Genome-wide global detection of genes involved in horizontal gene transfer (HGT) remains an active area of research in medical microbiology and evolutionary genomics. Utilizing the explicit evolutionary method of comparing topologies of a total of 154,805 orthologous gene trees against corresponding 16S rRNA “reference” trees, we previously detected a total of 660,894 candidate HGT events in 2,472 completely-sequenced prokaryotic genomes. Here, we report an HGT-index for each individual gene-reference tree pair reconciliation, representing the total number of detected HGT events on the gene tree divided by the total number of genomes (taxa) member of that tree. HGT-index is thus a simple measure indicating the sensitivity of prokaryotic genes to participate (or not participate) in HGT. Our preliminary list provides HGT-indices for a total of 69,365 genes (detected in >10 and <50% available prokaryotic genomes) that are involved in a wide range of biological processes such as metabolism, information, and bacterial response to environment. Identification of horizontally-derived genes is important to combat antibiotic resistance and is a step forward toward reconstructions of improved phylogenies describing the history of life. Our effort is thus expected to benefit ongoing research in the fields of clinical microbiology and evolutionary biology. Prokaryotic organisms often exchange genetic material directly with other organisms not necessarily related by vertical descent , we produced lists of frequently and non-frequently transferred genes in prokaryotic genomes  gene sets where either all pair-wise neighbor-joining (NJ) distances were close to zero, genomes include only one species, or harbored <10 genomes, from the downstream analysis. Of the remaining, 69,365/117,345 (59%) gene sets were sufficiently large  and yielded detectable gene-species tree conflict indicative of horizontal evolution , transport , transcription , transduction , and translation (875), along with antibiotic and toxin (27 each), stress (285), homeostasis (221), rRNA (211), and transposition (174) processes indicating the wide range of functions mediated by HGT in prokaryotes.Because the likelihood of HGT events increases for larger gene trees, next we studied the HGT-indices of genes harboring the maximum number of taxa  method of HGT detection is expected to be 100% accurate owing to the inherent complexity underlying the myriad of ways microorganisms interact with each other in natural environments and considering the billions of years of evolutionary timespan for the age of some of the microbial genomes. Therefore, the reported lists of genes that are frequently Dataset  or non-fSaccharomyces cerevisiae was added and treated as “outgroup” to root the reference tree. Gene and reference trees were reconciled using RANGER-DTL-U algorithm (ver. 1.0) (Bansal et al., Orthologous genes and HGT event mappings were retrieved directly from Jeong et al. . In brieBoth authors contributed to the design and analysis of the study and wrote, edited, and approved the final manuscript.The 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.


Background and objective: hearing impairment is common in older adults and has been implicated in the risk of disability and mortality. We examined the association between hearing impairment and risk of incident disability and all-cause mortality.Design and setting: prospective cohort of community-dwelling older men aged 63–85 followed up for disability over 2 years and for all-cause mortality for 10 years in the British Regional Heart Study.Methods: data were collected on self-reported hearing impairment including hearing aid use, and disability assessed as mobility limitations , difficulties with activities of daily living (ADL) and instrumental ADL (IADL). Mortality data were obtained from the National Health Service register.Results: among 3,981 men, 1,074 (27%) reported hearing impairment. Compared with men with no hearing impairment, men who could hear and used a hearing aid, and men who could not hear despite a hearing aid had increased risks of IADL difficulties . The associations remained after further adjustment for covariates including social class, lifestyle factors, co-morbidities and social engagement. Associations of hearing impairment with incident mobility limitations, incident ADL difficulties and all-cause mortality were attenuated on adjustment for covariates.Conclusion: this study suggests that hearing problems in later life could increase the risk of having difficulties performing IADLs, which include more complex everyday tasks such as shopping and light housework. However, further studies are needed to determine the associations observed including the underlying pathways. Hearing impairment increases with age and has been associated with chronic conditions including cardiovascular disease (CVD) and poor quality of life , 2. DemoIt is important to understand the influence of hearing impairment on disability including activities of daily living to establish the impact of hearing impairment on functional independence in later life. Therefore, we investigated the association of self-reported hearing impairment with subsequent mobility limitations, ADL, IADL and all-cause mortality in a representative sample of older British men aged 63–85 followed up for 2 years for disability and 10 years for mortality. We also examined whether these associations were independent of age, social class, lifestyle factors and co-morbidities known to be associated with hearing impairment including CVD, hypertension and diabetes .This study uses data from the British Regional Heart Study (BRHS), a prospective study in a socioeconomically and geographically representative sample of 7,735 middle-aged men drawn from 24 general practices representing all major British Regions . The menQuestions on hearing impairment included ‘Do you use a hearing aid?’ and ‘Using a hearing aid if needed, is your hearing good enough to follow a TV programme at a volume others find acceptable?’ with answer options yes/no , and allAll men were followed up for mobility limitations and activities of daily living (ADL) and instrumental ADL (IADL) from 2003 to 2005 and for all-cause mortality from 2003 to 2013. Two questions asking whether they had problems taking the stairs and problems walking 400 yards with answer options yes/no were used to assess mobility limitation. Reporting problems with one or both was classified as having mobility limitations. ADL was classified as having some difficulty or in need of help undertaking one or more of the following activities: bathing, dressing, eating, getting in or out of bed or chair, toileting and/or walking across a room . IADL wa2 and over [Covariates included socioeconomic and lifestyle factors including social class, social engagement, cigarette smoking, obesity and physical activity. Co-morbidity-related covariates included doctor-diagnosed CVD , hypertension and diabetes analysed dichotomously. Participants were divided into manual and non-manual social class based on the longest held occupation of subjects at study entry using the Registrar Generals' Social Class Classification. The men were grouped into non-smokers, ex-smokers and current smokers. Being obese was defined as having a body mass index (BMI) of 30 kg/mand over . Physicaand over , where nand over . Depressn = 38) were excluded. Participants free from mobility limitations and difficulties in ADLs and IADLs at baseline were followed up for each of these types of disability. Models were adjusted for age, social class, lifestyle factors and co-morbidities. A Bonferroni correction was applied for multiple comparisons, and findings remained significant. All analyses were carried out using SAS version 9.3 software .Logistic regression was used to assess the associations of hearing impairment with incident mobility limitations and difficulties in ADLs and IADLs. Odds ratios (OR) with 95% confidence intervals (CI) were obtained using no hearing impairment  as reference group. Survival analysis was used to examine the association between hearing impairment and mortality, and Cox proportional hazards regression was used to calculate hazard ratios (HR) with 95% CIs. We also performed Cox regression using age as the time variable. The test confirmed that the proportionality hazards assumption was met. Participants who did not answer any of the hearing-related questions . Of these, 3,108 men had no previous mobility limitations, 3,346 men had no previous ADL and 3,410 men had no previous IADL. At 2-year follow-up, there were 238 (8%) new cases of mobility limitations, 260 (8%) new cases of ADL and 207 (6%) new cases of IADL. All 3,981 men were also followed for all-cause mortality over 10 years during which 1,463 (37%) deaths occurred. Characteristics of participants by hearing impairment are shown in Table Table This study investigated the association of hearing impairment with incident disability  and all-cause mortality in older British men. Our findings show that men with hearing impairment had greater risks in particular of disability affecting IADLs. The associations observed between hearing impairment and incident mobility limitations, incident ADL and all-cause mortality were attenuated on further adjustment for covariates.The association between hearing impairment and mobility limitations was attenuated particularly on adjustment for social engagement. Communication problems due to hearing impairment may restrict social engagement . Being sMen who could not hear and did not use hearing aid had greater risks of all-cause mortality compared with men with no hearing impairment. However, the association was attenuated after adjustment for social class, lifestyle factors and co-morbidities. This is consistent with earlier studies demonstrating no association after adjustment for potential confounders including social class and physical functioning , 13.The major strengths of this study are that it was a large socioeconomically representative cohort with negligible loss to follow-up for disability and mortality . In addiLimitations include that hearing impairment was self-reported rather than objectively measured. However, the questions used have been validated against objective measures . Furtherper se underlying the association. Future longitudinal studies are required to further assess the association between hearing impairment and incident disability, taking cognitive impairment and inflammation into account.Hearing problems in later life may increase the risk of having difficulties performing IADLs.Inconsistent findings across the hearing impairment groups suggest that something may be underlying the association with IADLs.The association of hearing impairment and all-cause mortality was attenuated on adjustment for covariates.In summary, our study shows that older men who could follow TV and used a hearing aid have greater risks of disability affecting IADLs, which are important for maintaining functional independence in later life. The inconsistent findings across the hearing impairment groups further suggest that it may not be hearing None declared.


To investigate the relationship between mumps and the extent of hearing impairment and otolith organ damage.A total of 27 patients with unilateral hearing impairment following mumps were enrolled. The degrees of hearing loss and otolith organ damage were confirmed by audiometric and vestibular evoked myogenic potential [VEMP] tests. All the results were compared and analyzed using Stata 13.0 software for Windows.The VEMP thresholds of the affected ears were significantly higher than those of the unaffected ears in both tests . The mean cVEMP and oVEMP threshold values of the affected ears with hearing impairment for ≤10 years were significantly lower than those of affected ears with hearing impairment for >10 years .Deafness resulting from mumps is usually profound and permanent, which indicates severe damage to the cochlea due to the disease. The functions of otolith organs in the vestibular system are also impaired. Over time, the function of the otolith organs or their neural pathway may suffer secondary damage. Mumps, an acute respiratory infectious disease, is caused by the mumps virus, which is a single-stranded RNA virus of the family Paramyxoviridae. Mumps is easily transmitted among humans by oropharyngeal secretions. It is, therefore, endemic worldwide and has a high risk of outbreaks in schools and childcare facilities, as children and adolescents are highly susceptible to mumps . BetweenZamir et al. reported that 13.4% of the patients exhibited systemic symptoms and/or complications  with a short tone burst  at 500 Hz. The electromyography signals were amplified by a Bio-Logic Navigator PRO system , and the band pass-filtered sounds between 10–1500 Hz. In total, 120 stimuli were applied to each ear. The sound stimuli (500 Hz) were presented via calibrated headphones. The starting intensity was 95 dB nHL, which was decreased in decrements of 5 dB nHL until the VEMP response was abolished. This process was repeated three times. The intensity of the last characteristic waveform of VEMP was defined as the response threshold.An active electrode on the middle of the sternocleidomastoid muscle (SCM), with a reference electrode on the lower part of the suprasternal fossa and a ground reference on the middle of the forehead. The cVEMP waveforms were recorded after a total of 120 responses, induced by the stimuli of ACS with a 500-Hz short tone burst transmitted via the insert headphones, were averaged. The presence of cVEMP was registered when the amplitude of the first positive-negative-positive peak (P1-N1-P2) was recorded. The absence of cVEMP was recorded in cases where the waveforms could not be recognized or were not repeatable. Before placing the electrodes, the skin was scrubbed off in order to decrease the electrode impedance to < 5 KΩ. During the test, patients were instructed to elevate their heads to activate the SCM electrode when they heard a sound on the calibrated insert headphones.In the oVEMP test, an active electrode was placed about 1 cm below the center of the inferior eyelid of the right eye contrary to the side of sound stimulation, with a reference electrode 2cm below the active electrode and a ground reference on the middle of the forehead. The oVEMP waveforms were recorded after a total of 120 responses, induced by ACS stimuli with a 500-Hz short tone burst transmitted via the insert headphones, were averaged. The presence of oVEMP was registered when the peaks of the first negative and positive biphasic wave, designated as N1 and P1, respectively, were recorded. The absence of oVEMP was recorded in cases where the waveforms could not be recognized or were not repeatable. Before placing the electrodes, the skin was scrubbed off in order to decrease the electrode impedance to <5 KΩ. During the test, patients were instructed to fix their gaze on a point about 30° above the horizontal line when they heard a sound on the calibrated insert headphones.Statistical analysis was performed using the Stata 13.0 software for Windows. Descriptive statistical methods were used to describe the frequencies of the evaluated variables. Chi-square tests were used to compare the response rates of the VEMPs. Wilcoxon’s signed-rank test was used to compare the threshold values of the VEMPs between the affected and unaffected ears. A two-sample Wilcoxon rank-sum [Mann-Whitney] test was performed to evaluate the difference in the degree of damage to otolith organs between patients with hearing loss lasting ≤ 10 years and those with hearing loss lasting > 10 years. Values of p < 0.05 were considered statistically significant.The average age of the 27 patients at the time of infection with mumps was 8.56 ± 4.03 years. All of the patients exhibited unilateral hearing impairment after mumps. While 13 patients suffered hearing impairment in the right ear, 14 were affected in the left ear. Among the affected ears, 21 (77.8%) exhibited no response during audiometric examination, but six (22.2%) exhibited profound sensorineural hearing loss . All 27 Only one patient suffered from vertigo and exhibited mild abnormalities in the caloric test and sensory organization test. The other 26 patients did not have any complaint of dizziness and imbalance or any syndrome of vestibular dysfunction.In the cVEMP test, 22 patients (81.48%) exhibited a bilateral response, five (18.52%) exhibited no response in the affected ear. The response rate of the affected ears was lower than that of the unaffected ears  exhibited a bilateral response, 13 patients (48.15%) exhibited no response in the affected ear. The response rate of the affected ears was significantly lower than that of the unaffected ears Click here for additional data file.


STZ-induced diabetic mice exhibited distinct cardiac dysfunction, dampened intracellular calcium handling, alterations in cardiac morphology, and elevated cardiomyocyte apoptosis, which were mitigated in the cathepsin K knockout mice. Additionally, cathepsin K knockout mice attenuated cardiac oxidative stress and calcineurin/NFAT signaling in diabetic mice. In cultured H9c2 myoblasts, pharmacological inhibition of cathepsin K, or treatment with calcineurin inhibitor rescued cells from high-glucose triggered oxidative stress and apoptosis. Therefore, cathepsin K may represent a potential target in treating diabetes-associated cardiac dysfunction.Diabetes is a major risk factor for cardiovascular disease and the lysosomal cysteine protease cathepsin K plays a critical role in cardiac pathophysiology. To expand upon our previous findings, we tested the hypothesis that, knockout of cathepsin K protects against diabetes-associated cardiac anomalies. Wild-type and cathepsin K knockout mice were rendered diabetic by streptozotocin (STZ) injections. Body weight, organ mass, fasting blood glucose, energy expenditure, cardiac geometry and function, cardiac histomorphology, glutathione levels and protein levels of cathepsin K and those associated with Ca Despite the availability of new drugs to control diabetes, the prevalence of diabetic cardiomyopathy continues to rise. Thus, novel therapeutics directed at the etiology and pathophysiologies underlying diabetic cardiomyopathy are needed.Diabetes mellitus is an independent risk factor for heart failure and is and is characterized by dilated ventricles, hypertrophic cardiomyocytes, pronounced interstitial fibrosis, diastolic dysfunction, and impaired/preserved systolic function, which ultimately leads to heart failure6. Cathepsin K is the most potent mammalian cysteine protease with strong elastase and collagenase properties. Physiologically cathepsin K has been shown to mediate cellular protein turnover, collagen degradation, and the remodeling of the extracellular matrix7. In addition, increased expression and activity of cathepsin K has been reported in the hypertrophic and failing heart8. We have previously shown that knocking out cathepsin K in mice alleviates obesity and pressure overload–associated cardiac dysfunction in mice10. However, the explicit role of cathepsin K in diabetic cardiovascular complications or the potential mechanisms remains unknown. Accordingly, in this study, we hypothesized that cathepsin K knockout protects agains cardiac structural and functional alterations induced by diabetes. We also assessed the effect of deletion of cathepsin K on cardiomyocyte Ca2+ handling, oxidative stress, apoptosis and calcineurin/NFATs (nuclear factor of activated T-cells) signaling.Recent evidence suggests cathepsin K plays a significant role in the progression of cardiovascular diseases, as well as in the modulation of adiposity and glucose intoleranceAs shown in Table 2), carbon dioxide production (VCO2), and respiratory exchange ratio (RER = VCO2/VO2), under both light and light-dark cycles. Whereas cathepsin K knockout mice reconciled STZ-induced attenuation of VO2 and VCO2, which it did not alter RER    in isolated cardiomyocytes Fig. . HoweverFI) Fig. , and redit) Fig. , and an ion Fig. . In contTo evaluate the impact of genetically knocking out cathepsin K on myocardial histology in diabetic mice, cardiomyocyte cross-sectional area and cardiac fibrosis were examined  condition in the absence or presence of a cathepsin K inhibitor II (Cat K I-II). Protein levels for AKT, phosphorylation of AKT, apoptotic markers, as well as reactive oxygen species (ROS) were evaluated. Furthermore, we evaluated the contribution of NFAT pathway, which included a pharmacological inhibitor of calcineurin cyclosporine A (CsA) in these studies. Western blot results in Fig. 2. In addition, diabetes is associated with structural and functional changes within the heart. These adaptations result in diastolic and systolic dysfunction and oxidative damage ending in a decompensated state. Despite the availability of several therapeutic options to treat diabetes and cardiac dysfunction, the prevalence of diabetic cardiomyopathy is on the rise. Therefore, identifying and characterizing pharmacological agents to treat or alleviated diabetes-associated cardiac dysfunction therefore represents an important clinical need. To our knowledge, this is the first study to show that knocking out cathepsin K confers protection against diabetes-associated cardiac complications. Specifically, we showed that knocking out cathepsin K alleviates diabetes-induced hyperglycemia, alterations in energy metabolism, and “normalizes” cardiac structure and functional anomalies. Furthermore, cathepsin K deficiency suppressed diabetes-associated myocardial apoptosis, oxidative stress and calcineurin/NFATs signaling. In cultured cardiomyocytes, glucose toxicity perturbed intracellular Ca2+ homeostasis due to alterations of calcium-related proteins. These changes were reconciled by deletion of cathepsin K. At the molecular level, cathepsin K activated calcineurin, facilitating the nuclear translocation of transcription factors NFAT and subsequent apoptosis.Dilated cardiomyopathy is a cardiovascular complication in diabetic subjects, which eventually leads to heart failure10. However, in both these models, it is uncertain whether cathepsin K knockout has a direct effect on cardiac anomalies associated with overt hyperglycemia as the former is a model for insulin resistance whereas the latter is a model for pressure-overload. In this study, we treated the mice with streptozotocin, which results in the destruction of the pancreatic beta cells leading to type I diabetes. Streptozotocin-induced diabetic mice typically exhibit dilated cardiomyopathy, unlike the high-fat diet and abdominal aortic constriction models used previously, which results in hypertrophic cardiomyopathy. Previous studies have shown that inhibition of cathepsin K attenuates obesity-induced increase in serum glucose and insulin, and improves systemic glucose utilization12, which is confirmed by our current study wherein cathepsin K knockout mice had lower serum glucose levels compared to the wildtype mice following STZ injection. Metabolic analysis revealed that the volume of O2 consumption, volume of CO2 production, the total activity and heat generation were significantly diminished in STZ-challenged mice, and these effects were dramatically attenuated in cathepsin K knockout mice. Thus indicating that cathepsin K deletion may contribute to the augmentation of metabolism, which has been suggested in studies from the group of Funicello and coworkers11. Additionally, STZ-injection significantly reduced the body weight gain, and enlarged cardiomyocyte cross-sectional area, which were mitigated by cathepsin K knockout. Consistent with previous observations, in our studies, STZ-treatment resulted in enlarged ventricular chambers14. Genetic deletion of cathepsin K attenuated diabetes-associated changes in cardiac posterior wall thickness, increase in LVEDD and LVESD, as well as the reduction in fractional shortening and heart rate. This suggests there may be a protective role of knocking out cathepsin K activity on diabetes-induced cardiac changes. In addition, our data suggests cathepsin K has a critical role in cardiac pathology, as diabetic mice lacking cathepsin K did not develop abnormalities in cardiomyocyte contractility, intracellular Ca2+ homeostasis, and dysregulated calcium-related proteins such as SERCA2a and PLB. While glucose toxicity elevated the levels of cathepsin K and calcineurin A and the phosphorylation of its downstream effector NFATc3, which were attenuated in the cathepsin K knockout mice. The contribution of reductions in blood glucose (by cathepsin K knockout) to the beneficial cardiac profile in the diabetic mice cannot be ruled out. A limitation of the present study is that we did not have a conditional knockout to delineate the difference. Also, previous studies have shown that streptozotocin can have direct cardiotoxic effects when administered systemically, another limitation of our study is that we cannot fully rule out the contribution of cathepsin K knockout in countering this direct effect16.We had previously shown that genetic knockout of cathepsin K protects against high-fat diet and abdominal-aortic constriction-induced cardiac hypertrophy and dysfunction17. Interstitial fibrosis can lead to both structural and functional alterations of the heart. Therefore, proteases and antiproteases have been the target of extensive investigation in a bid to understand the progression of cardiac fibrosis and hence cardiovascular disease19. Among the proteases studied for their role in cardiac fibrosis are the matrix metalloproteinases (MMP2 and MMP9), plasminogen activator inhibitor-1, calpains, caspases and plasmin and their endogenous inhibitors20. In addition, cathepsin D has been shown to be play detrimental role in diabetic cardiomyopathy whereas cathepsin L has a protective effect, underscoring the unique role of each protease21. Our current study adds cathepsin K to the repertoire of proteases that play a critical role in regulating cardiac function, thus broadening the potential target for treating cardiovascular disease.Cardiac injury often triggers a fibrotic response which may be attributed to the activation of proteases found in cardiomyocytes, cardiac fibroblasts or enmeshed in the extracellular matrix22. Calcineurin has two subunits, a catalytic subunit, calcineurin A, and a regulatory subunit calcineurin B with four intrinsic Ca2+-binding sites. Calcineurin A has a catalytic domain and three regulatory domains including a calmodulin (CaM) binding domain, a calcineurin B binding domain and an autoinhibitory domain23. Conventionally, calcineurin is activated by increased intracellular Ca2+ concentration, which in turn triggers a Ca2+/CaM complex associated with calcineurin A through CaM binding domain and calcineurin B binding domain to expose the substrate binding cleft by releasing the autoinhibitory domain24. Our results showed higher levels of intracellular Ca2+ and calcineurin A together with an upregulation of cathepsin K in diabetic heart, which could potentially trigger CaM binding to its effector domain to increase the activity of calcineurin A25.Calcineurin is a serine/threonine-protein phosphatase that plays a pivotal role in eliciting hypertrophic responses by activating NFAT transcription factors and contributes to the development of dilated cardiomyopathy and heart failure27. Activation of calcineurin dephosphorylates cytosolic NFATs, which translocate to the nucleus leading to transcription of genes29. NFATc2, NFATc3 and NFATc4 (NFAT3) have been demonstrated as downstream targets of calcineurin that trigger hypertrophic signaling, especially NFATc332. NFATc1 (NFAT2) exhibits a critical role in ECM remodeling, development of heart valves and coronary vessels during cardiac maturation, and has been shown to be an essential regulator of cathepsin K by RANKL signaling35. Our data using cultured H9c2 cells suggest that cathepsin K regulates the calcineurin/NFATc3 cascade in the progression of cardiac remodeling. In addition, calcineurin also affects Ca2+ fluxes by mediating various Ca2-related proteins and channels in the heart, specifically, SERCA2a, RyR/Ca2+-release channels and the L-type Ca2+ channel, the alteration of which is important for initiating hypertrophic processes and cardiac remodeling36. Finally, Ca2+ disorder can also cause cardiomyocyte contractile dysfunction and contribute to the pathophysiology of heart failure37. In our studies, calcineurin inhibitor CsA attenuated high glucose mediated impairment of SERCA2a (data not shown), suggesting a crosstalk between calcineurin and Ca2+ regulation. A major shortcoming of our approach is that we have used H9c2 cells  rather than primary cultures for these signaling studies owing to the challenges in maintaining viable primary cells for over 24 h in culture.Inhibition of calcineurin/NFATs activity protects cardiomyocytes from hypertrophy by suppressing of hypertrophic genes10, and other cell types such as osteoclasts and lung fibroblasts38. However, the precise molecular mechanism by which cathepsin K induces apoptosis in the heart remains unclear. It was reported that high-levels of calcineurin activity is closely associated with ischemia and caspase-3-mediated apoptosis, which can be reduced by pre-incubation with calcineurin inhibitors FK506 or cyclosporine A39. Furthermore, overexpression of Bcl-2 suppresses activation of calcineurin/NFAT–mediated apoptosis40. Additionally, constitutive overexpression of truncated calcineurin A, which lacks an autoinhibitory domain, is capable to induce apoptosis in different cell types41.We also found that knocking out cathepsin K reversed high-glucose-induced upregulation of Bax and cleaved caspase-3, and prevented the alterations in Bcl-2, which is in agreement with earlier observations that cathepsin K is involved in modulating apoptotic signaling in cardiomyocytes2+ is an important contributor to cellular apoptosis42. Our in vivo and in vitro studies demonstrated that knocking out cathepsin K significantly attenuated STZ-enhanced calcineurin/NFAT signaling and reduced GSH/GSSG. Pharmacological inhibition of cathepsin K and calcineurin decreased high glucose-induced ROS generation, apoptosis, and alterations in phospho-AKT. These observations favor the notion that the targeting cathepsin K activity lowers glucose-induced oxidative stress and apoptosis which may be mediated by the remission of intracellular Ca2+ disturbance and restored calcineurin-induced AKT dephosphorylation, thus triggering a pro-survival mechanism. Despite the documented role of cathepsin K as a protease, we did not observe distinctive changes in cardiac fibrosis, collagen content and TGF-β in cathepsin K knockout mice. Therefore, our data suggests a potentially nontraditional role of this protease in mediating its beneficial effects. The increased activation of GSK3β through its dephosphorylation is consistent with the studies, which showed that AKT inhibits GSK3β43 and calcineurin dephosphorylates GSK3β at Ser944. The activated GSK3β further suppresses the glycogen synthesis from glucose45.Alteration of intracellular CaIn summary, these data suggest that knocking out cathepsin K deficiency mitigates diabetes-induced cardiac anomalies due to attenuation in calcineurin/NFAT signaling and subsequent reduction in cardiac oxidative stress and apoptosis. Thus, targeting cathepsin K may represent a novel strategy to treat or prevent diabetes-associated cardiac complications.Ctsk−/−) and C57BL/6 mice (wildtype) were subjected to intraperitoneal injections of streptozotocin  dissolved in 0.1 M sterile citrate buffer or vehicle for 2 consecutive days. Following 4-weeks, fasting blood glucose levels were monitored using a glucometer. Mice with fasting blood glucose levels > 13 mM were deemed diabetic47.All studies were performed in accordance to the relevant guidelines and was approved by the University of Wyoming’s Institutional Animal Care and Use Committee. Three-month-old male cathepsin K knockout mice , and end-systolic dimension (ESD) were measured. All measurements were done from leading edge to a leading edge in accordance with the Guidelines of the American Society of Echocardiography49. Heart rates were averaged over 10 cardiac cycles. The percentage of LV fractional shortening and LV mass (g) were calculated as [(EDD − ESD)/EDD] × 100 and 0.8(1.04[([LVEDD + IVSd + PWd]3 − LVEDD3)]) + 0.6 respectively. Normalized LV mass was calculated as LV mass (mg)/body weight (g)51.Cardiac geometry and function were evaluated in anesthetized mice using a two-dimensional guided M-mode echocardiography  equipped with a 15-6 MHz linear transducer as reported previously52. Four weeks after STZ-injection, WT and Ctsk−/− mice were individually placed in the CLAMS metabolic cages with 100 ml water and ad libitum access to food. Following acclimation, metabolic parameters including volume of carbon dioxide produced (VCO2), the volume of oxygen consumed (VO2), the respiratory exchange ratio (RER = VCO2/VO2), the caloric (heat) value and physical activity  were determined. All the parameters were measured every 15 min for 10 hours during daytime and 10 hours during nighttime. Results that were recorded in the first and last 2-hours were not used.Metabolic measurements were made using the Comprehensive Laboratory Animal Monitoring System 53. After ketamine/xylazine sedation, hearts were removed and perfused with Ca2+-free HEPES-buffered Tyrode’s solution containing (in mM): NaCl 120, KCl 15, KH2PO4 0.6, Na2HPO4 0.6, NaHCO3 4.6, MgSO4 1.2, HEPES 10, taurine 30, glucose 10, butanedione monoxime 10 at pH 7.4, and gassed with 95% oxygen and 5% carbon dioxide. Hearts were digested with 1 mg Liberase TH  in 20 ml perfusion buffer for 10–15 min to harvest the cells. Only rod-shaped myocytes with clear edges were selected for mechanical study.Murine cardiomyocytes were isolated as described previously® system . Cardiomyocytes were placed in a chamber mounted on the stage of an inverted microscope (Olympus IX-70) and superfused with a HEPES buffer containing 1 mM CaCl2. Myocytes were field stimulated with suprathreshold voltage at a frequency 0.5 Hz using a pair of platinum wires placed on opposite sides of the chamber connected to a FHC stimulator . IonOptix SoftEdge software was utilized to capture cell shortening and relengthening changes. Cell shortening and relengthening were assessed using the following indices: resting cell length, peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), and maximal velocity of shortening/relengthening (±dL/dt)54.Mechanical properties of cardiomyocytes were evaluated using a SoftEdge Myocam2+ clearance54.Cardiomyocytes were loaded with fura-2/AM (0.5 μM) for 15 min, and fluorescence intensity was recorded with a dual-excitation fluorescence photomultiplier system (IonOptix). Cardiomyocytes were placed onto an Olympus IX-70 inverted microscope and were imaged through a Fluor × 40 oil objective. Cells were exposed to light excited by a 75 W lamp and passed through either a 360 or a 380 nm filter, while being stimulated to contract at a frequency of 0.5 Hz. Fluorescence emissions were detected between 480–520 nm and qualitative change in fura-2 fluorescence intensity (FFI) was inferred from the FFI ratio at the two wavelengths (360/380 nm). Fluorescence decay time  was calculated as an indicator of intracellular Ca55. Slides were then washed with PBS and mounted with Fluoromount-G mounting media . Cardiomyocyte cross-sectional area was measured and quantified from 90 random cardiomyocytes by using a digital microscope (×400) and the Image J (version1.39 u) software. The Masson’s trichrome staining was used to detect fibrosis in heart sections using a Masson’s Trichrome stain kit56.Following anesthesia, hearts were excised and immediately placed in 10% neutral-buffered formalin at room temperature for 24 h. White adipose tissues were collected from both sides of their lower abdomen and the weight was determined. Following sectioning, heart tissues were dehydrated through serial alcohols and cleared in xylenes. The specimen was embedded in the paraffin and cut into 5μm sections. Slides were deparaffinized, washed once in PBS, and stained with 0.1 mg/ml Lectin-FITC conjugate  for 2 h at room temperature in the dark53.Tissue samples (~50 mg) were sonicated in picric acid and centrifuged at 13,500 × g for 20 min. One aliquot of the supernatant was used to directly measure total GSH assay and the other for GSSG. 100 μl of supernatant fractions were treated with 2 μl vinyl pyridine to scavenge GSH for the GSSG determination. The GSSG was then subtracted from the total glutathione to evaluate the GSH levels. GSH was determined by the DTNB-glutathione reductase recycling mechanismCardiac collagen type I level was measured using a mouse Type I Collagen Detection Kit  per manufacturer’s specifications.2DCFDA) 57. H9c2 cells were grown in a clear bottom and black side 96-well plate in the Dulbecco’s Modified Eagle Medium (DMEM) and incubated for 72 h with media containing high glucose (25 mM) with or without cathepsin K inhibitor-II  or calcineurin inhibitor cyclosporine A , and loaded with 10 µM CM-H2DCFDA in the culture medium for 30 min at 37 °C in the dark. DCF fluorescence was measured using fluorescent plate reader  with excitation wavelength at 485 nm and emission wavelength at 530 nm.Intracellular ROS levels were evaluated by measuring the changes in fluorescence intensity resulting from intracellular probe oxidation, using a membrane-permeable probe 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate , anti-phospho-phospholamban , anti-SERCA2a , anti-calcineurin A , anti-NFATc1 (1:250), anti-phospho-NFATc3 (1:250), anti-glycogen synthase kinase 3 beta , anti-phospho-GSK3β , anti-AKT , anti-phospho-AKT , anti-transforming growth factor-β , anti-cleaved caspase-3 (1:500), anti-Bcl-2 (1:1000), anti-Bax , anti-GAPDH  and anti-α-tubulin  antibodies. Blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000). Antigens were detected by the luminescence method. Band densities were determined using Quantity One software Data are presented as mean ± SEM. Statistical significance (p < 0.05) for each variable was estimated by a one-way analysis of variance (ANOVA) followed by a Turkey’s post-hoc analysis.The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.Supplementary Figure 1.


Assessment of activities of daily living (ADL) is paramount to determine impairment in everyday functioning and to ensure accurate early diagnosis of neurocognitive disorders. Unfortunately, most common ADL tools are limited in their use in a diagnostic process. This study developed a new evaluation by adopting the items of the Katz Index (basic [b-] ADL) and Lawton Scale , defining them with the terminology of the International Classification of Human Functioning, Disability and Health (ICF), adding the scoring system of the ICF, and adding the possibility to identify underlying causes of limitations in ADL.n = 79), mild cognitive impairment (MCI) (n = 73), and Alzheimer disease (AD) (n = 71) underwent a diagnostic procedure for neurocognitive disorders at the geriatric day hospital of the Universitair Ziekenhuis Brussel . Additionally, the ICF-based evaluation for b- and i-ADL was carried out. A global disability index (DI), a cognitive DI (CDI), and a physical DI (PDI) were calculated. The i-ADL-CDI showed high accuracy and higher discriminative power than the Lawton Scale in differentiating HC and MCI , MCI and AD , and HC and AD . The b-ADL-DI showed significantly better discriminative accuracy than the Katz Index in differentiating HC and AD . This study was conducted in a clinically relevant sample. However, heterogeneity between HC, MCI, and AD and the use of different methods of reporting ADL might limit this study.The construct validity, interrater reliability, and discriminative validity of this new evaluation were determined. From 2015 until 2016, older persons (65–93 y) with normal cognitive ageing   is seen as a transitional zone between normal aging and dementia.Assessment of activities of daily living (ADL) is paramount to underpin accurate diagnostic classification in MCI and dementia.Unfortunately, most common report-based ADL tools have limitations for diagnostic purposes.We set out to develop and validate a new tool to evaluate basic  and instrumental  ADL in an older population with cognitive disorders.We developed a tool based on the framework of the International Classification of Functioning, Disability and Health (ICF).A global disability index (DI), a Cognitive DI (CDI) (a disability index taking into account solely activities impaired because of cognitive reasons), and a Physical DI (PDI)  were calculated for both b- and i-ADL, based on the number of activities performed and the severity and causes of the functional problem.n = 79), (2) patients with MCI (n = 73), or (3) mild to moderate dementia (n = 71) were included and underwent the new ICF-based evaluation of b- and i-ADL.223 community-dwelling older persons diagnosed as (1) cognitively healthy participants , MCI, and AD, and the b-ADL-DI showed high discriminative accuracy in differentiating HC and AD.The new ICF-based evaluation of b- and i-ADL showed a good ability to distinguish normal and pathological cognitive aging and might enable the diagnosis of MCI and mild dementia.The discriminative power of the ICF-based evaluation of b- and i-ADL for underlying causes of limitations is an advantage.Since an evaluation of ADL is experienced as less invasive by older persons, this evaluation offers directions for clinical use in diagnosing cognitive disorders and might offer possibilities for clinical treatment, rehabilitation, advising, and coaching.This study was conducted in a clinically relevant sample at a geriatric day hospital. However, heterogeneity in the participants might limit this study. Health services are dealing with an increasing number of older patients. Although most seniors are in reasonably good health and living an active life, a considerable number of them are at risk of developing major chronic conditions and mental disorders such as dementia. Worldwide, it is estimated that dementia affects 46.8 million persons, which causes great stress to medical, social, and informal care ,2. SeverIn clinical practice, information about ADL is most commonly ascertained by asking a patient or his or her caregiver to report about the everyday functioning . Report-Over the years, multiple report-based ADL scales have been developed in order to contribute to the early diagnosis of NCD . Tools sTo address the concerns of report-based ADL scales, performance-based scales such as the Assessment of Motor and Process Skills ,52 and tCurrently, the most commonly used tools for assessing b- and i-ADL are respectively the Katz Index  and the Therefore, this study set out to develop a new tool to evaluate b- and i-ADL for diagnostic purposes in a geriatric population with NCD. This evaluation is based on the International Classification of Functioning, Disability and Health (ICF) developed by the World Health Organization (WHO) . The ICFThe study protocol was based on the STARD criteria, developed to improve the completeness and transparency of reporting of studies of diagnostic accuracy .The Ethical Committee of the Universitair Ziekenhuis Brussel approved this study (B.U.N. 143201523678). All data were collected in accordance with the ICH-GCP guidance and the declaration of Helsinki. All participants and informants gave written informed consents.Three groups of community-dwelling older persons (≥65 y) were recruited consecutively through the geriatric day hospital of an academic teaching hospital : (1) HC, (2) patients with MCI, and (3) with Alzheimer disease (AD). Patients with MCI and AD underwent a procedure for the diagnosis of cognitive disorders that was performed by a multidisciplinary team and is considered as good clinical practice . This prn = 79) were a history of NCD, a score <26/30 on the MMSE, and any self- or informant-based complaint of functional or cognitive deficits, which were suggestive of MCI or AD. Exclusion criteria were a score of <80/105 on the total CamCog score, <18/27 on the memory section of the CamCog score, and <8/12 on the MIS and VAT ) see . FurtherExample (for b-ADL): A person previously performed 6 b-ADL (TNA = 6). ICFScore 0 is assigned to four activities, ICFScore 1 is assigned to one activity because of cognitive problems, and ICFScore 3 is assigned to one activity because of physical factors. This person has two limited activities, and the maximal disability is 24 (TNA*4). His b-ADL-DI is 16.6% , the b-ADL-CDI is 4.2% (only the activities limited because of cognitive reasons are taken into account), and the b-ADL-PDI is 12.4% .p < 0.05 for all analysis. Demographic and clinical characteristics  and the MMSE, Katz Index, Lawton Scale were evaluated between groups by one-way ANOVA with Bonferonni post hoc tests or chi-square analysis. The construct validity was checked, in absence of a true golden standard, by determining the new evaluation’s ability to distinguish between HC, MCI, and AD. We hypothesised HC would show less disability than MCI and the latter less than AD, and that the CDI for both b- and i-ADL would differ more than DI and PDI. The indices for b- and i-ADL were compared across the groups using analysis of covariance (ANCOVA) in which age, number of used medications, number of comorbidities, level of education, and gender were included in the model as covariates. Secondly, in checking the construct validity, we calculated correlations between the indices and the MMSE. We hypothesised that (1) the CDI for both b- and i-ADL would show higher correlations with the MMSE than the DI and PDI since the CDI expresses solely deficits caused by cognitive disorders and (2) the i-ADL-CDI would show higher correlations than b-ADL-CDI because performing i-ADL is more vulnerable to cognitive disorders. Correlation analyses were performed using Pearson correlation between the MMSE, Katz Index, Lawton Scale, and the indices. To interpret the correlations, the guideline by Evans (1996) . As illustrated in p < .001; F = 88.9, p < .001). The i-ADL-CDI had a moderate correlation with the MMSE . The i-ADL-PDI expressed no significant differences between the groups and had a weak correlation with the MMSE .For i-ADL, participants performed an average of eight activities . There were no significant differences between the groups on TACT i-ADL [F = 0.01, p = < .05) (p < .001). The b-ADL-PDI showed significantly more severe deficits in AD than HC  = 3.9, p = .016). No significant differences were found between HC and MCI or MCI and AD. All indices of b-ADL had a weak correlation with the MMSE .For b-ADL, participants always performed all activities. Persons with AD and MCI expressed significantly more severe deficits in b-ADL-DI than HC  = 12.6, = < .05) . The b-An = 25) was excellent for b-ADL-DI ; b-ADL-CDI ; b-ADL-PDI ; i-ADL-DI ; i-ADL-CDI ; and i-ADL-PDI  . No significant differences between raters were observed.The interrater reliability (p < .05). The i-ADL-CDI showed best accuracy, expressed by AUCs ranging from 0.805 to 0.968 and a significantly higher discriminative power than the Lawton Scale with DeLong’s test . The i-ADL-PDI did not show a better accuracy than the Lawton Scale.The AUC of i-ADL-DI ranges from 0.736 to 0.968 and has a significantly better discriminative accuracy than the Lawton Scale for differentiating between HC and MCI and between HC and AD with DeLong’s test  a significantly better discriminative accuracy than the Katz Index in differentiating between HC and AD with an AUC of 0.828. The b-ADL-CDI and b-ADL-PDI showed no better diagnostic accuracy.The AUC of b-ADL-DI showed with DeLong’s test  adopting the activities of the Katz Index and Lawton Scale and linking them to the definitions and codes of the ICF, (2) by developing a scoring system based on the performance qualifiers of the ICF, and (3) by adding the possibility to take causes of limitations in performance into account. This new evaluation takes the person as his or her own reference. By doing so, it is possible to compute a set of indices. This study determined the construct validity, discriminative validity, and interrater reliability of this new evaluation in a geriatric population.The new evaluation showed more accuracy in evaluating b- and i-ADL compared to the Katz Index and Lawton scale and subsequently has the potential to improve diagnostic differentiation between HC, mild NCD , and major NCD . As hypothesised, this evaluation followed the hierarchical continuum of functional decline ; b-ADL-DUntil now, self- and informant-report scales did not show sound psychometrical properties ,43 and wAlthough many studies have already tried to improve the use of the Katz Index and the Lawton Scale, not all improvements were relevant for the diagnosis of cognitive disorders. In this study, we attempted to achieve more clarity, transparency, and nuance by maintaining the activities of the Katz Index and the Lawton Scale but by adopting the terminology and the scoring system of the ICF. A first advantage is that each activity is clearly defined by definitions according to the ICF. In contrast to the content of the original Katz Index and Lawton Scale—which varies depending on setting and circumstances ,95—the IAlthough the results of our study are promising and may imply a change in the evaluation of everyday functioning in clinical practice, some considerations need to be made. First, a measurement bias might have occurred by using different methods of reporting ADL in HC and patients with MCI and AD. Although a report-based method has the clinical advantages of being easy to obtain, minimally disturbing, and of low cost, proxy and patient-based measures can be biased by mood status, social desirability, diminished awareness, denial, and other cognitive deficits ,97. But As a result, the contrast between groups might be larger than would be expected in a clear clinical sample. However, in the statistical analysis, our data was controlled for possible confounders such as age, medication use, number of comorbidities, level of education, and gender. Lastly, another consideration is that HC were—apparently—cognitively healthy persons. Yet, it is still possible that mild cognitive problems were present in some of them. However, we used strict cutoffs of MMSE—which can be considered as a valuable instrument for cognitive screening—in order to rule out cognitive deficits.Based on the results of this study, we argue that this evaluation can contribute to the diagnostic differentiation between cognitively healthy ageing, mild NCD , and major NCD . Particularly, the i-ADL-CDI might be useful. Since it is likely that decline in everyday functioning occurs over time, and this change leads to a conversion from mild to major NCD, further research—a longitudinal prospective follow up study—should address the predictive validity of this evaluation as follow-up assessment .In conclusion, this new ICF-based evaluation for b- and i-ADL addresses important issues in assessing everyday functioning by (1) providing an operationalization of the evaluated activities by ICF codes and definitions, (2) providing a detailed scoring system that is based on the ICF qualifiers, and (3) by making a differentiation in causes of limitations. With validation in longitudinal prospective cohorts, this evaluation might offer a useful addition to the common diagnostic process and be of added value in a multidisciplinary approach with established cognitive and mood measures and biomarkers.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S1 STARD Checklist(DOCX)Click here for additional data file.


Erwinia amylovora is a Gram-negative bacterial plant pathogen in the family Enterobacteriaceae and is the causal agent of fire blight, a devastating disease of apple and pear. Fire blight is traditionally managed by the application of the antibiotic streptomycin during bloom, but this strategy has been challenged by the development and spread of streptomycin resistance. Thus, there is an urgent need for effective, specific, and sustainable control alternatives for fire blight. Antisense antimicrobials are oligomers of nucleic acid homologs with antisense sequence of essential genes in bacteria. The binding of these molecules to the mRNA of essential genes can result in translational repression and antimicrobial effect. Here, we explored the possibility of developing antisense antimicrobials against E. amylovora and using these compounds in fire blight control. We determined that a 10-nucleotide oligomer of peptide nucleic acid (PNA) targeting the start codon region of an essential gene acpP is able to cause complete growth inhibition of E. amylovora. We found that conjugation of cell penetrating peptide (CPP) to PNA is essential for the antimicrobial effect, with CPP1 [(KFF)3K] being the most effective against E. amylovora. The minimal inhibitory concentration (MIC) of anti-acpP-CPP1 (2.5 μM) is comparable to the MIC of streptomycin (2 μM). Examination of the antimicrobial mechanisms demonstrated that anti-acpP-CPP1 caused dose-dependent reduction of acpP mRNA in E. amylovora upon treatment and resulted in cell death . Anti-acpP-CPP1 (100 μM) is able to effectively limit the pathogen growth on stigmas of apple flowers, although less effective than streptomycin. Finally, unlike streptomycin that does not display any specificity in inhibiting pathogen growth, anti-acpP-CPP1 has more specific antimicrobial effect against E. amylovora. In summary, we demonstrated that PNA–CPP can cause an effective, specific antimicrobial effect against E. amylovora and may provide the basis for a novel approach for fire blight control. Erwinia amylovora, is one of the most serious diseases of apple and pear in the United States and worldwide. Fire blight infection can occur in flowers, leaves, shoots, and fruits, resulting in yield reduction; the pathogen also can spread systemically through trees to the rootstock, ultimately resulting in tree death , mechanisms of antimicrobial activity, and documented effectiveness of PNA–CPP in limiting pathogen growth on detached apple flowers.Although PNA–CPPs have shown some promising applications in controlling bacterial infections in animal models, to our knowledge, no research has explored the use of PNA–CPP in controlling plant diseases. Regulatory small RNAs (sRNAs) play important roles in modulating gene expressions in bacteria . We prevunctions . These fE. amylovora Ea110, which was isolated from an apple orchard in Michigan  for 20 h. During the incubation, the OD 600 of each well was measured every 10 min. The lowest concentration of a PNA–CPP that prevented growth after 20 h represented the MIC. Three replicates were included in each testing, and the experiment was repeated twice.E. amylovora Ea110 were adjusted to 5 × 105 CFU / ml in LB broth. Forty microliters of anti-acpP-CPP1 (100 μM), streptomycin (100 μM), or H2O were added into 460 μl of the LB broth containing E. amylovora cells to reach a final concentration of 8 μM of the compounds mentioned above. The cultures were incubated at 28°C with 200 rpm of continuous shaking. Samples were taken at 0, 1, 2, 3, 4, 5, and 6 hours post inoculation. Bacterial cells from each sample were collected by centrifugation (6500 rpm for 8 min), washed with sterile water to remove the residual compounds, diluted 102 to 105-fold, and plated on LB agar plates. Colonies formed after 48 hour-incubation at 28°C are counted and original cell concentration was calculated. Five replicates were included in each assay and the experiment was repeated twice with similar results observed.Overnight cultures of Bacterial cells were washed with sterile water to remove residual medium from the culture. LIVE/DEAD BacLight bacterial viability kit  components A and B were added to the cells following the manufacturer’s instructions. Green fluorescence and red fluorescence of cells stained were observed using a Zeiss Scope A1 fluorescence microscope equipped with a FITC filter and a DAPI filter . Images were acquired with a Spot RT3 camera using the Spot Advanced software . Statistical analysis was performed using ANOVA with α = 0.05. The level of significance labeled in figure (denoted by different letters) was calculated using LSD (α = 0.05). ANOVA and LSD analyses were conducted in R ver. 3.3.1 .Erwinia amylovora Ea110 was inoculated into LB broth at the final concentration of 5 × 105 CFU/ml, and was treated with various concentrations of PNA–CPP, water, or streptomycin. The inoculated cells were incubated at 28°C with orbital shaking, and were collected for RNA isolation at 15.5 h post inoculation. Total bacterial RNA was isolated using the RNeasy Protect Bacteria Mini Kit  following the manufacturer’s instructions. The quality and quantity of RNA was measured with a Nanodrop 2000c .acpP detection in Northern blots was synthesized from polymerase chain reaction (PCR) using acpP primers (forward primer 5′-TGG GCG TTA AGC AGG AAG AAG-3′ and reverse primer 5′-TAC GCC TGG TGA CCA TTG AT-3′). The PCR product was purified using a QIAquick PCR Purification kit (Qiagen) and labeled using a Biotin DecaLabel DNA Labeling Kit  as per instruction of kit manual. Thirty micrograms of total RNA from different treatments were loaded to a formaldehyde denaturing gel in MOPS buffer. 16s rRNA was quantified using a transluminator as internal control of RNA quantity. BrightStar®-Plus positively charged nylon membranes (Thermo Fisher Scientific) were used for transfer of RNA from gel to membrane. Transfer, pre-hybridization, and hybridization were performed following the manufacturer’s instructions of the Northern Max kit (Thermo Fisher Scientific). The biotin signal was detected using a Biotin Chromogenic Detection Kit (Thermo Fisher Scientific). The band intensity was quantified by ImageJ  in May 2016. The pedicel of the flowers was detached from the flower cluster, and each flower was placed in a 7 ml plastic tube (containing 10% sucrose solution) through a hole created in the plastic cap. One microliter of E. amylovora (Ea110) at the concentration of 107 CFU/ml was inoculated onto the five stigmas of each flower (approximately 0.2 μl per stigma). Twenty microliters of the PNA–CPP at different concentrations were evenly applied onto the stigmas of each flower 2 h before and 19 h after the inoculation. Water and streptomycin (100 μM) were used as negative and positive controls. In the PNA–bacteria mix treatment, PNA at the concentration 20 μM was mixed with an equal volume of E. amylovora cells at the concentration of 107 CFU/ml in a microcentrifuge tube and incubated for 30 min at 22–25°C. Two microliters of the mixture were applied evenly onto the five stigmas of the flowers. The pathogen population on the apple stigmas was quantified at 42 h post inoculation, by dissecting the stigmas from the flowers, and suspending them in 1 ml of 0.5x PBS. A Taqman probe realtime PCR assay was used to quantify the pathogen amount. Primers used in this assay are: amsk120F: 5′-CAT GCA ATT TCC AGT TTC CT-3′; amsk120R: 5′-GCA TGA CGG TTA ACC AAA TC-3′, amsk120Probe: 5′-TGC GTG ACC TGA TTC AGC ACA A-3′. The reaction was performed on a Biorad CFX96 Realtime PCR machine. The cell concentrations were calculated by f (Cq) = –3.47 Cq + 45.513. A standard curve was generated using five predetermined concentrations of Ea110  cells. Six flowers were used in each treatment. The experiment was repeated twice with similar results observed, and results from one representative experiment are displayed. The box plot was generated by R ver. 3.3.1  and plated on LB agar medium and incubated at 28°C for 48 h. On the basis of morphological colony characteristics, 200 bacterial colonies were shortlisted, sub-cultured, and identified using16s rRNA sequencing using the forward primer 63f (5′-CAG GCC TAA CAC ATG CAA GTC-3′) and reverse primer 1387r (5′-GGG CGG WGT GTA CAA GGC-3′). Fifteen different species were selected from the 200 bacterial cultures for testing against anti-acpP-CPP1 and streptomycin. Biological control agents Pseudomonas fluorescence A506, Bacillus amyloliquefaciens D747 and Pantoea agglomerans E325 were isolated from the original products on LB agar plate.Fifty flowers were collected from apple trees E. amylovora, we first synthesized a 10-nucleotide PNA oligomer with antisense sequence to the start codon region (–5 to +5) of a previously discovered essential gene acpP (encoding an acyl carrier protein), anti-acpP PNA (Table 1) .To explore the possibility of using PNAs as antimicrobials against the fire blight pathogen Table 1) . The effacpP PNA on E. amylovora growth could be due to the inefficient entry of PNA into the cytoplasm, we determined whether conjugation of the anti-acpP PNA with CPP would result in enhanced antimicrobial effect. Three formulations of CPP  that were demonstrated to have promising delivery efficiency of PNAs in animal pathogenic bacteria were then individually conjugated to the anti-acpP PNA, resulting in anti-acpP-CPP1, anti-acpP-CPP2, and anti-acpP-CPP3 (Table 1). The effect of the CPP-conjugated PNAs on E. amylovora growth was tested under the same conditions. Compared to the water control, addition of anti-acpP-CPP1 or anti-acpP-CPP2 both resulted in complete growth inhibition of E. amylovora at the 20 μM concentration, whereas addition of anti-acpP-CPP3 did not cause any growth inhibition at any concentrations . The growth inhibition caused by anti-acpP-CPP1 was more potent than the inhibition caused by anti-acpP-CPP2, suggesting that CPP1 [(KFF)3K] was the most efficient CPP in delivering PNA into E. amylovora. To determine if the growth inhibition caused by PNA–CPP was through specific targeting of acpP, a PNA with random nucleotide sequence in conjugation with CPP1 (nontarget PNA–CPP1) was also tested (Table 1). The nontarget-CPP1 did not cause any inhibition of E. amylovora growth .As the ineffectiveness of the anti-acpP-CPP1 and streptomycin was 2.5 and 2 μM, respectively . In addition, we showed that the growth inhibition caused by anti-acpP-CPP1 is positively correlated with the concentrations of anti-acpP-CPP1, in a linear manner . Taken together, these results suggest that CPP-conjugated PNA with antisense sequence targeting the start codon of acpP is able to cause potent growth inhibition of E. amylovora under in vitro conditions. This inhibition was caused by the antisense sequence of acpP on PNA. CPP was essential in delivering PNA into E. amylovora and causing the antimicrobial effect. Among the three CPPs previously used in animal pathogens, CPP1 was the most efficient for PNA delivery into E. amylovora.We further determined that the minimal inhibitory concentration (MIC) of anti-acpP mRNA in E. amylovora cells treated with water, anti-acpP-CPP1 at two sub-lethal concentrations (1 and 2 μM), and streptomycin at sub-lethal concentrations (1 and 3 μM) using Northern blot. Compared to the water-treated cells, cells treated with anti-acpP-CPP1 showed a visual reduction in the level of acpP mRNA. This reduction is positively correlated with anti-acpP-CPP1 concentrations . In contrast, streptomycin treatment did not affect the acpP mRNA abundance. These observations suggest that anti-acpP-CPP1 binding to acpP mRNA caused the degradation of acpP mRNA. It also indicates that the growth inhibition caused by anti-acpP-CPP1 is through a gene specific interaction with acpP mRNA.After entering the bacterial cytoplasm, PNA–CPP is believed to bind to the start codon region of the target mRNA and block its translation . HoweverE. amylovora by anti-acpP-CPP1 was bactericidal or bacteriostatic, we tested the viability of E. amylovora cells in LB broth at different time points after treatment of anti-acpP-CPP1 (8 μM), streptomycin (8 μM), or water, using a serial dilution plating method. In the water-treated sample, we observed an exponential increase in the amount of viable cells after inoculation . Following treatment with anti-acpP-CPP1, the viable cell count remained the same during the first 3 h, displaying a bacteriostatic effect . After 3 h, the viability of cells treated with anti-acpP-CPP1 drastically declined, which suggested that anti-acpP-CPP1 caused a bactericidal effect during this period. After 6 h of treatment with anti-acpP-CPP1, the number of viable cells was reduced by more than 1000-fold , at a slightly faster rate than streptomycin. These observations suggest that the growth inhibition of E. amylovora caused by anti-acpP-CPP1 is bactericidal, similar to the traditional antibiotic streptomycin.To determine whether the growth inhibition of acpP-CPP1 was also studied using a bacterial viability kit and fluorescence microscopy. At 6 h after inoculation, cells treated with water were green fluorescent but not red fluorescent when stained with a mixture of fluorescent dye SYTO 9 and propidium iodide, suggesting that these cells were viable . However, a large number of cells treated with anti-acpP-CPP1 at a concentration above the MIC (8 μM) were both green fluorescent and red fluorescent, suggesting that many of these cells lost viability . Interestingly, cells treated with anti-acpP-CPP1 showed an elongated cell shape compared to the water-treated cells . No cell lysis  was observed following treatment with anti-acpP-CPP1, which suggests that the bactericidal effect may not be caused by physical disintegration of the cell structure . The fluorescence microscopy observation is consistent with the plating method, together suggesting that the growth inhibition of E. amylovora caused by anti-acpP-CPP1 is bactericidal.The mechanism of the growth inhibition caused by anti-acpP-CPP1 has potent growth inhibition of E. amylovora under in vitro conditions, we further evaluated the effectiveness of anti-acpP-CPP1 in inhibiting E. amylovora growth on apple stigmas using a detached flower assay. Compared to the water-treated control, stigmas treated with 100 μM anti-acpP-CPP1 showed a significant reduction of E. amylovora population . However, this reduction was less potent in comparison to the reduction by streptomycin . At lower concentrations, anti-acpP-CPP1 treatment did not result in significant reduction of the pathogen population when directly applied to the apple stigmas . However, premixing anti-acpP-CPP1 (20 μM) with E. amylovora cells for 30 min before applying the cell-compound mixture onto the apple stigmas resulted in excellent reductions of pathogen populations on apple stigmas . The results above suggest that anti-acpP-CPP1 is able to inhibit pathogen growth on apple stigmas when applied at 100 μM. It also suggests that the efficacy of anti-acpP-CPP1 may be improved if optimal cell-compound contact can be established.Following demonstration that anti-acpP-CPP1 affects the growth of environmental microbes associated with apple flowers, bacteria were first cultured from freshly collected ‘McIntosh’ apple flowers. Two hundred bacterial colonies were subcultured, and the identities of these colonies were determined by 16s sequencing. The sequencing and Blast analyses suggest that these bacteria belong to 15 different species (Table 2). Representative strains from each species were tested for their susceptibility to anti-acpP-CPP1 and streptomycin at concentrations above the MICs for E. amylovora . Our results suggest that anti-acpP-CPP1 did not affect the growth of most species (12/15), such as Pseudomonas ssp., Bacillus muralis, Curtobacterium plantarum, and multiple species in the Pantoea genus. However, three species, P. ananatis, P. agglomerans, and Lysobacter oligotrophicus displayed susceptibility to anti-acpP-CPP1 similar to E. amylovora. Comparison of the start codon sequences of acpP in species with genomes available in NCBI revealed that many species contain sequence discrepancies with E. amylovora in this region, although some other species contain similar sequences to E. amylovora . P. agglomerans that has an identical sequence as E. amylovora was also susceptible to anti-acpP-CPP1. In addition to the bacterial species isolated from the apple flower, we also tested the susceptibility of three microorganisms from fire blight biological control products to anti-acpP-CPP1. Similar to the environmental isolates, Pseudomonas fluorescens A506 and Bacillus amyloliquefaciens D747 (active ingredients of BlightBan A506 and Double Nickel) were not affected by anti-acpP-CPP1 whereas P. agglomerans E325  was susceptible (Table 2). All strains tested are susceptible to streptomycin with the exception of P. fluorescens A506 (Table 2). Finally, some other plant and animal pathogens, such as Dickeya dadantii and Pectobacterium carotovorum are susceptible to anti-acpP-CPP1 (Table 2) and further examination of their sequences suggests they have similar sequences targeted by anti-acpP-CPP1 as E. amylovora.Compared to the traditional antibiotics that kill microbes with little selection, antisense antimicrobials inhibit bacterial growth through a sequence specific manner. To determine whether anti-E. amylovora. We showed that a 10-bp oligomer of PNA containing antisense sequence of the essential gene acpP is able to cause complete growth inhibition of E. amylovora at a similar efficacy of streptomycin under in vivo conditions. Although there are two additional sites on the chromosome (in hrcU and EAM_0144) that could be potentially bound by the 10-bp oligomer PNA, hrcU, and EAM_0144 (encoding a T3SS protein and a fatty acid synthesis regulator) are not known as essential genes in bacteria and reduction of acpP mRNA was observed upon treatment of anti-acpP-CPP1 , which suggests that the growth inhibition of anti-acpP-CPP1 should be through the interaction with acpP mRNA. We also showed that conjugation of the PNA with a CPP is essential for the PNA to exert antimicrobial effect in E. amylovora and identified the most effective CPP sequence for E. amylovora. We provided evidence that the antimicrobial effect observed is bactericidal. And finally, no phytotoxicity  was observed in flowers treated with PNA–CPP molecules only during the flower assay. These qualities suggest that PNA–CPPs are good alternatives for streptomycin in future fire blight management. Conditions generated from this research will add to our knowledge base for the practical development of formulations, rates, and timing for future applications, although many other factors, such as the toxicity and the stability of PNA–CPP in the natural environment, need to be determined before application.In this study, we demonstrated the proof-of-concept of using PNA–CPPs as antimicrobials against the fire blight pathogen acpP-CPP1 has a potent antimicrobial effect against E. amylovora with higher strain specificity than traditional antibiotics. We hypothesize that the antisense antimicrobials may also have good sustainability: if resistance is developed through mutations, the mutated nucleotide(s) could be identified by sequencing, and new antisense molecules could be synthesized to target the mutated sequence. Some bacteria may be able to generate resistance by acquiring multi-drug transporters. However, it is not very likely in this case as previous work demonstrated that PNA–CPPs cannot be readily transported out of the cells due to the large molecular weight (E. amylovora may provide more potent and robust growth inhibition, although further actions need to be taken to enhance the entry of PNA–CPP molecules into bacterial cells. Currently, the biggest limitation of agricultural application of PNA–CPP is the cost. With the current synthesis method, the cost of PNA–CPP is about $1000USD/μMol. Thus, research in developing new synthesis technologies and improving the synthesis efficacy is essential for future large-scale application of PNA–CPP.Considerations that are often taken into account when developing antimicrobials include effectiveness, specificity, sustainability, and cost. We demonstrated that anti-r weight . FurtherPrior to this research, the use of PNA–CPPs in controlling bacterial infections has been explored in human medicine. Compared to human medicine application, there are essential differences in agricultural use. First, the delivery method and locations where the antimicrobial activity occurs are different in the two systems. Antimicrobials for human medical purposes are usually delivered through the circulation system and the antimicrobial activity occurs mostly internally in the human body. For agricultural use, the delivery is mostly achieved through aerial spray to the plant surface, and the antimicrobial activity occurs mostly externally on the plant surface. Second, non-pathogenic, beneficial bacteria are not readily used as a disease control strategy in human medicine, but the beneficial, pathogen-antagonistic microbes are of great use in plant disease management as biological controls. This emphasizes a need of selectively targeting the pathogen without affecting the non-pathogenic, beneficial bacteria in agricultural settings.With the differences between the two systems, our study demonstrated that the application of a PNA–CPP can reduce the bacterial pathogen population on plant surfaces. Our results also emphasized the importance of ensuring the compound-bacteria contact for the optimization of antimicrobial results, as we showed that premixing bacteria with PNA–CPP ensured the full bacteria-compound contact and had the most potent antimicrobial effect. It is possible that the use of a nonionic surfactant may be able to improve the antimicrobial effect of PNA–CPP on plant surfaces. In addition, whether the reduction in pathogen population on stigmas would also result in the reduction in blossom blight infection incidence needs to be further evaluated in future orchard trials. Finally, the stability of PNA–CPP in natural environment, when exposed to UV radiation, rain, and temperature changes, is not known. Understanding the stability will help us formulate the product rates, and timing of sprays to achieve the optimal control efficacy in the field.E. amylovora and less toxic to other environmental bacteria than traditional antibiotics. Not affecting the environmental microbiome has many benefits. First, healthy microbiota serves as competitors of the pathogens for nutrients, space and can have a positive effect in preventing and limiting disease occurrence. Second, the selective pressure on the total microbiome of antibiotics often facilitated the development and spread of antibiotic resistance into pathogen population (strA–strB that confer streptomycin resistance in the fire blight pathogen E. amylovora are believed to be acquired from saprophytic bacteria isolates such as P. agglomerans through horizontal gene transfer (Our study also showed that the antimicrobial effect of PNA–CPP molecules is more specific to pulation . For exatransfer . Thus, cacpP-CPP1 is compatible with multiple biocontrol agents such as P. fluorescens and B. subtilis. The combined application of biological control agents with PNA–CPP may further enhance the control efficacy.One strategy of reducing the usage of antibiotics in tree fruit disease management is to combine biocontrol agents with antibiotics. However, as antibiotics and biocontrol agents are often non-compatible, the antibiotics and biocontrol agents often have to be applied separately, which not only adds labor cost and time constraints, but also may reduce the efficiency. Here we showed that anti-E. amylovora. This suggests that the antimicrobial effect of PNA–CPP on E. amylovora is potent and permanent. It also suggests that besides the potential application in disease management, PNA–CPPs may also be used as an effective approach to modulate gene expression in E. amylovora and potentially other bacteria. As the mRNA inhibition and dose of PNA–CPP is positively correlated, gene expression can be easily repressed at different levels for molecular research purposes. Thus, the gene expression repression by PNA–CPPs may have potential advantages over the gene knockout approach. This is specifically true in situations such as studying the functions of essential genes, when the gene knockout approach is not an option.We observed that PNA–CPP antisense antimicrobials caused degradation of the target mRNA of an essential gene with a subsequent bactericidal effect against In summary, we performed the first exploration of using PNA–CPPs in controlling a bacterial plant disease. Plant diseases caused by bacteria have a long history of control difficulties, and the lack of effective management options results in significant economical losses worldwide . The resRP performed most experiments and analyses. GS and C-HY helped with the experiment design and edited the manuscript. JW, RH, and XY helped with some experiment procedures the analyses. QZ designed this study, performed some experiments, and wrote the manuscript. All authors read and approved the manuscript.The 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.


Cathepsin D has been recently implicated in insulin resistance and cardiovascular disease. This study was designed to investigate the relationship between cathepsin D and newly diagnosed type 2 diabetes.  Circulating cathepsin D levels and metabolic variables were measured in 98 cases and 98 controls. Myocardial performance index “Tei index” that reflects both left ventricular systolic and diastolic function was measured with Doppler echocardiography in cases. P < 0.01). In newly diagnosed type 2 diabetes, a significant correlation was found between cathepsin D levels and HOMA-IR (homeostatic model assessment of insulin resistance) . In contrast, no significant correlation was found between cathepsin D levels and clinical parameters in the control group . Interestingly, correlation analysis revealed a positive association between cathepsin D levels and Tei index in type 2 diabetes .  Newly diagnosed type 2 diabetes demonstrated significantly higher circulating cathepsin D concentrations than controls (median level: 227 ng/ml versus 174 ng/ml,  Increased levels of circulating cathepsin D are closely linked with the presence of type 2 diabetes, and cathepsin D might serve as a novel biomarker for cardiac dysfunction in newly diagnosed type 2 diabetes. Emerging technologies have allowed the feasibility of acquiring high-throughput proteomic blood profiling from a blood specimen . These tRecently, altered circulating cathepsin D levels have been described in two large community cohorts with prevalent insulin resistance by using proximity extension assay . ExtraceDespite the important roles of cathepsin D in many physiological and pathological conditions, whether its circulating levels associated with diabetes and clinical variable remain to be established. Accordingly, we set out to investigate circulating cathepsin D concentrations and their associations with indexes of insulin resistance and clinical variables in normal individuals and newly diagnosed type 2 diabetic patients.In order to examine the relationship between circulating cathepsin D concentrations and indexes of insulin resistance and various metabolic variables, 98 newly diagnosed type 2 diabetic patients and 98 age- and sex-matched healthy controls were recruited from individuals undergoing routine health examinations at Tongji Hospital in Wuhan  between December 2012 and May 2017. Oral glucose tolerance test (OGTT) was performed in all of the included participants. They had no history of drug intake including antihypertensive agent or lipid-lowering medication. Subjects with cardiovascular diseases, gestational diabetes, chronic renal failure, and active liver cirrhosis were excluded from the study. All participants did not received antidiabetic medication or insulin therapy, therefore, did not have glycemic control. Type 2 diabetes was diagnosed based on the American Diabetes Association guideline [Blood samples were collected after an overnight fast in the morning in order to avoid potential confounding influences. Serum and plasma were stored in aliquots without preservatives at −80°C. Serum and plasma parameters were determined at the Department of Medical and Chemical Laboratory Diagnostics of Tongji Hospital according to routine procedures. The homeostasis model assessment of insulin secretion (HOMA-IS) and the homeostasis model assessment of insulin resistance (HOMA-IR) were calculated from fasting insulin and glucose levels with the following equations: HOMA‐IS = [20 × fasting insulin(IU/ml)]/[fasting glucose(mmol/l)–3.5] and HOMA‐IR = fasting glucose(mmol/l) × fasting insulin(mU/l)/22.5.Circulating cathepsin D concentrations were determined using commercially available enzyme-linked immunosorbent assays  according to the manufacturer's instructions. All samples were analyzed in duplicate. Inter- and intra-assay coefficient of variation was <10%.GE Vingmed Vivid 7 or Vivid 9, Horten, Norway). Left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), and fractional shortening (FS) were measured using M-mode in the parasternal LV long axis view. Left ventricular biplane Simpson method ejection fraction (EF) was measured in apical 4- and 2-chamber views. The parameters of LV diastolic function were measured by recording transmitral flow velocity using Doppler echocardiography. The peak velocities of early (E velocity) and late (A velocity) transmitral flow velocities were measured, and the E/A ratio was calculated. The Tei index, which reflects both systolic and diastolic function, was obtained from tissue Doppler as previously published [A standard echocardiographic examination was performed in all type 2 diabetic patients , median (25th and 75th percentiles) for continuous variables, or as percentage for categorical variables. The distribution of quantifiable variables was tested for normality using a one-sample Kolmogorov–Smirnov test. Comparisons of quantitative variables among groups were performed by one way ANOVA with post hoc Tukey's test. However, data that were not normally distributed were logarithmically transformed before analysis. Categorical variables were examined by Chi-squared test. Correlation analysis between continuous variables was performed by Pearson's analysis, and multiple testing was adjusted using Bonferroni correction. Statistical and association analyses were performed using SPSS 15.0 . All tests were two-sided, and The clinical characteristics of the study population for both nondiabetic subjects and newly diagnosed type 2 diabetic patients are shown in P < 0.01) (P < 0.05). However, most of these associations disappeared after stratification analysis. In newly diagnosed type 2 diabetic patients, a highly significant correlation was found between cathepsin D levels and HOMA-IR . Furthermore, cathepsin D levels tended to marginally correlate positively with fasting glucose in the case group . In contrast, no significant correlation was found between cathepsin D levels and clinical parameters in the healthy control group .Of note, compared with the control group, circulating cathepsin D concentrations in newly diagnosed type 2 diabetic patient group were significantly increased  . Next, wr = 0.22, P = 0.03) (P > 0.05).The echocardiographic parameters of type 2 diabetic patients are summarized in  = 0.03) . Other eThe present study provides the first evidence that increased levels of circulating cathepsin D are associated with newly diagnosed and untreated type 2 diabetic patients. Furthermore, our study not only suggested the findings of cathepsin D associated with metabolic variables but also provided several novel findings. Our results showed that circulating cathepsin D concentrations were positively correlated with indexes of insulin resistance. Especially among newly diagnosed type 2 diabetic patients, cathepsin D were strongly correlated with insulin resistance. In agreement with previous finding , our datPreviously, data have shown that cathepsin D is a critical contributor to many steps of tumor progression, including the stimulation of cancer cell proliferation, the inhibition of tumor apoptosis, and growth of micrometastasis . Recent Accumulating evidence demonstrated that obesity, low-grade chronic inflammation, and increased production of proinflammatory cytokines play crucial roles in the development of insulin resistance . It has Increasing evidences point to a strong link between cathepsin D and cholesterol-mediated inflammation . LikewisIn conclusion, we provide the first evidence that increased level of cathepsin D serves as a novel determinant of type 2 diabetes and measurement of circulating levels may be helpful for assessment of diabetes risk. Moreover, the present study shows that circulating cathepsin D concentrations not only positively correlated with indexes of insulin resistance but also correlated with myocardial performance index. Therefore, cathepsin D may have clinical significance for the early identification of individuals at high risk of insulin resistance and cardiac dysfunction. Further studies are required to elucidate the role of cathepsin D in the pathogenesis of insulin resistance and cardiac dysfunction.


The 2016 National Academies of Sciences report “Hearing Health Care for Adults: Priorities for Improving Access and Affordability” included a call to action for government agencies to strengthen efforts to collect, analyze, and disseminate population-based data on hearing loss in adults.CDC analyzed the most recent available data collected both by questionnaire and audiometric tests of adult participants aged 20–69 years in the 2011–2012 National Health and Nutrition Examination Survey (NHANES) to determine the presence of audiometric notches indicative of noise-induced hearing loss. Prevalence of both unilateral and bilateral audiometric notches and their association with sociodemographics and self-reported exposure to loud noise were calculated.Nearly one in four adults (24%) had audiometric notches, suggesting a high prevalence of noise-induced hearing loss. The prevalence of notches was higher among males. Almost one in four U.S. adults who reported excellent or good hearing had audiometric notches . Among participants who reported exposure to loud noise at work, almost one third had a notch.Noise-induced hearing loss is a significant, often unrecognized health problem among U.S. adults. Discussions between patients and personal health care providers about hearing loss symptoms, tests, and ways to protect hearing might help with early diagnosis of hearing loss and provide opportunities to prevent harmful noise exposures. Avoiding prolonged exposure to loud environments and using personal hearing protection devices can prevent noise-induced hearing loss. Hearing plays an important role in communication, health, function, and quality of life. Hearing loss is the third most common chronic physical condition in the United States and is twice as prevalent as diabetes or cancer . The weighted prevalence of an audiometric notch among U.S. adults aged 20–69 years was 39.4 million or 24.4%  .Differences were identified by age, sex, and race/ethnicity, and by whether participants were exposed to loud noise at work. The presence of an audiometric notch increased with age (p<0.01), ranging from 19.2% among persons aged 20–29 years to 27.3% among persons aged 50–59 years . The preTwenty-one million U.S. adults (19.9%) who reported no exposure to loud or very loud noise at work had an audiometric notch  . PersonsNoise-induced hearing loss is a significant health problem among U.S. adults, is more prevalent among males, and increases with age. Persons with auditory damage caused by noise frequently do not recognize it; one in four U.S. adults who reported excellent or good hearing had an audiometric notch. Among persons who reported work exposure to loud noise, one third had a bilateral or unilateral notch.Noise exposure is the second most common cause of acquired hearing loss (after aging) ; using quieter products ; reducing equipment noise by replacing worn or unbalanced machine parts; moving as far as possible from the loudest sound-producing source, such as loudspeakers or cannons at college stadiums; and using hearing protection devices  among persons aged 20–29 years suggests that early life interventions need to be developed.Hearing screenings can help reduce delays in diagnosis and improve access to hearing aids for those with hearing loss, thus improving health-related quality of life  and WHO are raising awareness about noise-induced hearing loss. DHHS is collecting data on hearing status and risk factors, as well as developing guidelines on hearing aids. WHO is developing guidelines on noise exposure. Other public entities, such as states and counties, partner with community groups to reduce noisy environments and use evidence to inform policies that decrease noise exposures. Other ways to reduce environmental noise exposure include using sound-absorbent materials in office buildings and public venues, erecting highway barriers, and passing noise ordinances. Managers and owners of public venues can decrease the loudest sound levels at those locations to help decrease noise exposure.The findings in this report are subject to at least two limitations. First, this is a report of audiometric notches as a proxy for noise-induced hearing loss, and it is possible that some of the hearing loss observed through this method could be caused by factors other than noise. Second, establishing prevalence rates of hearing damage attributed to risk factors such as noise is confounded by multiple data limitations, such as reliance on self-reported rather than measured noise exposures, complexity of categorizing hearing loss; and co-occurrence of risk factors, including genetic predisposition, and aging.This study examines evidence of hearing loss related to noise exposure in a single NHANES 2-year data cycle. It does not provide a longitudinal assessment of persons over time, nor does it compare the results of hearing examinations across different NHANES cycles. A need exists for longitudinal data that measure cumulative effects of noise exposure on hearing over time. These data also show high prevalence of audiometric notches in young adults. Recent studies have shown an increase in the number of young persons exposed to loud noise and music via personal listening devices and at entertainment venues. Future work is needed on early life exposure to noise and its relation to hearing later in life.• Noise exposure at home and in the community can permanently damage hearing.• Almost one in four adults who reported excellent to good hearing already have measurable hearing loss.• The presence of noise-induced hearing loss increased from one in five among young adults aged 20–29 years to one in four among adults aged 50–59 years.https://www.cdc.gov/vitalsigns/.• Additional information is available at