Therefore, the incorporation of gold nanoparticles into a commercial TMB-based ELISA kit could generate dual-color responses: the solution color varied gradually from wine red (absorption peak located at ~530 nm) to colorless, and then from colorless to yellow (absorption peak located at ~450 nm) with the increase amount of targets. These dual-color responses effectively improved the sensitivity as well as the accuracy of visual inspection. For example, the proposed dual-color plasmonic ELISA is demonstrated for the detection of prostate-specific antigen (PSA) in human serum with a visual limit of detection (LOD) as low as 0.0093 ng/mL.Colorimetric enzyme-linked immunosorbent assay utilizing 3′-3-5′-5-tetramethylbenzidine(TMB) as the chromogenic substrate has been widely used in the hospital for the detection of all kinds of disease biomarkers. Herein, we demonstrate a strategy to change this single-color display into dual-color responses to improve the accuracy of visual inspection. Our investigation firstly reveals that oxidation state of 3′-3-5′-5-tetramethylbenzidine (TMB Jiang and coworkersIn the past two decades, the unique optical properties of noble metal nanoparticles have attracted intense attention to both the academic and industrial communities1718192021142324In this work, we demonstrate another alternative to conduct plasmonic ELISA based on enzyme mediated etching of gold nanoparticles. Our approach displays two distinct colors in responses of different concentrations of analytes, namely, the solution gradually changed from wine red to colorless, and then from colorless to yellow with an increase amount of analytes. Therefore, during the whole detection process, the absorption spectra of the solution only shows two peaks; one located at ~530 nm, which corresponds to the wine red solution; the other located at ~450 nm, which corresponds to the yellow solution. These two distinct absorption peaks are easily to be detected with the microplate reader. Comparing with the conventional ELISA, this dual color plasmonic ELISA can effectively improve the accuracy of visual inspection.+. The blue TMB+ would turn into yellow TMB2+ after the addition of a stop solution containing hydrochloric acid. The concentration of TMB2+ was proportional to the concentration of antigen in the assay samples, which was the basis of the commercial ELISA for quantitative determination of the concentration of proteins. The second step was the colorimetric reaction process, and this step was the main difference between our method and the conventional ELISA. An appropriate amount of CTAB-capped AuNPs was added to the yellow solution containing TMB2+ which was produced in the first step. The solution color turned into red after the addition of AuNPs ; they showed an orange red color (the tertiary color of red and yellow) which was the same as the control . On the contrary, when CTAB or CTA+ and Br− existed (sample c and d), a sharp decrease appeared and the color of the mixture correspondingly changed from red to pink. These results demonstrate that (1) neither the mixture of TMB2+ and Br− nor the mixture of TMB2+ and CTAC can induce significant etching of AuNPs; and (2) either the presence of the mixture of TMB2+ and CTAB or the mixture of TMB2+, CTAC, and Br− can generate significant color variations.To show the experimental evidence of TMB2O2 can be hydrolyzed and therefore generates some powerful oxidant which can efficiently etch bulk Au27282+ solution used in this work was produced by HRP-H2O2-TMB system, namely, the TMB2+ solution may contain H2O2 which may influence the process of TMB2+ etching. In this section, another comparative study was conducted to demonstrate whether H2O2 could influence the etching effect of TMB2+. As presented in 2O2, CTAB, and AuNPs shows seldom change in color and absorption value (530 nm). The absorption variations of AuNPs with different reaction time were compared between TMB2+ and H2O2 , that is, the residual H2O2 in TMB2+ solution has no influence on TMB2+ etching process. Many studies have reported the etching mechanism by various oxidizing agents in the past years. It has reported that the potential of AuBr2−-CTA+/Au is no more than 0.2 V + and Br−, many reagents can induce the etching of AuNPs. Zhang et al.et al.+. Nevertheless, TMB2+ may be a stronger oxidant than hydrogen peroxide and can efficiently etch AuNPs in the presence of CTAB in this work.It has been reported that Hand H2O2 . The resn value 5 nm. The 2+ and AuNPs was also studied by UV-vis spectrum. The maximum absorption peak of TMB is at 285 nm2O2) and TMB2+ solution both exhibited the characteristic absorption peak, and the absorption intensity of pure substrate was nearly three times than that of TMB2+. When TMB2+ mixed with AuNPs, the absorption intensity at 285 nm rose, but the absorption intensity at 450 nm decreased. This phenomenon suggests the TMB generated after mixing TMB2+ with AuNPs. The reaction relationship may follow the equation (1) shown below:The reaction process of TMB2+. In the absence of TMB2+, the initial size of CATB-capped AuNPs is ~45 nm, and they are quasi-spherical; the solution color is deep wine red  and 0.75 mM (TMB). Thus the optimum concentrations of H2O2 and TMB were 1.2 mM and 0.75 mM, respectively. The CTAB concentration is another key factor for TMB2+ etching process. 530 increased with the increment of CTAB concentration and reached a platform at 0.15 M. Therefore, 0.15 M CTAB was used for the following study.To obtain the best etching effect, we investigated some experimental conditions. The colorimetric assay is based on the reaction between TMB530) increased linearly with the HRP concentration ranging from 4 to 20 mU/mL , which is similar to traditional ELISA.The feasibility of the proposed method was evaluated for visual detection of PSA in human serum. A commercially available human PSA ELISA kit was adopted for the detection of PSA. All operation procedures were followed by the manual of the kit with the only difference in the color display step. The conventional ELISA was added a stop solution to turn the TMBow TMB2+ , while wow TMB2+ . In the ow TMB2+ . Compareow TMB2+ ; whereas2+ can quantitatively etch AuNPs, thus the addition of AuNPs to the solution of conventional HRP-TMB based ELISA can turn the single color (e.g. yellow) into dual color (e.g. red and yellow) display. This vivid color display improves the accuracy of visual inspection with the naked eyes. The dual-color plasmonic ELISA might have a potential promising market for the detection of many disease biomarkers that are presently detected with the conventional ELISA.In summary, we have developed a dual-color plasmonic ELISA for the sensitive detection of disease biomarkers with the naked eyes. Our investigation reveals that TMB4) and TMB liquid substrate system were purchased from Aladdin . The sodium citrate, ascorbic acid, and sodium bromide were obtained from Fu Chen Chemistry , and CTAB was purchased from J & K Chemical Technology . HRP and human total PSA ELISA kit were purchased from Sigma-Aldrich (USA). All other reagents were used as received. UV-vis absorption spectra were recorded with Tianmei UV 2310II  or Multiskan spectrum microplate spectrophotometer . Ultrapure water was from Direct-Q3 UV system , and all photos were taken by Canon EOS 600D digital camera.Gold chloride solution (HAuCl4 aqueous solution (2.5 × 10−4 M) was refluxed in the oil bath at 120 °C with vigorous stirring for 30 min, followed by adding 10 mL of sodium citrate (1%). The mixture solution was continued refluxing for 20 min. Then the gold seed solution was cooled down to the room temperature. CTAB-capped AuNPs were synthesized with a seed mediated method reported previously4  and the mixture solution was incubated in water bath (30 °C). Then 3 mL of 0.1 M freshly prepared ascorbic acid was added followed by gentle stirring for 2 min. The color of the liquid turned into colorless when the ascorbic acid was added. Finally, 30 mL of Au seed solution was added and the mixtures were kept at 30 °C in a water bath for at least 6 h. The absorption peak of CTAB-capped AuNPs was ~530 nm, and the average diameter was ~45 nm calculated by the UV-vis adsorptionAu seeds were prepared according to previous method2+ stock solution was prepared by HRP-H2O2-TMB system. Firstly, 100 μL of HRP (0.15 U/mL) was added to a test tube, followed by adding H2O2 (1 mL) and TMB (1 mL). The mixture turned into light blue quickly and then blue color deepened as time extended. When the enzymatic reaction was about 10 min, HCl  was added to the mixture to end the reaction. The color changed to yellow immediately, indicating that the TMB2+ is produced. According to previous literature2+ is 5.9 × 104 M−1 cm−1.The TMB2+ (150 μL) mixed with 100 μL of CTAB-capped AuNPs  and TMB (100 μL), followed by adding HCl  after 10 min. The absorption value was measured at 450 nm.To verify the effectiveness of this method, we investigated the relationship between HRP concentration and the color. Different concentrations of TMBA human total PSA ELISA kit was adopted for the detection of PSA. Firstly, different concentrations of human PSA standard solution (100 μL) were added to well, followed by incubation for 2.5 h at room temperature. Aspirate and wash the wells three times with wash buffer. Then, 100 μL of biotinylated detection antibody was added to each well, followed by incubation for 1 h at room temperature with gentle shaking. After that, add 100 μL of HRP-Streptavidin conjugate to each well and incubate 45 min at room temperature with gentle shaking. Aspirate and wash the wells three times with wash buffer. Next, 100 μL of ELISA colorimetric TMB reagent was added to each well and incubate for 30 min at room temperature with gentle shaking. Then 50 μL of stop solution was added. Finally, 100 μL of AuNPs was added to each well and incubated for 5 min with gentle shaking. The absorption intensity was obtained with a microplate reader, and the photos were took in photo studio.How to cite this article: Guo, L. et al. Dual-color plasmonic enzyme-linked immunosorbent assay based on enzyme-mediated etching of Au nanoparticles. Sci. Rep. 6, 32755; doi: 10.1038/srep32755 (2016).


Staphylococcus aureus, the SaeRS two-component system (TCS) plays a major role in controlling the production of over 20 virulence factors including hemolysins, leukocidins, superantigens, surface proteins, and proteases. The SaeRS TCS is composed of the sensor histidine kinase SaeS, response regulator SaeR, and two auxiliary proteins SaeP and SaeQ. Since its discovery in 1994, the sae locus has been studied extensively, and its contributions to staphylococcal virulence and pathogenesis have been well documented and understood; however, the molecular mechanism by which the SaeRS TCS receives and processes cognate signals is not. In this article, therefore, we review the literature focusing on the signaling mechanism and its interaction with other global regulators.In the Gram-positive pathogenic bacterium  Staphylococcus aureus is a major cause of morbidity and mortality. This bacterium causes a variety of diseases ranging from soft-tissue infections to life-threatening invasive diseases such as endocarditis, toxic shock syndrome, and necrotizing pneumonia ,19saeP aur genes A. When Pand hla) , and (2)and hla) .hla without induction  promoter activity and hemolytic activity of culture supernatant. Based on those results, Flack et al. proposed that the conserved amino acids are critical for SaeS kinase activity. On the other hand, Liu et al. . F. FS. auragocytes . The Saetes [efb . α-hemolmonocyte . The Saemonocyte ,42,129.Considering the protective roles of the SaeRS TCS described above, it is not surprising that the SaeRS TCS is required for disease progression in several animal models of infection including silkworm and nematode killing ,131, murS. aureus, the SaeRS TCS plays a key role in production of over 20 virulence factors (S. epidermidis, which also has the SaeRS TCS [S. aureus seems to have evolved its virulence capacity by acquiring those virulence genes and placing them under the control of the SaeRS TCS. Despite intense research on the Sae system over the past two decades, we still do not know how cognate Sae signals such as HNPs are sensed by SaeS. Do HNPs directly bind SaeS? Or do they activate SaeS via a hitherto-unidentified receptor molecule? On top of that, HNPs do not activate the SaeRS TCS in certain strain backgrounds, and the reason is completely unknown. As discussed in In  factors , and pataeRS TCS ,137. The


Background: Hearing loss is one of the most common developmental disorders identifiable at birth with its prevalence increasing throughout school years. However, early detection programs are mostly unavailable in low- and middle-income countries (LMICs) where more than 80% of children with hearing loss reside.Objective: This study investigated the feasibility of a smartphone-based hearing screening program for preschool children operated by community healthcare workers (CHWs) in community-based early childhood development (ECD) centers.Method: Five CHWs were trained to map ECD centers and conduct smartphone-based hearing screenings within a poor community in South Africa over a 12-month period. The hearScreenTM smartphone application employed automated test protocols operating on low-cost smartphones. A cloud-based data management and referral function allowed for remote monitoring for surveillance and follow up.Results: 6424 children (3–6 years) were screened for hearing loss with an overall referral rate of 24.9%. Only 39.4% of these children attended their follow-up appointment at a local clinic, of whom 40.5% referred on their second screening. Logistic regression analysis indicated that age, gender and environmental noise levels (1 kHz) had a significant effect on referral rates (p < 0.05). The quality index reflecting test operator test quality increased during the first few months of testing.Conclusion: Smartphone-based hearing screening can be used by CHWs to detect unidentified children affected by hearing loss within ECD centers. Active noise monitoring, quality indices of test operators and cloud-based data management and referral features of the hearScreenTM application allows for the asynchronous management of hearing screenings and follow-ups. Hearing loss is one of the most common developmental disorders identifiable at birth which, if left undetected, has a negative impact on a child’s speech, language, cognitive, educational and socio-emotional development ,2. ApproUnfortunately, there are limited prospects of identifying hearing loss in children, particularly within developing regions such as sub-Saharan Africa where an estimated 6.8 million children suffer from permanent disabling hearing loss ,8,9. ThiCommunity-based hearing programs have been proposed as a way to improve access to ear and hearing care . WHO priTM solution that allows a low-cost alternative to conventional hearing screening equipment whilst adhering to required acoustic calibration standards [With the widespread penetration of 4.92 billion mobile phones worldwide, of which more than 3.74 billion are smartphones, mHealth hearing applications are demonstrating promise to improve access to hearing services in low- and middle-income countries (LMICs) –20. One tandards ,22.TM mHealth solution allows for pre-specified screening protocols with automated sequences to be employed by non-specialist personnel [The hearScreenersonnel ,23. ThisTM software employs noise monitoring algorithms which provide operators with real-time feedback on ambient noise levels, thereby providing a guide to minimize the effect of noise levels when testing in varying noise conditions [TM automatically retests frequencies where maximum noise levels were exceeded [A smartphone-based application may also offer other benefits essential when testing in informal settings, including environmental noise monitoring, quality control indices of test operators and data management ,22. The nditions ,22. The exceeded ,22. FurtTM application has demonstrated a low-cost, accurate and efficient asynchronous screening solution that could be facilitated by non-specialist personnel with limited training [Pure tone audiometry screening in schools using the hearScreentraining . Howevertraining . With thThe study was conducted in the community of Mamelodi, City of Tshwane, Gauteng, South Africa. Mamelodi is situated approximately 20 km east of the city. The unofficial population of Mamelodi is currently close to one million. Census indicated 110,703 households within the community of which only 61% are formal dwellings .TM application. This feature allowed CHWs to record the name of the ECD facility, geolocation, contact person and number of children enrolled. CHWs were thereafter trained to conduct hearing screenings using the hearScreenTM application. These participants had no formal training on hearing healthcare. Prior to implementation of the project, CHWs received a training session during which they were provided with information regarding ear and hearing healthcare, and its importance, as well as training and hands-on practice with the hearing screening smartphone application.Non-probability purposive sampling was used to select participants. Initially, three CHWs were trained to map ECD centers and conduct hearing screenings. An additional two CHWs joined the project during the last three months to assist with the workload. CHWs were first trained to map ECD centers using the facility-mapping feature of the hearScreenTwo hundred and fifty ECD centers were mapped in the community of Mamelodi East and West. ECD centers (crèches) included both public and private facilities that provided learning and support to children between the ages of three to six years. These ECD centers were often informal in nature and based in the homes of local community members. Once consent was obtained from the principal of the ECD centers to conduct hearing screenings, consent letters were sent to the parents/caregivers. Data was collected over a 12-month period with the exception of 3 vacation periods.TM smartphone application was initially operated on Samsung Trend Plus (S5301) smartphones . In July 2016, the Samsung Trend plus smartphones were replaced with Samsung J2 Galaxy smartphones , which operated an upgraded version of the hearScreenTM software. Smartphones were connected to supra-aural Sennheiser HD280 Pro headphones . The hearScreenTM calibration function was used to calibrate the headphones according to prescribed standards (ISO 389–1:1998) adhering to equivalent threshold sound pressure levels determined for this headphone [The hearScreeneadphone .TM application records a quality index of test operators, which gives an objective measure of their screen performance. During each test, a randomized false presentation of a sound is presented. The purpose of this presentation is to determine if the screener correctly records the response by the person tested as ‘no response.’ The hearScreenTM then calculates a quality index on the tests completed by each screener to provide an indication of the reliability of the tests conducted by the test operator. This quality index was monitored throughout the project in order to guide and retrain testers when needed.The hearScreenTM solution has been validated to monitor noise accurately [Noise levels are also recorded by the smartphone hearing screening application for each child during testing. In order to minimize false-positive results caused by exceeded noise levels, testing was not conducted at 0.5kHz. The hearScreencurately . Data coOnce mapping was completed, hearing screenings were only conducted on a set test date if consent was granted by both the ECD center and the parent/caregiver. ECD center staff allocated a room with the least noise possible for testing. Children were provided with a simple explanation and demonstration of what was required of them. The hearing screening application employed automated test protocols. In order to ensure that the child understood what was expected, testing began in the left ear with an initial conditioning tone at 1 kHz at an intensity level of 35 dB HL. Thereafter, a sweep was performed at the test frequencies of 1, 2 and 4 kHz bilaterally at a screening intensity of 25 dB HL . The smaFailure to respond at 25 dB HL at any frequency in any ear constituted an initial fail. In such cases, children were reconditioned and an immediate rescreen was initiated which followed the same procedure [Data were extracted from the cloud-based server to an MS Excel (2011) sheet and analyzed using SPSS v24 . Referral rates, test times, noise levels and quality indices of testers were analyzed using descriptive statistical measures. Binomial logistic regression analysis was used to determine the effects of age, gender and exceeded MPANLs on referral rates in children, with p < 0.05 used to indicate a significant effect. Frequency distributions were also used to analyze the quality indices of tester.A total of 6424 children  between the ages of 3 and 6 years were screened at ECD facilities. Initial screen referral rates were 34.8% , with noAverage noise levels recorded during the initial screen at ECD centers were 44, 41 and 40 dB for 1, 2 and 4kHz respectively. MPANLs were exceeded occasionally during the initial and immediate rescreens conducted at 1, 2 and 4 kHz at both ECD centers and clinics .10.1080/2(8) = 185.412, p < 0.001) and correctly classified 75.1% of the cases. Referral rates were significantly affected by age , gender , and noise levels at 1kHz in the left ear . Females were 1.26 times more likely to fail compared to males, and increasing age was associated with a decreased likelihood of failure (Table 2).A binomial logistic regression was performed to ascertain the effect of gender, and noise levels at each frequency, as well as age on overall results obtained at the ECD centers. The logistic regression model was statistically significant  and 21.3% (3.5–6 years) [TM smartphone application indicated a significantly lower referral rate of 4.3% for older children aged 5 to 7 [The referral rate in the preschool-aged population (3–6 years) using the hearScreen6 years) ,28. In cd 5 to 7 .Referral rates of the current study were greatest in children aged 3 years (45.8%) as opposed to older children, which prompted the researchers to discontinue testing this age group. A study by Sideris and Glattke  found thTM utilizes integrated noise monitoring, providing operators with real-time feedback on noise levels to allow testers to minimize noise levels before continuing with tests. Results and corresponding noise levels analyzed on the centralized cloud-based server indicated that only noise levels at 1 kHz had a significant effect on referral rates obtained in comparison to 2 and 4 kHz test frequencies. Previous studies using the hearScreenTM application also reported similar effects when testing at the lower frequency of 1 kHz [Environmental noise can also have an effect on referral rates, particularly when testing within an ECD setting where noise levels often fluctuate due to the absence of a sound-treated room, children leaving or entering the test environment, testers providing instructions, or groups of children walking past the test room ,21,29. Aof 1 kHz .Increasing screening intensities to 30 dB HL at 1 kHz to compensate for high noise levels in future community-based studies could reduce the incidence of exceeded MPANLs and false-positive results, but may decrease the validity of the screening process as mild losses may be missed ,29. The Gender effects were evident in smartphone hearing screening outcomes, with females more likely to refer than males. Mahomed-Asmail et al.  also repDuring follow-up appointments at clinics, ECD screening results were initially pulled from the cloud-based data system and analyzed. Thereafter, children received a second rescreen at the clinic. This was done in order to avoid unnecessary diagnostic assessments and to reduce the workload on already strained audiologists. Less than half (40.5%) of the children who failed their ECD screening failed the second screen at the clinic. The referral rate dropped by a further 5% (35.7%) when excluding more difficult to test children aged three to four years. Some influences which may have contributed to the difference in referral rates include ambient noise levels, headphone placement, visual distractions, and examiner instructions and expertise .Mean test durations, including the initial and immediate rescreen of both ears, were 177.8 and 174.3 seconds when testing at ECD centers and clinics respectively. Wu et al. , reporteAlthough parents were notified of their child’s result via an SMS sent automatically from the cloud-based system, a low follow-up return rate of 39.4% was found. We suspect that this rate was affected by a long waiting period before follow-up appointments, parents changing their mobile phone number and not notifying the ECD center, and difficulties with taking leave from work, which may result in loss of income for informal workers. Other reasons that may account for non-attendance include lack of transportation, fear and uncertainty about the referral clinic, lack of education regarding hearing loss, and a lack of visibility of services . More prImmediate onsite automated audiometry could motivate parents to attend follow-up appointments by providing an immediate indication of the severity of a hearing problem and thereby also reduce the number of appointments that parents need to attend at clinics . Using oAsynchronous cloud-based monitoring and surveillance allowed for quality indices of test operators to be monitored throughout the test period to ensure quality control . This guECD hearing screening programs using an mHealth point-of-care diagnostics and cloud-based data management and referral systems can be successfully implemented by CHWs within LMICs to identify children prior to school entry. This mHealth model provides a means to improve the cost-effectiveness, quality and efficiency of, and access to, hearing health services in poorer communities, particularly where hearing healthcare providers are unavailable. Quality control features including integrated noise monitoring, quality control indices of test operators and data management allow for asynchronous remote management to ensure reliable testing and to intervene when necessary. Age contributed significantly to high referral rates, suggesting an optimal screening age of five to six years of age. Environmental noise also posed a challenge when testing at the frequency of 1 kHz. Methods to improve the parental follow-up rate should be explored in future studies.


The recent success of anti-PD1 drugs in metastatic colorectal cancer patients with mismatch repair deficiency generated overwhelming enthusiasm for immunotherapy in the disease. However, patients with mismatch repair deficient colorectal cancer represent only a small subset of the metastatic population. Current research focuses on advancing immunotherapy to earlier stages of the disease including adjuvant and first-line metastatic settings, and on inducing sensitivity to immune checkpoint inhibitor therapy through a combinatorial approach. Here, we review the contemporary understanding of the immune and molecular landscape in colorectal cancer and discuss ongoing clinical trials evaluating novel combination regimens based on immune checkpoint inhibitors. Treatments focused on altering the immune system have recently made their way broadly into clinical oncology practice, based upon the successes seen with immune checkpoint inhibitors. While multiple cancers of the digestive tract have seen preliminary evidence of efficacy, colorectal cancers remain the steadfast exception. By and large, colorectal cancer has not benefited from immunotherapeutics; however, emerging data demonstrates that subsets of patients, those with hypermutated colorectal cancers, may benefit from immune checkpoint inhibitors. In addition, combinatorial approaches are evolving which may ultimately overcome this relative resistance across colorectal cancers. This article will provide an overview of the molecular and immunologic landscape, as well as a survey of immunotherapeutics currently under clinical evaluation in colorectal cancer.APC. Relevant to therapeutic targeting, in metastatic disease RAS (KRAS or NRAS), mutations are seen in over 50% of patients, with BRAF mutations seen in 5–10% [In part due to the presence of clear precursor lesions, the step-wise pathogenesis of colorectal cancer was well described over two decades ago . The effin 5–10% ,4. Additin 5–10% .A subset of colorectal cancers possesses markedly elevated mutational rates. Predominantly, these tumors are characterized by dysfunction of the mismatch repair genes (microsatellite high or MSI-H). MSI-H tumors make up a minority of colorectal cancers, with decreasing frequency in more advanced stage disease. The prevalence of MSI-H in stage II, III and IV colorectal cancers stands at 22%, 12%, and 3%, respectively ,8. A smaFour consensus molecular subtypes (CMS) of colorectal cancer have recently been agreed upon in a unification of prior classification criteria . This clA recent analysis examined several independent cohorts of colorectal cancers, with the goal of better describing the tumor microenvironment as it pertains to the CMS subtypes . While CAs a major component of the CMS1 category of colorectal cancers, MSI-H colorectal cancers deserve individual attention, as they represent the initial subset of colorectal cancers where immunotherapies have seen success. Microsatellite instability (MSI) is a marker of dysfunctional mismatch repair proteins within a tumor; MSI was first utilized on a clinical level to identify patients who should undergo germline testing for Lynch Syndrome . HoweverMSI is a PCR based assay wherein typically five microsatellites are evaluated for instability; if ≥2/5 are unstable, the sample is deemed to be MSI-H. Alternatives results are MSI-L (1/5) or MSS (0/5), with MSI-L and MSS being equivalent for the purposes of this discussion. An alternative assay, which is a similarly sensitive screening test for Lynch Syndrome involves assessment of the MMR proteins by IHC: MLH1, MSH2, MSH6, and PMS2. An absence of staining or a ‘negative’ result is equivalent to an MSI-H tumor; a tumor that is negative for one or two MMR proteins is referred to as mismatch repair deficient (dMMR). The MMR assay has some potential advantages over the true MSI assay, as this can be performed in the vast majority of pathology laboratories, is considerably less expensive, and can allow for more focused mutational testing when considering Lynch Syndrome work-up.Relevant to the present discussion, MSI-H tumors have long been recognized to carry a better prognosis in early stage colorectal cancer . While sThe TCGA data in 2013 highlighted that a sizeable proportion of hypermutated tumors are not MSI-H: 7/30 23%) samples tested. Rather, the majority of these hypermutated, MSS tumors are characterized by mutations in POLE ,22. Muta% samplesAn early study utilizing a CTLA-4 antagonist mAb, Tremelimumab, demonstrated the possibility of activity of immune checkpoint inhibitors in colorectal cancer, producing one response durable to 6 months . HoweverBased upon knowledge of the immunogenic tumor microenvironment in MSI-H tumors, as well as the documented dramatic response in such a patient with anti-PD-1 therapy, a trial of pembrolizumab (Keytruda) 10 mg/kg q3week was undertaken, enrolling three cohorts of patients: MSI-H colorectal cancers, MSI-H non-colorectal cancers, and MSS colorectal cancers . In the Further efforts have been undertaken utilizing the PD-1 inhibitor, nivolumab, in MSI-H tumors. Here, 70 patients with MSI-H tumors were enrolled and treated with nivolumab 3 mg/kg q2week . At the As in multiple other cancers, the combination of nivolumab and ipilimumab has been evaluated for clinical efficacy. In CheckMate 142, Nivolumab 3 mg/kg q2week was combined with ipilimumab 1 mg/kg q3 week × 4 doses, and followed by nivolumab monotherapy . Thirty With this compelling data, the most recent NCCN guidelines (1.2017) have been modified to include both pembrolizumab and nivolumab as options for metastatic and unresectable MSI-H colorectal cancer. Equally important, in November 2015, pembrolizumab was granted Breakthrough Therapy Designation for MSI-H CRC by the FDA. One would hope and expect that both pembrolizumab and nivolumab will see approval in the months ahead, while data begins to trickle in on various additional approaches.Multiple studies are ongoing or planned to evaluate the role of PD-1/L1 inhibition in MSI-H colorectal cancer, including the three potentially practice-changing studies described here. A summary of key studies is depicted in The cooperative group system is currently in the process of initiating a similar study, NRG GI004/SWOG1610 (NCT02997228) or COMMITT. This study will utilize a monoclonal antibody which inhibits PD-L1, atezolizumab. In this study, 439 patients with MSI-H/dMMR metastatic colorectal cancer who are treatment naïve will be enrolled and randomized to FOLFOX and bevacizumab, atezolizumab alone, or atezolizumab combined with FOLFOX and bevacizumab. The primary endpoint is progression-free survival. Overall survival, response rates, duration of response, disease control rates and quality of life measures will be among the secondary endpoints.Another important trial will evaluate the role of PD-L1 inhibition in early Stage MSI-H colon cancer. Also developed through the cooperative group system, Alliance A021502 plans to evaluate FOLFOX ± atezolizumab in 720 patients with Stage III colon cancer (NCT02912559). In the experimental arm, patients will receive 6 months of adjuvant FOLFOX concurrently with atezolizumab, which will then be followed by 6 additional months of atezolizumab monotherapy. The primary endpoint is disease-free survival. Overall survival and adverse events will be the secondary endpoints.One informative arm of the pivotal pembrolizumab study enrolled a cohort of patients specifically with MSS colorectal cancer . In thisAs PD-1 and CTLA-4 have demonstrated synergy in multiple tumor types, recent efforts to explore these combinations have also been pursued in MSS colorectal cancer. From a preliminary report of the Checkmate 142 study, there appears to be only limited efficacy in MSS tumors via this therapeutic strategy . FollowiWhile there is clearly a developing role for PD-1 inhibition in MSI-H colorectal cancer, for MSS colorectal cancer, alternative approaches will be required. CTLA-4 and PD-1/L1 may yet prove beneficial in some subsets of MSS colorectal cancers. However, in the remainder, new drugs and new combinations are sorely needed. Pre-clinical data has suggested multiple opportunities. In melanoma, colorectal, and breast cancer models, MEK inhibition upregulates IFN-gamma mediated HLA molecule and PD-L1 expression ,39. In aClassic cytotoxic therapies are thought to have impact on the tumor microenvironment. It is thought that therapy-induced cell death can be immunogenic, promoting the presentation of tumor antigens which might provoke an adaptive immune response. Both chemotherapy and radiation therapy have been construed to have such properties, though the optimal regimens for induction of immunogenic cell death (ICD) remain to be determined and the true magnitude of this effect remains unclear. Both 5-FU and oxaliplatin have been thought to have a favorable effect . Based oThe potential immune-modulatory role of anti-angiogenic agents was observed in a melanoma study of bevacizumab and ipilimumab. In this study, the blockade of VEGF signaling by bevacizumab combined with ipilimumab increased CD163+ dendritic cell trafficking and and CD8+ T-cell trafficking across the tumor vasculature beyond what was achieved via ipiliumumab alone . When coIn a study combining atezolizumab  and bevacizumab with or without chemotherapy (NCT01633970), of the 14 refractory patients who received bevacizumab and atezolizumab, one (7%) experienced response and nine (64%) had stable disease (including two (14%) that were durable for 24 weeks). Another similar study is the phase I trial of the VEGF-TRAP, Aflibercept, and pembrolizumab (NCT02298959).The combined effects of chemotherapy and anti-angiogenic agents on immune checkpoint therapy are being evaluated clinically. The combination of FOLFOX, bevacizumab and atezolizumab has been investigated in a cohort of 30 patients. In this arm, 23 patients were treatment naïve; in this population, 11 (48%) demonstrated partial response 48% with 20/23 (87%) achieving response or stable disease. Tumor biopsies and peripheral blood demonstrate immune activation . It remaIn a biomarker-driven, multi-arm study, atezolizumab is combined with 5-FU and bevacizumab as maintenance following 8 weeks of FOLFOX and bevacizumab induction (NCT02291289). Separately, the BACCI study is evaluating the impact of adding atezolizumab to Capecitabine and bevacizumab in refractory colorectal cancer (NCT02873195).Radiotherapy and thermal therapies have been similarly touted as being immunogenic. For years, rare abscopal responses have been described to radiotherapy, with more recent pre-clinical models suggesting molecules such as PD-1 can otherwise prevent abscopal responses . MultiplNumerous additional combinations are being studied. Agents that might block suppressive immune factors, such as indoleamine 2,3-dioxygenase (IDO) or LAG-3, are being investigated in phase I trials, combined with PD-1 or PD-L1 inhibitors . Drugs that are capable of acting as direct immune stimulators, such as KIR and 4-1BB (CD137), are also being studied in multiple combinations, including with PD-1 inhibition . There are multiple additional checkpoints and immunomodulatory compounds in development and in phase I investigation. A few of interest are detailed below and a summary is depicted in n = 19) with an objective response rate of 58% and complete responses in 26% [IDO is an enzyme which breaks down non-dietary tryptophan, potentially having a very important impact in the tumor microenvironment. IDO is upregulated during inflammation by interferons, prostaglandin E2 (PGE2) and multiple other stimuli. In the microenvironment, IDO depletes tryptophan and increases the concentration of breakdown products, kynurenines; the combination potently induces T cell apoptosis. Further, evidence suggests that IDO drives FoxP3+ Treg differentiation, MDSC accumulation and activation . All of s in 26% . This coAnother potentially important immunosuppressive signaling molecule is colony-stimulating factor 1 (CFS1). Macrophage colony-stimulating factor 1 (CSF1R) is a receptor for CSF1, representing a hub which controls differentiation and function of macrophages. Preclinical models have linked the inhibition of CSF1/CSF1R signaling to the reprogramming of the monocytoid population, shifting the population from tumor promoting monocytes (MDSCs) to that of tumor suppressive, antigen presenting macrophages. In these pancreatic cancer models, combinatorial CSF1R and PD-1 or CTLA-4 blockade substantially improved responses . On thisFinally, Cetuximab is a monoclonal antibody which binds to the epidermal growth factor receptor (EGFR) and is approved for use in RAS wt colorectal cancer. As cetuximab is an IgG1 monoclonal antibody, it carries potential to induce antibody dependent cellular cytotoxicity (ADCC). In clinical investigations, cetuximab has been demonstrated to induce an EGFR-specific T-cell response as well as induce antigen spreading in head and neck cancers . In patiThe American Society of Clinical Oncology (ASCO) declared Immunotherapy to be the 2016 Clinical Cancer Advance of the Year. In 2017, the advance of the year has already been announced to be Immunotherapy 2.0. Despite years of frustration, we are beginning to see some success through the use of the immunotherapy approach in colorectal cancer, namely PD-1 inhibition in MSI-H cancers. However, the successful targeting of MSS cancers and non-hypermutated tumors appears to be not too far off on the horizon. MEK and PD-L1 combinations are being rigorously tested, multiple agents and combinations are in development and multiple companies have shifted their focus and investments toward immunotherapeutics. Neglected in this review, but also of note, a case of remarkable success has been witnessed utilizing adoptive cell therapy via tumor infiltrating lymphocytes (TILs) in colorectal cancer . Thus, c


The classic view of sensorineural hearing loss has been that the primary damage targets are hair cells and that auditory nerve loss is typically secondary to hair cell degeneration. Recent work has challenged that view. In noise-induced hearing loss, exposures causing only reversible threshold shifts (and no hair cell loss) nevertheless cause permanent loss of >50% of the synaptic connections between hair cells and the auditory nerve. Similarly, in age-related hearing loss, degeneration of cochlear synapses precedes both hair cell loss and threshold elevation. This primary neural degeneration has remained a “hidden hearing loss” for two reasons: 1) the neuronal cell bodies survive for years despite loss of synaptic connection with hair cells, and 2) the degeneration is selective for auditory nerve fibers with high thresholds. Although not required for threshold detection when quiet, these high-threshold fibers are critical for hearing in noisy environments. Research suggests that primary neural degeneration is an important contributor to the perceptual handicap in sensorineural hearing loss, and it may be key to the generation of tinnitus and other associated perceptual anomalies. In cases where the hair cells survive, neurotrophin therapies can elicit neurite outgrowth from surviving auditory neurons and re-establishment of their peripheral synapses; thus, treatments may be on the horizon. Our ears were not designed to withstand long and repeated exposure to the high sound pressures produced by the machinery that surrounds us in modern industrialized society, be it work-, leisure- or combat-related. Correspondingly, with increasing life expectancy, the prevalence of age-related hearing loss (AHL) is also on the rise. The National Institute on Deafness estimates that 33% of people over the age of 65 have significant hearing impairment. The two types of hearing loss are likely interrelated, as people in minimally industrialized areas (e.g. the Sudanese desert) do not show the inexorable age-related deterioration of hearing seen in the developed world4, as defined by the audiogram, which measures the minimal sound pressure required for pure-tone detection in a quiet test booth. For decades, it was assumed that cochlear neural loss occurred only after hair cell death5 and thus was rarely of functional significance in NIHL or AHL.Both NIHL and AHL are known as sensorineural hearing loss because the dysfunction arises in the inner ear, or cochlea, where sound-induced vibrations are transduced by sensory hair cells into electrical signals in cochlear neurons that relay the encoded information to the brain . For dec6. This synaptic loss silences large numbers of cochlear neurons but is invisible in routine histological material and does not affect tests of threshold detection, so long as the loss is not complete. This cochlear synaptopathy, also known as "hidden hearing loss", compromises performance on difficult listening tasks such as understanding speech in a noisy environment, which is the classic complaint of those with NIHL and AHL. In animal models, post-exposure treatment with neurotrophins, delivered locally to the inner ear, can repair or replace the damaged synapses7, suggesting possible future therapies for some of the most disabling sensory impairments in sensorineural hearing loss.Recently, my lab showed, in both NIHL and AHL, that synaptic connections between hair cells and cochlear neurons can be destroyed well before the hair cells are damagedThe mammalian cochlea is a spiraling, fluid-filled tube within a particularly dense bone . In cros8. The two hair cell types have different functions. The OHCs have been called the "cochlear amplifier" because they possess electromotility, which is driven by molecular motors containing a membrane protein called prestin9. Prestin undergoes a voltage-sensitive conformational change that turns sound-driven hair-cell receptor potentials back into mechanical motion that is powerful enough to vibrate the entire sensory epithelium, including the IHC stereocilia. The IHCs are more conventional sensory receptors, generating the pre-synaptic drive for all the myelinated sensory fibers of the auditory nerve. Each ANF has a bipolar "type I" cell body in the spiral ganglion that sends a myelinated peripheral axon towards the sensory epithelium, where its unmyelinated terminal contacts a single IHC, and a myelinated central axon to the cochlear nucleus  of thin, unmyelinated fibers13. These "type II" ANFs also project to the cochlear nucleus14. Their function is unclear, but they may be nociceptors17.In humans, the cochlear spiral is ~32 mm long and contains roughly 3,200 IHCs and 10,000 OHCsnucleus , the fir–5 newtons/m2, which is defined as 0 dB SPL (decibels sound pressure level). The dB scale is logarithmic, and each 20 dB increment corresponds to a 10-fold increase in the amplitude of the sound wave. The ear has an enormous dynamic range: loudness grows monotonically over at least a 100 dB range (105 × threshold pressure) and the threshold of pain is cited as 140 dB SPL (107 × threshold pressure)18.As for most types of hearing dysfunction, characterizing NIHL and AHL in humans begins with the threshold audiogram, which measures the lowest audible sound pressure for pure tones at octave-frequency intervals, typically 0.25, 0.5, 1, 2, 4, and 8 kHz . Human hearing is normally most sensitive near 1 kHz, where average sound pressure at threshold in young adults is 2 × 1019, where, prior to federal regulation of workplace noise, SPLs were in excess of 100 dB SPL (current regulations limit an 8-hour workday exposure to 85 dB SPL A-weighted). In its early stages, NIHL is often seen as a "notch" (i.e. threshold elevation) in the audiogram at 4 kHz. As exposure-time accumulates, the hearing loss extends to 8 kHz, and ultimately the audiogram can reveal no hearing sensation above 1 or 2 kHz, even at the highest sound pressures tested. AHL, as documented in cross-sectional studies, also affects the high frequencies first and can often lead to high-frequency deafness similar to that in advanced NIHL20.Cross-sectional studies in the 1960s documented the rise in audiometric thresholds with increasing years of exposure in noisy factories21. However, for continuous-noise exposures at sound pressures like those in even the noisiest pre-regulation factories, damage is restricted to the inner ear, and OHCs are particularly vulnerable2. Complete loss of OHCs will elevate thresholds by 40–60 dB22. Loss of IHCs will silence all sound-evoked activity from affected cochlear regions2. Many surviving IHCs and OHCs suffer stereocilia damage that compromises function and can produce larger threshold shifts than predicted by the number of lost hair cells alone23. In animal studies, a single 2-hour exposure at 115 dB SPL can destroy all IHCs and OHCs throughout the basal (high-frequency) half of the cochlear spiral24. Fortunately, humans may be somewhat less vulnerable to noise than are smaller mammals, requiring higher SPLs or longer exposures to produce comparable damage25. Nevertheless, human ears with advanced NIHL or AHL also show extensive hair cell loss throughout the high-frequency cochlear regions26.At extremely high sound pressures, such as in blast injury (>180 dB SPL peak), there can be eardrum rupture and disarticulation of the ossicles27. This observation led to the dogma that hair cells are the primary target of noise damage and that neurons die only secondarily to loss of their peripheral synapses5. It has been known since the early 1980s that noise can lead to severe swelling of ANF terminals at their IHC synapses when examined within 24 hours post exposure29. This swelling, often accompanied by membrane rupture and loss of cytoplasmic contents, appears to be a kind of glutamate excitotoxicity, as it can be mimicked by cochlear perfusion of glutamate agonists and partially blocked by perfusion of glutamate antagonists31. However, ANF terminal swelling can be observed in ears with temporary threshold shifts (TTSs), and it disappears within a few days as thresholds recover. This threshold recovery led to the idea that neural connections recover or regenerate after noise damage, so long as the hair cells survive32.Although hair cell damage and death can be seen in minutes to hours after acoustic overexposure, death of spiral ganglion cells (the cell bodies of ANFs) is delayed by months to years33. Furthermore, threshold recovery,per se, is not proof of synaptic recovery. Cochlear function in these animal experiments is measured by recording ensemble ANF activity in response to brief tone bursts via metal electrodes on the cochlear capsule  or in the skin of the external ear (auditory brainstem responses [ABRs]). Thresholds for these "gross" neural potentials are very sensitive to OHC damage, which can severely attenuate sound-evoked cochlear vibrations, but extremely insensitive to subtotal neural degeneration. This is because loss of ANFs and their contributions to the ensemble response can be readily compensated for, especially near threshold, by small increases in stimulus level, which recruit more responding fibers by spreading sound-evoked vibrations farther along the mechanically tuned cochlear spiral.However, for years, no one counted ANF terminals in recovered ears because these unmyelinated endings and their synaptic connections are invisible in routine light-microscopic material , and the34. Thinking back to the work on acute noise-induced ANF terminal swelling, we speculated that the exposure in our mice might be causing immediate and irreversible synaptic damage, which was revealed only by the extremely slow death of the disconnected spiral ganglion cells.Thus, for many years, the question of whether or not noise destroys ANF synapses on surviving IHCs was not pursued. Then, my colleague Sharon Kujawa asked me to collaborate on a study of the interaction between NIHL and AHL. She exposed mice as young adults to a noise designed to produce a modest (40 dB) permanent threshold shift (PTS) and then let them age for 2 years to see if the cochlea deteriorated more rapidly in exposed versus unexposed animals. No prior work, to our knowledge, had followed animals for so long post exposure. Two years later, the noise-exposed mice showed ~50% loss of spiral ganglion cells in the basal half of the cochlea versus <5% in age-matched controls despite no significant loss of IHCs or OHCs in either group35. Each ANF contacts a single IHC via a single terminal bouton  and a glutamate receptor subtype  show pairs of closely apposed red and green, pre- and post-synaptic puncta 41, and surviving IHCs at the end of the mouse lifespan have lost ~50% of ANF synapses41. Normal-aging humans, i.e. those without explicit otologic disease, can also show dramatic cochlear neuropathy in regions of minimal hair cell loss42: e.g. one 89-year-old ear retained only ~20% of the normal complement of ANF contacts despite minimal loss of either IHCs or OHCs12.We now know that even exposures producing only a TTS, and leaving all hair cells intact, can destroy up to 50% of IHC synapses across large cochlear regions . The dam31. Recent work showing that synaptopathy also occurs after a single high-intensity shockwave43 suggests that prolonged overexposure of the post-synaptic membrane to glutamate may not be required. Furthermore, it is unclear whether noise-induced, age-related, and drug-induced synaptopathy all share the same mechanism.The mechanisms underlying noise-induced synaptic damage have not been clarified beyond the cochlear perfusion studies of glutamate excitotoxicity in the 1980s44 because the damage is not visible in routine cochlear histopathology and because primary neural degeneration does not significantly affect the threshold audiogram until it exceeds ~80%46. Although not needed for pure-tone detection in quiet environments, a full complement of ANFs is likely required for more difficult listening tasks.Regardless of underlying mechanisms, emerging evidence suggests that surviving IHCs are partly or largely disconnected from their primary sensory fibers in many types of acquired sensorineural hearing loss. This synaptopathy has been called "hidden hearing loss"49. Although both high- and low-SR fibers can contact the same IHC  and high-threshold fibers with low SRs , constitame IHC , their same IHC  and thei61, but their loss should not affect threshold detection of stimuli in an otherwise quiet environment. In the presence of continuous masking noise, however, their contributions become more critical, and their loss becomes more handicapping. By virtue of their higher thresholds, low-SR fibers are more resistant to "masking" by continuous noise62. As the noise level rises, low-threshold, high-SR fibers are driven to "saturated" discharge rate, leaving only the high-threshold, low-SR fibers to carry information about stimuli embedded in the noise.As shown in63. However, the inner ear cannot be biopsied, so enhanced diagnostic tests are needed to screen living subjects.Difficulty hearing in noise is a major complaint of people with sensorineural hearing loss, and it has long been known that two people with the same audiogram, whether normal or abnormal, can perform differently on speech-in-noise tests. Prior to the discovery of hidden hearing loss, these differences were ascribed largely to differences in central auditory processing. A few human histopathological studies suggest that cochlear synaptopathy is an important component of human sensorineural hearing loss, and one even suggests that it is correlated with word-recognition scores41. Once thresholds are elevated, it is difficult to separate changes due to synaptopathy from those due to hair-cell damage. Auditory evoked potentials such as ABRs are easily measured in human subjects from scalp and/or ear-canal electrodes. In a recent study of young adults with normal audiograms, we found a correlation between performance on a difficult speech-in-noise test and alterations in auditory evoked potentials that were consistent with cochlear synaptopathy64. Having purposely sought out subjects who abused their ears (aspiring musicians who never wore ear protection) and those who routinely protected their ears, we also noted a correlation between ear abuse and poorer performance on speech-in-noise tests. Other studies have shown correlations among normal-threshold young subjects between the ability to perform complex listening tasks and alterations in ABRs that suggest a peripheral rather than a central origin65. A recent study of military veterans with normal audiograms has also found a correlation between ABR wave 1 amplitudes and noise-exposure history66, while another recent study of "normal-hearing" subjects in the UK failed to find such a correlation67. However, different metrics of noise history were used, and neither study correlated the electrophysiological results with performance on speech-in-noise tasks.In mouse studies, we showed that suprathreshold amplitudes of ABR wave 1, the summed onset responses of ANFs, were well correlated with the degree of cochlear synaptopathy, so long as cochlear sensitivity was not compromised due to OHC dysfunction68, a careful re-evaluation of these guidelines is warranted if hidden hearing loss is to be prevented as well.Clearly, more work is needed in this area. However, existing data from humans and animals make it clear that significant cochlear neural damage can occur without hair cell damage and thus can hide behind a normal audiogram. This neural damage is likely to be a handicap in difficult listening situations, especially as overt hearing loss (i.e. threshold elevation and hair cell damage) is added to the mix. Since existing federal guidelines on workplace noise exposure were derived based on the assumption that exposures producing no PTSs are benign69. In mammalian cochleae, including those in humans, hair cells and cochlear neurons are post-mitotic, and damaged or lost elements are never replaced70. Although limited hair cell regeneration via transdifferentiation of remaining support cells has been demonstrated in animal models71, the repair of cochlear synaptopathy is arguably simpler because there is an extended therapeutic window in which the hair cell targets as well as the spiral ganglions and their central axons survive6. Multiple animal studies have shown that local delivery of neurotrophins, endogenous players in the signaling pathways involved in neuronal development and maintenance, can elicit neurite extension from spiral ganglion cells even in the adult mammalian ear72. Several recent studies in the mouse and guinea pig have shown that at least within 24 hours post exposure, neurotrophin delivery can repair the noise-induced synaptic damage as it restores ABR amplitudes74. Regeneration is likely more difficult at longer post-exposure times, but even in humans the distance from cell body to hair cell is <0.5 mm, and spiral ganglion cell death must be extremely slow because cochlear implants inserted years after deafness onset still provide useful hearing75. Thus, it does not seem too far-fetched to imagine that there could be therapies for hidden hearing loss on the horizon.An exciting aspect of this work is the notion that some of the hearing handicap in sensorineural hearing loss might be treatable or preventable


Escherichia coli contains two LytS/LytTR-type histidine kinase/response regulator systems, BtsS/BtsR (formerly YehU/YehT) and YpdA/YpdB, which have been identified as pyruvate-responsive two-component systems. Since they exhibit remarkable similarity, we analyzed their phylogenetic distribution within the γ-proteobacteria, and experimentally characterized them in a set of representative species. We found that BtsS/BtsR is the predominant LytS/LytTR-type two-component system among γ-proteobacteria, whereas YpdA/YpdB primarily appears in a supplementary role. Based on our observations in E. coli, we used the highly conserved DNA-binding motifs to test the in vivo functionality of both systems in various genera, including Salmonella, Enterobacter, Citrobacter, Xenorhabdus, Yersinia, Aeromonas and Vibrio. The results suggest that, in all cases tested, BtsS/BtsR and YpdA/YpdB respond to different levels of pyruvate in the environment.Bacterial histidine kinase/response regulator systems operate at the interface between environmental cues and physiological states.  Escherichia coli harbors two LytS/LytTR-type HK/RR systems: BtsS/BtsR (formerly named YehU/YehT) and YpdA/YpdB  were determined in a microplate reader (Tecan Infinite F500). Cultures, inoculated at a starting OD600 of 0.05, were grown in LB or M9 minimal medium with 0.4% (wt/vol) pyruvate as C-source and the appropriate antibiotic, at the indicated temperature under agitation, while OD600 and luminescence were monitored continuously over time.600 = 0.05 in LB medium and aerobically grown at either 30°C or 37°C, as described above. At selected time points , the supernatants were harvested by centrifugation, and subsequently analyzed by hydrophilic interaction liquid chromatography (HILIC). Acetonitrile (ACN), ammonium acetate and ammonium hydroxide  were obtained from Sigma-Aldrich (Sigma-Aldrich GmbH). Water was purified using a Merck Millipore Integral water purification system to a resistance of 18 MΩ and TOC < 5 ppb. Sodium pyruvate was obtained from Sigma-Aldrich, dissolved in water at a concentration of 100 mM and further diluted with ACN. Supernatants were mixed with ACN (1:1) and transferred to autosampler vials.Each of the tested strains was inoculated to an ODPyruvate excreted into the culture medium was quantified by a modified version of a previously published method . SeparatMetabolites were eluted as follows. After 3 min of 85% eluent B, a linear decrease to 2% B was applied over 9 min, followed by an isocratic hold for 3 min, return to initial conditions for 1 min and a 7-min re-equilibration time. Sample (5 μl) was injected via partial loop injection.For quantification, a calibration curve was constructed using various concentrations of pyruvate . Quantification was performed with Quant Analysis 4.4 .E. coli, BtsS and YpdA, were then used as branch markers, which also provided a reliable demarcation line. The majority (77.3%) of the sequences recovered were assigned to the (left) BtsS-type branch of the tree. The remaining 22.7% were YpdA-type HKs and are represented on the right-hand branch. While some genera, like Escherichia, Citrobacter or Serratia, harbor both BtsS and YpdA, others, including Salmonella, Photorhabdus or Vibrio have only one homolog, which is almost invariably assigned to the BtsS-type branch -type membrane-spanning input domain . In ordee branch . Interesbranches . In the branches . OverallE. coli, all identified LytS-type HKs could be unambiguously assigned to one or other of these categories , Salmonella enterica (99.4%) and Enterobacter aerogenes (93.6%) in comparison to E. coli] , a condition that can be brought about by cultivating bacteria in amino acid-rich (LB) medium until the end of exponential growth. Under this condition yjiY expression is transiently induced [yhjX, which is controlled by the YpdA/YpdB TCS, is activated in cells which are exposed to external pyruvate concentrations of at least 600 μM, and expression then lasts as long as pyruvate is available [In light of the high degree of conservation, the selected species were transformed with the luciferase-based promoter reporter plasmids pBBR ectively ,10. Subs induced . In contvailable .yjiY (yhjX promoter(s) (E. coli. Only S. marcescens (Enterobacteriaceae) and A. hydrophila (Aeromonadaceae) failed to induce yjiY or yhjX promoter activity  of the yjiY  and/or ymoter(s)  basicallity Figs  and 5, mity Figs . Based oents see .E. coli excretes pyruvate during growth in LB medium, and the onset of accumulation coincides with the activation of BtsS/BtsR and YpdA/YpdB [freundii, E. aerogenes, and Y. enterocolitica we observed a transient accumulation of external pyruvate, as in E. coli  and subsequently activates yjiY induction [yhjX expression [E. coli, the functional link between the two systems provides for positive feedback from YpdA/YhjX to yjiY transcription, while yhjX is negatively regulated by BtsS/YjiY activity [S. enterica serovar Typhi (S. Typhi), the target gene of the BtsS/BtsR-type TCS has been identified as cstA1 [Saccharomyces cerevisiae, where individual cells express predominantly either a low- or a high-affinity transporter, each of which can confer a similar phosphate uptake capacity [In nduction . The Ypdpression . In E. cactivity  Fig 7)..E. coli,as cstA1 ; howeveras cstA1 , pyruvatas cstA1 , pathogeas cstA1 ,21 and oas cstA1 ,23, whicas cstA1  and is fas cstA1 . The comcapacity .E. coli were found to be highly conserved, and were recognized in six out of eight (BtsR) and three out of five (YpdB), respectively, of the tested species Click here for additional data file.


Resistive patch array incorporating with metallic backplane provided an effective way to achieve broadband metamaterial absorbers (MAs) in microwave frequency, and the outstanding construction contributed more flexible and diversified broadband absorption. In this paper, we attempted to load metallic resonators (MRs) to three-dimensional resistive MA to further enhance the lower-frequency absorption performance. Simulation showed that the partial absorption peak was separated to the lower frequency, while the rest of broadband absorption was unaffected. Meanwhile, after combining multi-unit of the proposed MAs, the stair-stepping broadband absorption was also achieved. Finally, three samples were fabricated. The agreements between simulations and experimental results demonstrated that resistive MA loaded with MRs provided an effective way for further enhancement of lower-frequency absorption with almost no change of the absorbing structure and lightweight characteristic. Thus, it was worthy to expect a wide range of applications to emerge inspired from the proposed attempt. Metamaterials consist of periodic subwavelength unit cell which can provide more possibility to construct the desired constitutive parameters. As new artificial medium, metamaterials have great applications in many fields, such as superlenses ,2,3, invTo achieve broadband MAs, many attempts were carried out. First, the combination of multi-unit exciting contiguous resonances can be thought as a direct way to construct a continuous broadband absorption ,17,18,19In this paper, aiming at the target of lower-frequency absorption enhancement in resistive MA, we attempted to load MRs to the resistive MA to reconfigure the broadband absorption performance. As a proof, the three-dimensional resistive patch array standing on the metal backplane was firstly introduced for broadband absorption. Then, the metallic bamboo joints as MRs were adhered to the resistive patches. The strong resonance inspired by the MRs at low frequency broke the original broadband absorption. Simulation showed that the proposed MA can effectively separate partial absorption peak to the lower frequency, while keeping the broadband absorption at rest frequencies unchanged. Meanwhile, after combining multi-unit, the stair-stepping broadband absorption was also achieved. Finally, three samples were fabricated for experimental demonstration. It is expected that a wide range of applications will emerge from the proposed attempt.d, a and fz, respectively. The resistive patch arranged along x-axis direction on the square backplane with the size of p. The gap between adjacent resistive patches was s. The F4B3 substrate  was used here as a supporter for the construction of the resistive patch array with height d, width p, and thickness t. The relative permittivity and loss tangent of the F4B3 substrate are 2.65 and 0.001, respectively. The copper plate was used here as backplane with a frequency independent conductivity σ = 5.8 × 107 S/m and the thickness tc = 0.017 mm. In the simulation, the electric field of the incidence should be set along x-axis, while the magnetic field was along y-axis to obtain the broadband absorption. The absorptive efficiency of the three-dimensional resistive MA under normal incidence can be defined as A(ω) = 1 − R(ω) − T(ω) = 1 − |S21|2 − |S11|2, where A(ω), |S11|2 and |S21|2 are the absorbance, reflectivity and transmissivity, respectively. Due to the existence of metal backplane, the transmission (S21) is zero. Thus, the absorbance can be calculated by A(ω) = 1 − |S11|2 in this paper. After optimization, when the thickness was given as d = 8.0 mm, the three-dimensional resistive MA can achieve broadband electromagnetic wave absorption with efficiency more than 90% ranging from 4.6 to 21.4 GHz, as shown in a of resistive patch from 10.1 to 10.7 mm or decreasing the gap s between adjacent resistive patches from 0.9 to 0.3 mm, the broadband absorption would further move to the lower frequency with very slight extent. The sheet resistance of the resistive patch can be seen as an assistant factor for the improvement of absorption during the operating frequencies. As a comparison, the height of resistive patch played an important role in the enhancement of broadband absorption. With the increase of height from 7.0 to 10.0 mm, the low boundary frequency of absorption bandwidth with efficiency more that 90% would further expand from 7.3 to 4.6 GHz. Thus, it can be concluded that there was less space for further enhancement of lower-frequency absorption when the height of the three-dimensional resistive MA was given.Using resistive patch to construct broadband MA has been widely adopted. Here, our method attempted to use resistive patch array stand on the metal backplane as three-dimensional resistive MA. As shown in d, width p and thickness t were introduced here to stand up on the squared metal backplane with the size p. On the one side of the substrate, the aforementioned resistive patches were adhered periodically. On the other side of the substrate, metallic bamboo joint was also printed accordingly. The height, width and sheet resistance of the resistive patch are d, a and zf, respectively. The width of the wire, width of the bamboo joint, and the gap between adjacent bamboo joints are w, l and s, respectively. The metallic bamboo joint units were arranged along x-axis with the gap of g above the metal backplane. In the simulation, the electric field of the incidence was also set along x-axis to achieve optimal broadband absorption performance. When the three-dimensional resistive MA is loaded with MRs, the originally continuous broadband absorption will be affected. Instead, the partial absorption peak was separated to the lower frequency. As shown in l of metallic bamboo joint from 2.0 to 6.0 mm, the separated absorption peak would gradually move to the lower frequency from 4.4 to 2.4 GHz, while the continuous broadband absorption during higher frequencies was still unaffected. Here, it should be noted that optimized results demonstrated the gap g should be constant with metallic bamboo joint out of the center. The minimized value of g benefited the broadband absorption during the rest frequencies, as shown in l of the metallic bamboo joint, the absorption bandwidth radio would further increase. Thus, it was obvious that loading MRs to the three-dimensional resistive MA provided an alternative way to enhance the lower-frequency absorption, which we were more concerned with, and sacrificed the absorption in the middle frequency band, which we were less concerned with. Meanwhile, the limited bandwidth radios of the proposed MA were also calculated based on the theoretical Rozanov limit [Here, based on the aforementioned design of three-dimensional resistive MA, we attempted to load the metallic bamboo joint as MR to reconfigure the broadband absorption. The structure diagram in shown in ov limit . During ω is the angular frequency, ε″ is the imaginary part of permittivity, σ is the conductivity and E is the total electric field. First, the electric field intensity distributions and the surface current distributions of the proposed MAs with different widths l = 2.0, 4.0 and 6.0 mm of metallic bamboo joints at the corresponding frequency f = 4.4, 3.3 and 2.5 GHz of separated absorption peaks are given in l of the bamboo joint was seen as a great factor which will influence the equivalent capacitance C as well as the resonance frequency. Therefore, the width l of the bamboo joint was seen as a vital parameter directly determining the separated absorption, as shown in l = 2.0, 4.0 and 6.0 mm at the frequencies of the lower-frequency absorption peaks f = 4.4, 3.3 and 2.5 GHz are given in Based on the reconfigured broadband absorption performance, the physical principles deserve exploration. Different from meta-surfaces  and the l = 1.0 and 2.0 mm, respectively. The simulation showed that the two resonances were excited at the frequencies of 4.2 and 5.8 GHz. A stair-stepping broadband absorption was achieved with the efficiency more than 70% from 3.4 to 6.5 GHz and 90% from 6.5 to 21.0 GHz. l = 1.0, 2.0 and 3.0 mm. The simulation showed that the three resonances were excited at the frequencies of 3.6, 4.4 and 6.0 GHz. A stair-stepping broadband absorption was achieved with the efficiency more than 70% from 3.2 to 6.7 GHz and 90% from 6.7 to 21.0 GHz. The combination of four-units with the widths of bamboo joints l = 1.0, 2.0, 3.0 and 4.0 mm is shown in Three-dimensional resistive MA loaded with MRs succeeded in separating partial absorption peak to the lower frequency for the achievement of reconfigurable broadband electromagnetic wave absorption. Due to the strong resonance inspired by the MRs, highly effective absorption band was achieved at low frequency together with the discontinuousness absorption at the middle frequencies. To eliminate the discontinuous absorption, the multi-unit combination of the proposed MA to construct the continuous broadband absorption was also proposed. Different from the similar construction applied in meta-surfaces  and gradl = 1.0, 2.0, 3.0 and 4.0 mm. Then, the resistive patches were adhered to the other side of the F4B3 substrate periodically, and the basic components of the proposed MA were achieved. Meanwhile, the implemented 297 × 297 mm2-sized copper plate with foam-fabricated card slots was also fabricated. The multi-unit MA components  were selected here and then were inserted into the card slots to construct the finished samples, as shown in To fabricate the three samples in The experimental measurements of the fabricated three samples were performed by the arch measurement system in a microwave anechoic chamber. The system is based on an Agilent E8363B network analyzer  with two pair of broadband antenna horns, respectively, working in the frequency bands of 2–8 GHz, 8–12 GHz, and 12–20 GHz. As shown in In conclusion, we showed that resistive MA loaded with MRs can be optimized for the enhancement of lower-frequency absorption. As a proof, we loaded the metallic bamboo joint to the three-dimensional resistive MA. Simulation showed that the partial absorption peak of the proposed MA was separated to the lower frequency, while the remaining absorption band was unchanged. The separated absorption band can be adjusted with different size of MRs. Then, after combining the multi-unit of the proposed MA, the stair-stepping broadband absorption was also achieved. Finally, three samples were fabricated, and the experimental results agreed well with the simulations. Inspired by the proposed attempt, the enhancement of lower-frequency absorption is expected to be popularized in the field of electromagnetic compatibility, stealth technology, sensors, etc. Meanwhile, it should be noted that the total absorbing efficiency was not fundamentally improved during the operating frequencies. The enhancement of lower-frequency absorption was achieved at the sacrifice of the absorption during other frequencies. However, the proposed attempt provided an effective way to enhance the lower-frequency absorption of resistive MA with no effect on original absorbing structure and lightweight characteristic. Never have attempts concentrated on the reconfiguration of the broadband absorption for further enhancement of lower-frequency absorption, nor has a resistive MA loaded with MRs been designed in three-dimensional construction. In the follow-up work, the outstanding combination between the resistive MA and metallic metamaterial will be investigated for diversified and outstanding absorption performance.


In patients with repeated exposure to cold water, such as cold water surfers and kayakers, the reactive exostoses can occur in the external auditory canal. The external auditory canal exostoses are multiple, benign bony growths. They can cause external auditory canal stenosis, leading to repeated otitis externa and potentially conductive hearing loss. It is vital to consider this entity in susceptible patients who report hearing loss, as timely intervention such as proper ear protection equipment can lower the risk of developing severe external auditory canal exostoses. We present a case of a 42-year-old male, cold water surfer with conductive hearing loss and bilateral external auditory canal (EAC) stenosis demonstrated on the computed tomography. External auditory canal (EAC) exostoses are multiple, benign bony growths within the external auditory canal in response to repeated exposure to cold water. They can cause conductive hearing loss and recurrent otitis externa . We presA 42-year-old male presented to our emergency department (ED) with “hearing problems” as the chief concern. The patient was admitted multiple times for the recurrent ear infections over several months. His ear infections typically began unilaterally, but usually progressed to bilateral involvement. The otorhinolaryngology was consulted in the ED. At this time, Rinne and Weber’s tests showed bone conduction greater than air conduction in both ears, suggesting conductive hearing loss. The patient’s tympanic membranes could not be visualized with an otoscope. The computerized tomography (CT) of the temporal bone was performed which revealed relatively symmetrical bilateral bony outgrowths from the anterior walls of the bilateral external auditory canal (EAC) causing significant canal stenosis, representing bilateral EAC exostoses, consistent with the surfer's ear Figure .Further investigation revealed that the patient surfed most of his life and has never worn protective ear plugs or neoprene hoods while surfing. Thus, resulting in multiple, bilateral EAC exostosis causing significant external auditory canal stenosis and conductive hearing loss. In concert with the otorhinolaryngology consultation, the final diagnosis was classic surfer’s ear.The external auditory exostosis (EAE) are multiple, benign bony growth towards the lumen of the external ear canal in response to repeated exposure to cold water. Exostoses form at the suture lines of the tympanic bone, temporal bone, and mastoid bone in the bony canal. Exostoses are commonly found in the patients such as surfers and kayakers, thus earning EAE the moniker of “surfer’s ear”. The external auditory exostosis (EAE) prevalence in cold water surfers ranges between 70% to 80%. Although usually asymptomatic, the complications of the EAE include cerumen impaction, conductive hearing loss, and otitis externa infection . ImagingThe cold-water theory suggests a causal relationship between the cold water exposure, length of exposure, and the severity of EAE. Possible pathophysiology may be periosteitis following cold water exposure leading to new bone formation. Initially, an anthropological study in 1986 stated that the highest prevalence of EAE occurs in middle latitudes where populations exploit marine and freshwater resources .It is important to cast a wide differential diagnosis when the patients present with hearing loss. The Rinne and Weber tests are clinical tests used to differentiate between sensorineural and conductive hearing loss. As in our case, the positive Rinne Test indicated the conductive hearing loss in our patient. The list of potential differential diagnoses for conductive hearing loss includes EAE, EAC osteoma, otitis externa, benign polyps, trauma, and the squamous cell carcinoma of the EAC. The EAE and EAC osteoma are both bony lesions of the EAC that may be confused clinically. The EAE was usually medial to the EAC isthmus and bilateral. Unlike EAC osteoma which was lateral, the EAC isthmus was unilateral -9.In cases related to the bilateral conductive hearing loss, the patient’s occupation, recreational activities, and exposure to cold water should be considered for diagnosis.


Noise-induced hearing loss is one of the most common forms of sensorineural hearing loss, is a major health problem, is largely preventable and is probably more widespread than revealed by conventional pure tone threshold testing. Noise-induced damage to the cochlea is traditionally considered to be associated with symmetrical mild to moderate hearing loss with associated tinnitus; however, there is a significant number of patients with asymmetrical thresholds and, depending on the exposure, severe to profound hearing loss as well.Recent epidemiology and animal studies have provided further insight into the pathophysiology, clinical findings, social and economic impacts of noise-induced hearing loss. Furthermore, it is recently shown that acoustic trauma is associated with vestibular dysfunction, with associated dizziness that is not always measurable with current techniques. Deliberation of the prevalence, treatment and prevention of noise-induced hearing loss is important and timely. Currently, prevention and protection are the first lines of defence, although promising protective effects are emerging from multiple different pharmaceutical agents, such as steroids, antioxidants and neurotrophins.This review provides a comprehensive update on the pathophysiology, investigations, prevalence of asymmetry, associated symptoms, and current strategies on the prevention and treatment of noise-induced hearing loss. It has been suggested that 12% or more of the global population is at risk for hearing loss from noise, which equates to well over 600 million people [volution .Without doubt, chronic noise exposure and the resultant cochlear trauma cause hearing loss and tinnitus. In the United States among workers not exposed to noise, 7% have hearing loss, 5% have tinnitus, and 2% are afflicted with both hearing loss and tinnitus. However, among noise-exposed workers the prevalence is significantly higher at 23, 15 and 9%, respectively . Within Despite its prevalence, there is still an ongoing debate about the consequence of the noise-induced damage. For many years, the maximum severity of NIHL was argued to be mild to moderate and symmetrical based on pure tone audiograms . The impThe objective of this review is to provide a comprehensive overview of NIHL including the fundamental and advanced pathophysiology, specific investigations, including detailed discussion on asymmetric NIHL, associated symptomatology, available interventions for prevention and treatment.NIHL is a complex disease that results from the interaction of genetic and environmental factors, but is generally still dictated by the extent of biological damage caused by noise exposure. The total amount of noise to which an individual is exposed can be expressed in terms of energy level. The energy level is a function of the sound pressure of noise (in decibels) and of the duration of exposure over time. The equal-energy principle effectively states equal energy will cause equal damage , such that similar cochlear damage may result after exposure to a higher level of noise over a short period of time as would occur after exposure to a lower level of noise over a longer period of time .For environmental exposure, hearing loss can be caused by long-term, continuous exposure to noise and is generally referred to as NIHL. However, hearing loss can also result from single or repeated sudden noise exposure, which is generally referred to as acoustic trauma. Exposure to sudden impulse noise is more detrimental than exposure to steady state noise . This reNoise trauma can result in two types of injury to the inner ear, depending on the intensity and duration of the exposure: either transient attenuation of hearing acuity a.k.a. temporary threshold shift (TTS), or a permanent threshold shift (PTS) . HearingThe recovery of TTS is probably a result of reversible uncoupling of the outer hair cell stereocilia from the tectorial membrane  and/or rThe characteristic pathological feature of NIHL with PTS is the loss of hair cells, particularly the prominent loss of outer hair cells at the basal turn, while loss of inner hair cells was limited. Degeneration of the auditory nerve followed the loss of outer hair cells in both temporal bone histopathology and in a mouse model . A cruciWith sufficient intensity and duration of noise, not only the hair cells but the entire organ of Corti may be disrupted . Destruc2+) in outer hair cells immediately after acoustic overstimulation contributed to by both entry through ion channels and liberation from intracellular stores [2+ overload can also trigger apoptotic and necrotic cell death pathways independent of ROS formation [Another consequence of noise exposure is an increase of free calcium  axis can modulate the sensitivity of the auditory system and be activated by acoustic stress . Mice laCdh23 [Pmca2 [Sod1 [Gpx1 [Trpv4 [Vasp [Hsf1 [The genetic susceptibility to NIHL has been clearly demonstrated in animals. Mouse strains (C57BL/6 J) exhibiting age-related hearing loss were shown to be more susceptible to noise than other strains –35. AlsoCdh23 , Pmca2 [3 [Pmca2 , Sod1 [3a2 [Sod1 , Gpx1 [3d1 [Gpx1 , Trpv4 [1 [Trpv4 , Vasp [4v4 [Vasp , and Hsfsp [Hsf1  were shoThe discovery of human genetic factors predisposing individuals to NIHL has been hindered by many difficulties. To date, no heritability studies have been performed, since families where all subjects are exposed to identical noise conditions are almost impossible to collect. Hence, another approach involving screening of Single Nucleotide Polymorphisms (SNPs) of different genes known to play a functional and morphological role in the inner ear has been adopted. SNPs are common point mutations in the genome (occurring every 100 – 300 base pairs), and their genotyping is believed to be a successful tool in analyzing the genetic background of complex diseases, such as NIHL. In such studies, a disease susceptibility allele is expected to occur more often among susceptible groups than resistant ones. The most promising results were obtained for the inner ear potassium (K+) ion recycling and heat shock protein (HSP) genes. K+ recycling genes are indispensable for the process of hearing, as evidenced by the fact that multiple mutations in these genes  lead to both syndromic and non-syndromic forms of hearing loss –46. HSPsEarly or moderately advanced NIHL usually results in the typical ‘boilermakers’ notch at 4 kHz, with spread to the neighbouring frequencies of 3 kHz and 6 kHz  and someHearing loss induced by noise exposure is quoted to be on average no greater than 75 dB in the high frequencies and no greater than 40 dB in the lower frequencies . HoweverTraditionally, pure tone thresholds were solely relied upon to determine the extent of NIHL, resulting in an underestimation of NIHL prevalence and functional impact. NIHL can be associated with a decrease in speech recognition scores in quiet as well as in background noise, even in the setting of a normal pure tone audiogram . This isOtoacoustic emissions have the necessary features to serve as an objective, sensitive, and easy-to-administer tool for the diagnosis of NIHL. In laboratory animals exposed to high noise levels, OAE amplitude reductions showed a good correlation with permanent threshold shift of more than 25 to 35 dB SPL measured by auditory evoked potentials and significant outer hair cell loss measured by histologic cochleograms . ParalleElectrophysiologic measurements such as ABR have been used to detect noise-induced synaptopathy . There iEmerging evidence suggests that acoustic reflex testing may be helpful for early detection of noise-induced synaptopathy in humans. Threshold shifts in acoustic reflexes, without audiometric hearing loss, might be caused by synaptopathy , 87. WheThe typical pattern of hearing loss resulting from acoustic trauma is symmetrical . HoweverA recent systematic review concluded that the evidence for asymmetrical noise-induced trauma was limited, however only studies that reported an asymmetry of more than 15 dB were included . In the It is worth considering some study findings in more detail. In a study of 208 patients, Fernandes et al. identified asymmetrical hearing loss in 22.6%, of which 6.4% had a definite history of asymmetrical noise exposure and in whom 60% had greater hearing loss in the left ear . Chung eStudies over the last two decades using industrial or continuous noise exposures have found that noise affects the left ear more than the right ear , 102. A Asymmetry in NIHL could theoretically be caused by ambient exogenous noise-exposure factors or by endogenous or anatomical factors. For instance, differentially shielding the right ear from noise or acoustic-energy emitting sources, termed the head shadow effect, may play a role in asymmetric hearing loss . SignifiAlternatively, the left ear may somehow be more susceptible to NIHL than the right ear, regardless of exogenous noise exposure factors, and this translates into an asymmetric pattern of hearing loss in both noise-exposure and general non-noise exposure populations , 110. ThUnilateral or asymmetrical sensorineural hearing loss is important to discern, as it can be a hallmark symptom/sign of a retrocochlear lesion (i.e. vestibular schwannoma), and in such cases further investigation is required (i.e. MRI scan) unless there is a known reason for the asymmetry . Hence, Conventional teaching suggests that a claimant for compensation who has occupational hearing loss with asymmetrical hearing thresholds is unlikely to have a noise-induced hearing loss in the worse ear, and like any other patient, should be investigated for the ‘other’ cause of the asymmetry. However, given the multitude of recent evidence in the literature, if the asymmetry under question cannot be explained by causes other than noise, and the MRI scan does not reveal another cause, then the decision given should be in favour of the worker, on the basis of benefit of doubt  as the aThe prevalence of tinnitus among noise-exposed workers is much higher (24%) than the overall population (14%) , and is There is increasing evidence for noise-induced vestibular deficiency, through a mechanism of noise-induced damage to the sacculocolic reflex pathway and/or damage to the vestibular hair cell cilia , 130. Thn = 20-30), showed that abnormal  cervical vestibular evoked myogenic potentials (VEMPs) and ocular VEMPs are associated with chronic or acute acoustic trauma [In humans, several studies, with relatively small sample sizes  semicircular canals in animal studies , 134. A In animals, noise exposure resulted in a reduction in stereocilia bundle density in vestibular end organs as well as a reduction in regular vestibular afferent baseline firing rates of the otolithic organs and the anterior semicircular canal . As a noThe United States Government Accountability Office report on noise (2011) indicated that hearing loss was the most prevalent occupational health disability in the Department of Defense (DoD) . The DoDThe consequences of occupational NIHL to the individual, although not life-threatening, can be dire. Hearing loss limits an individual’s ability to communicate with the surrounding world, which can lead to increased social stress, depression, embarrassment, poor self-esteem, and relationship difficulties . Social Occupational NIHL has been associated with an increased risk for work-related injuries. For each dB of hearing loss, a statistically significant risk increase was observed for work-related injuries leading to admission to hospital . IndividPrevention remains the best option for limiting the effects of acoustic trauma. Hearing conservation programs in elementary school children are potentially effective to increase the knowledge about the hazards of noise exposure early in life and this may result in behavioral changes towards noise reduction and ear protection . For indThe risk of NIHL can be minimized if noise is reduced to below 80 dB(A) (weighted decibel relative to human ear) . For higA recent Cochrane review concluded that in order to prevent occupational hearing loss, better implementation of legislation and better prevention programs are necessary . RegulatHearing protection offers a secondary level of protection. However, evidence for effective hearing loss prevention programs  is limited. The most effective hearing protection, including earmuffs and earplugs, can reduce loud noise trauma, but compliance may be limited due to the impact on one’s ability to communicate when they are worn and/or discomfort related to their use , 154. ToDifferent types of pharmaceutical agents have been shown to reduce the risk of hearing loss secondary to acoustic trauma. Steroids, specifically intratympanic dexamethasone, may have a therapeutic beneficial effect on NIHL when given before  or afterDifferent routes of delivery have been investigated in animals, including intratympanic, intraperitoneal and direct administration into the scala tympani, and all have demonstrated protective effects as evidenced by preserved hearing (15–20 dB lower hearing thresholds on auditory brainstem response (ABR) measurement and preserved cochlear architecture , 164. EaAntioxidants may be a safer alternative to steroids given a more favourable side effect profile. Free oxygen radicals and oxidative stress are important in the pathogenesis of the NIHL, and therefore antioxidants could theoretically constitute an effective treatment.N-acetylcysteine (NAC) has been reported to reduce the ototoxic effects of noise exposure in animal models –171. In Other antioxidants that can potentially play a protective role against noise-induced cochlear trauma include ginseng , co-enzyThere is evidence in animals that neurotrophins can offer protective effects against noise trauma –191. NeuOther pharmaceutical agents with possible protective NIHL effects include magnesium and statins. A human study  as well Statins might prevent NIHL by reducing oxidative stress and improving hair cell survival in animals , 198. A Due to the severity of the hearing loss and/or the poor speech recognition due to synaptopathy, some individuals with NIHL might eventually become candidates for cochlear implantation (CI) either with full electrical or with electro-acoustic stimulation (EAS). Studies have reported NIHL as the etiology of deafness in implanted individuals, with a prevalence ranging from 2% (CI) to 20% (CI with EAS) , 201. ThThe impact of noise-induced hearing loss is more widespread than has previously been recognized. Apart from a wide range of hearing frequencies that can be adversely affected by noise exposure, there is increasing evidence that noise-induced synaptopathy causes reduced speech perception in noise, even when pure tone thresholds are still preserved (“hidden hearing loss”). Evidence in the current literature further supports the notion that noise exposure can result in an asymmetric pattern of hearing loss due to unique differences in susceptibility to noise damage within individuals, increase frequency of tinnitus as well as vestibular dysfunction. The left ear  is more adversely affected by noise, even in the presence of symmetric noise exposure. Future studies should focus on underlying mechanisms that lead to the susceptibility of left-right asymmetry, and to understand the protective role of the efferent pathways to the cochlea as demonstrated in gender differences. Primary prevention with a focus on regulations, legislation and education in schools, in combination with proper hearing protection are important first lines of defense. Further human studies are needed to address the effectiveness of pharmaco-therapeutic options to prevent or mitigate noise-induced trauma.