumoniae strains to identify virulence-associated factors by two-dimensional electrophoresis (2-DE). Seven proteins were found to be ≥ 1.5-fold more abundant in 168, and protein–protein interaction network analysis revealed that all seven interact with putative virulence factors. Unexpectedly, six of these virulence-associated proteins are encoded by core rather than accessory genomic elements. The most differentially abundant of the seven, fructose-1,6-bisphosphate aldolase (FBA), was successfully cloned, expressed and purified. Flow cytometry demonstrated the surface localisation of FBA, recombinant FBA (rFBA) mediated adhesion to swine tracheal epithelial cells (STEC), and anti-rFBA sera decreased adherence to STEC. Surface plasmon resonance showed that rFBA bound to fibronectin with a moderately strong KD of 469 nM. The results demonstrate that core gene expression contributes to adhesion and virulence in M. hyopneumoniae, and FBA moonlights as an important adhesin, mediating binding to host cells via fibronectin.The online version of this article (10.1186/s13567-018-0610-2) contains supplementary material, which is available to authorized users. Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia, one of the most damaging respiratory diseases affecting pig farming. Despite low direct mortality, M. hyopneumoniae can lower the feed conversion efficiency and reduce the growth rate, resulting in large economic losses.M. hyopneumoniae pathogen colonises and destroys the epithelial surfaces of the respiratory tract [M. hyopneumoniae adhesion [M. hyopneumoniae [l-lactate dehydrogenase, an immunogenic cytoplasmic protein involved in the glycolytic process but also present at the cell surface with adhesin functions [M. hyopneumoniae by binding to multiple host proteins [M. hyopneumoniae remain poorly understood [The ry tract , and adhry tract , 3. Severy tract . Other ary tract , 6, P159ry tract , P146 [8ry tract , Mhp271 ry tract , Mhp683 ry tract , Mhp107 ry tract  and so oeumoniae ; and l-lunctions . Glyceraunctions . Finallyproteins , 20. Howderstood .M. hyopneumoniae is highly prevalent (ranging between 38 and 100%) in almost all areas of pig production worldwide, there are discrepancies in terms of pathogenicity among isolates of M. hyopneumoniae. Comparative proteomics research, which offers a systematic analysis, can reveal novel putative virulence factors between bacterial strains in which virulence differs. For example, the available proteome map of M. hyopneumoniae strain 7448 served as a reference for comparative analysis of differentially virulent M. hyopneumoniae strains [M. hyopneumoniae pathogenic strain 232 and avirulent strain J revealed 11 differentially abundant proteins [M. hyopneumoniae strains. For example, the size of the genome ranges from 892 758 bp  to 964 503 bp . Furthermore, differences are magnified after translation into proteins, due to variation in regulation and modification at both RNA and protein levels. Thus, the results obtained from comparative proteomics analyses of different sources of bacterial strains can be confusing, preventing the ability to focus on the most critical and common factors [M. hyopneumoniae strain 168L (F380) from continuous passage of pathogenic strain 168 (F107) in cell-free medium [M. hyopneumoniae.Although infection by  strains . A subse factors . Subjecte medium . Herein,All animal experiments were approved by the Committee on the Ethics of Animal Experiments and performed in Jiangsu Academy of Agricultural Sciences (License No. SYXK (Su) 2015-0019). The experimental procedures conformed to the guidelines of Animal Regulations of the Jiangsu Province (Government Decree No. 45) in accordance with international law.Mycoplasma hyopneumoniae strain 168 was isolated in Gansu Province, China, from a pig exhibiting typical characteristics of mycoplasmal pneumonia of swine (MPS) [was cultured in KM2 cell-free liquid medium (a modified Friis medium) containing 20% (v/v) swine serum at 37 °C, and was gradually attenuated by continuous passage to the 380th passage, yielding strain168L [ne (MPS) . This firain168L .M. hyopneumoniae strains 168 and 168L were free of sera IgG antibody recognising classical swine fever virus , porcine reproductive and respiratory syndrome virus , porcine pseudorabies virus , porcine circovirus type 2 , and M. hyopneumoniae . They were also free of secretory IgA antibody recognising M. hyopneumoniae [M. hyopneumoniae [8 colour change unit (CCU)/mL culture of strains 168 or 168L (or KM2 medium as a negative control). The piglets were observed daily for clinical signs of pneumonia, such as coughing and asthma. All animals were euthanised at 28 days after challenge, and lung lesions were scored using the rule of 28 method [p ≤ 0.05.Nine non-immunised cross-bred (Xiaomeishan × Landrace) 50-day-old snatch-farrowed, porcine-colostrum-deprived (SF-pCD) piglets were raised according to the methods described by Huang et al. , 28. Alleumoniae , and anteumoniae  and M. heumoniae . The pig8 method . A valueg for 20 min at 4 °C at the late exponential phase of growth. Pellets were washed three times with 10 mM TRIS–HCl (pH 7.4) and resuspended in protein extract consisting of 1.52 g thiourea, 4.2 g urea, 0.4 g CHAPS, 200 µL amphoteric electrolyte, 61.6 mg dithiothreitol , and protease inhibitor (Merck) dissolved in 10 mL ultrapure water. After vortexing and centrifugation, total protein in the supernatant was subjected to cleanup with a ReadyPrep 2-D cleanup kit (Bio-Rad). Purified proteins were redissolved in 350 µL rehydration solution  and centrifuged to remove insoluble components. Samples were finally loaded onto a 17 cm strip  and isoelectric focusing (IEF) was carried out at 20 °C by positively rehydrating at 50 V for 12 h, increasing slowly to 250 V for 1 h, rapidly to 1000 V for 1 h, 10 000 V for 3 h, rapidly to 10 000 V to a total of 90 000 Vh, then rapidly to 500 V. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was then performed according to a previous protocol [Cultures of strains 168 and 168L were harvested by centrifugation at 16 000 × protocol .t test ≥ 0.05) were excised from the 2-DE gel and subjected to matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF–MS/MALDI-TOF-TOF–MS) analysis. Peptide mass fingerprinting data were analysed using the MASCOT server. Peptides with a rank of 1 in the MASCOT search were considered significant and used for the combined peptide score.After Coomassie blue staining of the SDS-PAGE gel, the detected protein spots from each gel were matched automatically using PDQuest V8.0 software with additional visual analysis. The intensity of individual spot was normalised relative to the total valid spot intensity for each gel. Protein expression levels were calculated as the fold-change. Only spots for which the abundance in strain 168 was ≥ 1.5-fold higher than in strain 168L  of a pan-genome, defined as the entire genomic repertoire of a given species, can be divided into core , dispensable, and strain-/isolate-specific genes , 33. Theanalysis . The accanalysis . Graphs fba gene (MHP168_014) was cloned into the pET-28a(+) vector by site-directed mutation and overlap extension (SOE-PCR) due to the existence of rare codons, and rFBA was produced in Escherichia coli BL21 (DE3) and purified by Ni-chelating chromatography as described in a previous study [The full-length us study . A polycM. hyopneumoniae strain 168L is expressed as the percentage of that in M. hyopneumoniae strain 168. Three replicates were subjected to statistical analysis by SPSS 20.0.Equal amounts (40 μg) of each protein sample were separated on a 12% SDS-PAGE gel, and proteins were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes  and developed with Ponceau-S as the loading control. After blocking with TBST buffer comprising 20 mM TRIS–HCl (pH 7.6), 150 mM NaCl and 0.1% Tween-20 containing 5% skimmed milk, membranes were probed with anti-rFBA antibody (1:2000 dilution). Horseradish peroxidase (HRP)-conjugated secondary antibody (1:10 000 dilution) was used for final identification. ImageJ software was used to calculate the optical density (OD) of the corresponding bands. The OD of FBA from different samples was normalised to that of the Ponceau-S-stained membrane. The abundance of FBA in M. hyopneumoniae strain 168, and to probe FBA surface content differences between 168 and 168L, flow cytometry was performed. In brief, M. hyopneumoniae strains 168 and 168L (each 1 × 108 CCU/mL) were incubated with anti-rFBA serum at a 1:100 dilution (1:100 diluted preimmune serum was used as a negative control). M. hyopneumoniae cells were then stained with fluorescein isothiocyanate (FITC)-conjugated anti-IgG and the fluorescence intensity was measured using a BD Accuri C6 flow cytometer as described previously [M. hyopneumoniae incubated with anti-rFBA serum is expressed as the fold-change relative to the corresponding strain incubated with preimmune serum. The assay was performed in triplicate, and data were analysed using Student’s t tests in SPSS 20.0. For all tests, p ≤ 0.05 was considered statistically significant.To investigate whether FBA is present on the surface of eviously . The meaTo investigate the ability of rFBA to promote adherence to STEC, indirect immunofluorescence assays were performed. STEC were grown to confluence in 24-well plates with RPMI-1640 medium  supplemented with 10% (v/v) fetal bovine serum . After incubation with 100 μg of purified rFBA, the cells were washed three times with phosphate-buffered saline (PBS) and incubated with anti-rFBA antibody at a 1:1000 dilution, then with tetraethyl rhodamine isothiocyanate (TRITC)-tagged anti-IgG . Finally, cell nuclei were stained with 6-diamidino-2-phenylindole (DAPI). Fluorescence was detected using a fluorescence microscope . BSA was used instead of rFBA as a negative control .Mycoplasma hyopneumoniae cells (1 × 107 CCU/mL) were washed three times with PBS and pre-incubated with polyclonal antibody raised against rFBA or preimmune sera (1:20 dilution) at 37 °C for 30 min. Bacteria suspended in RPMI-1640 medium were added to 24-well cell plates containing confluent STEC, and plates were centrifuged at 800 × g for 10 min and incubated at 4 °C for 2 h. Bacteria counting including bacterial genome extraction and real-time PCR was performed according to a previous method [t tests with SPSS 20.0 .s method , 37. TheM. hyopneumoniae FBA could bind to fibronectin, the Far-Western blot (Far-WB) protein–protein interaction method was performed. A 20 μg sample of rFBA was separated by SDS-PAGE and transferred to a PVDF membrane [To explore whether membrane . After bThe interaction dynamics of rFBA and fibronectin were further investigated in real time by surface plasmon resonance (SPR) using a BIAcore X100 Plus instrument . Fibronectin was diluted to 10 µg/mL in 10 mM sodium acetate (pH 4.0) and covalently linked to the carboxymethylated dextran matrix of a CM5 sensor chip as the ligand using an amine coupling kit (Biacore AB). Immobilisation of soluble fibronectin generated resonance units (RU) of 2868. Binding kinetics were measured with increasing concentrations (0–100 μg/mL) of the analyte (rFBA) in running buffer (HBS-EP) consisting of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% (v/v) surfactant P20 (Biacore AB) at a flow rate of 30 μL/min for 180 s over immobilised fibronectin at 20 °C. The dissociation phase was monitored for 1000 s by allowing the buffer to flow over the chip. Association kinetics were analysed manually using Biacore X100 Control Software .M. hyopneumoniae strain 168 began to cough, while no clinical signs of pneumonia were observed from the group challenged with M. hyopneumoniae strain 168L or the control group. All pigs were alive during the entire experimental period. After euthanising, each of the seven pulmonary lobes was scored for severity of mycoplasmal lesions ranging from 0 to 4 . The results show that lung lesion levels indicated by the sum of seven pulmonary lobes in the group challenged with M. hyopneumoniae strain 168 (average lung lesion score 12) were significantly higher than those in the group challenged with strain 168L (average lung lesion score 1) , glyceraldehyde 3-phosphate dehydrogenase (GAPDH), adenine phosphoribosyltransferase (Apt), lactate dehydrogenase (LDH), heat shock protein GrpE, and FBA. DnaK, GAPDH and FBA were not detected in lysates of strain 168L by 2-DE analysis, indicating that they were expressed at very low levels in the attenuated strain. However, FBA showed relatively higher expression in M. hyopneumoniae 168 based on 2D gels, and received a higher score in MS analysis  between To explore the potential mechanism(s) by which surface localisation of FBA affects virulence, we used indirect immunofluorescence to determine whether rFBA could adhere to STEC. The results revealed significant fluorescence on the cell surface of STEC incubated with rFBA Figure A, but noM. hyopneumoniae strain 168. A polyclonal antibody against rFBA was found to decrease M. hyopneumoniae adherence to STEC relative to treatment with preimmune sera  reduction in the adherence efficiency of M. hyopneumoniae to STEC, further confirming that FBA plays an indispensable role in adherence of M. hyopneumoniae to host cells.Antibody inhibition assays were performed to further assess the contribution of surface-localised FBA to adhesion in KD = 468 ± 23 nM and ka = 3470 ± 580 M−1 s−1.To investigate the STEC components that interact with FBA, we examined the fibronectin-binding activity of rFBA using Far-WB analysis. The corresponding bands were observed in both reactions of rFBA to anti-FBA antibody (positive control) and to fibronectin, while no specific reaction was observed in the negative control. The analysis indicates that rFBA could specifically bind to fibronectin Figure A. Using Escherichia coli strains include 1200 genes in the conserved core genome, but more than 13 000 gene families in the pan-genome. This is in part due to several major virulence factors being located on transmissible genetic elements such as pathogenicity islands, bacteriophages, or plasmids, which are classed as accessory genes [stx, is a bacteriophage element [eae, which belongs to a pathogenicity island.The pan-genome includes the core genome containing genes present in all individuals, the accessory or dispensable genome containing genes present in two or more strains, and unique genes specific to single strains . The expry genes . Similar element , and theAlthough accessory genes encode many conspicuous determinants of virulence, core genes can also confer appreciable virulence. Indeed, more and more moonlighting proteins are being recognised with roles in bacterial pathogenesis . MoonligM. hyopneumoniae strain. A virulence-associated network constructed using these seven proteins and previously reported putative virulence factors showed that all seven novel differentially abundant proteins are involved in M. hyopneumoniae virulence. Since all seven are conserved enzymes, we explored a possible common pattern among these virulence-associated factors from a system and whole-genome perspective. A pan-genomics analysis with nine available M. hyopneumoniae genomes was therefore performed. Unexpectedly, six of the seven novel differentially abundant proteins are encoded by core genes, and the only protein potentially encoded by an accessory gene may not be in reality due to incomplete sequencing of strain 11. This indicates that core genes, which are often neglected, can play an important role in M. hyopneumoniae virulence.In the present study, 2-DE analysis revealed that seven proteins were significantly increased in abundance in the virulent M. hyopneumoniae strains, and this protein is a promising therapeutic and vaccine target in bacteria [Of the seven proteins, FBA showed the most fold change in different virulence bacteria . FBA is bacteria . Two difbacteria . Class-Ibacteria .Streptococcus pneumoniae in mice [S. pneumoniae FBA is a cell wall-localised protein, and anti-rFBA antibodies can inhibit S. pneumoniae adherence to epithelial cells. Flamingo cadherin has been identified as the host receptor [Streptococcus suis, which is reasonably well conserved among S. suis strains, was verified as an immunogenic cell wall protein, suggesting it could be developed as a vaccine candidate [Neisseria meningitidis is localised both in the cytoplasm and the outer membrane, and is required for adhesion to human cells [Mycobacterium tuberculosis, FBA is required for survival during the chronic phase of mouse infection [Toxoplasma gondii [Francisella novicida is important for bacterial multiplication in macrophages, and plays a regulatory role in pathogenesis [Despite lacking identifiable secretion signals, FBA are also localised at the bacterial cell surface, where they interact directly with host proteins and exhibit non-glycolytic functions. Remarkably, FBA has been recently reported to play a role in the pathogenesis of several pathogens. FBA is antigenic in humans, and affords significant protection against challenge with virulent  in mice . S. pneureceptor . FBA froandidate . FBA of an cells  by bindian cells . In Myconfection . FBA hasa gondii , and FBAogenesis .M. hyopneumoniae 168 strain, but less so on the attenuated 168L strain. Adherence of viable M. hyopneumoniae strain 168 to STEC was inhibited significantly by anti-rFBA antisera, which indicates that FBA is indispensable for adherence to STEC. These results demonstrate that M. hyopneumoniae FBA, in addition to its major cytoplasmic, biosynthetic, and metabolic roles, can translocate to the surface and moonlight as an important adhesion factor.Our flow cytometry analysis indicates that FBA was accessible on the surface of the highly virulent M. hyopneumoniae. Since adherence to host tissues is an important prerequisite for colonisation and subsequent disease development for pathogenic bacteria, adhesins are of crucial importance for M. hyopneumoniae infection [M. hyopneumoniae. The capacity to bind fibronectin is widespread in bacterial pathogens, and among mycoplasmas, the first fibronectin interaction was identified in M. penetrans [M. pneumoniae [M. hyopneumoniae [Streptococcus [Staphylococcus [M. hyopneumoniae adherence to the cell surface. Consistently, fibronectin was verified as one of the host cell receptors for M. hyopneumoniae adhesion [KD of 468 nM, signifying a specific and moderately strong interaction that may be physiologically relevant. This is the first report of an interaction between FBA of M. hyopneumoniae and host fibronectin contributing to adherence and therefore pathogenicity.Colonising the epithelial surfaces of the respiratory tract is an important part of the pathogenesis of nfection . Bacterinfection , 56, an nfection . Immunohnfection , the mosenetrans , followeeumoniae  and M. heumoniae . Many fitococcus  and Staplococcus . The mamadhesion . The disM. hyopneumoniae. Specifically, FBA is abundant on the surface of virulent M. hyopneumoniae strains, where it binds strongly to host fibronectin, thereby promoting adherence to STEC. FBA can be surface translocated and may play a role in the adhesion to host cells and colonisation, thereby serving as an important candidate virulence factor of M. hyopneumoniae. Thus, we can conclude that core enzymes may be important virulence determinants. To our knowledge, this is the first report describing the moonlighting function of FBA in M. hyopneumoniae virulence, and the first to present evidence of the involvement of core genes in virulence in this species. Intrinsic virulence functions represent potential targets for broad-spectrum drugs, and links between intrinsic gene functions and virulence traits are clearly worthy of further study. For organisms with relatively small genomes, multi-functional proteins may be particularly useful for optimising the potential of the genome [M. hyopneumoniae genomes are currently available, which may limit pan-genome analysis to some extent. Indeed, only partial inhibition of FBA was observed in competitive adhesion inhibition assays.In summary, our research indicates the importance of core genes in the virulence of e genome . HoweverM. hyopneumoniae. Much remains to be elucidated about how proteins lacking signal motifs are localised on the bacterial cell surface. The roles of core genome encoding proteins in infection and immunity in M. hyopneumoniae and other pathogenic organisms are clearly worthy of further investigation.In conclusion, the core genome encoding proteins located in the cytoplasm and the cell membrane appears to be associated with virulence in M. hyopneumoniae. Among these factors, FBA does not have a pig ortholog, making it a prime candidate for a swine pneumonia vaccine. Our comprehensive analysis revealed seven virulence-associated factors, at least six of which are encoded in the core genome of M. hyopneumoniae. The current lack of effective vaccine candidates and the global expansion of sequencing data make this a potentially powerful approach for the identification and development of effective vaccines against M. hyopneumoniae and other bacterial pathogens.The combined comparative proteomics and core genomics analyses employed herein successfully identified potential virulence-associated factors in Additional file 1.Protein–protein interaction network. Network analysis of novel differentially abundant proteins and known putative virulence factors of M. hyopneumoniae by String database.Additional file 2.Pan-genomic analysis. A pan-genomic analysis with nine available M. hyopneumoniae genomes.