A Clinically Viable Medial Olivocochlear Reflex Assay Using Transient-Evoked Otoacoustic Emissions.

OBJECTIVES: The contralateral medial olivocochlear reflex (MOCR) strength may indicate various auditory conditions in humans, but a clinically viable assay and equipment are needed for quick, accurate, and reliable measurements. The first experiment compared an earlier version of the assay, which used a nonlinear-mode chirp stimulus, with a new assay using a linear-mode click stimulus, designed to give reliable MOCR measurements in most normal-hearing ears. The second experiment extended the improved assay on a purpose-built binaural hardware platform that used forward-pressure level (FPL) calibration for both the stimulus and the contralateral MOCR elicitor. DESIGN: Transient-evoked otoacoustic emission (TEOAE) tests were measured with and without a 60-dB SPL MOCR-evoking contralateral broadband noise. The normalized MOCR strength (MOCR%) was derived from the TEOAE responses for each trial pair using the complex pressure difference weighted by the TEOAE magnitude. Experiment 1 compared MOCR% within-subject and across-day using two TEOAE stimuli: nonlinear-mode chirps (50 dB SPL, bandpass 1-5 kHz, 14 ms window delayed by 2 ms) and linear-mode clicks (50 dB SPL, bandpass 0.5-2.5 kHz, 13 ms window delayed by 5 ms). TEOAE responses were analyzed in the 0.5 to 2.5 kHz band. Thirty adult participants with normal hearing (30 ears) completed the study. The TEOAE stimulus was calibrated in situ using spectral flattening, and the contralateral noise was calibrated in a coupler. Twelve TEOAE trial pairs were collected for each participant and condition. Experiment 2 used a purpose-built binaural system. The TEOAE stimuli were linear-mode clicks (50 dB SPL, bandpass 1-3 kHz, 13 ms window delayed by 5 ms), analyzed in the 1 to 3 kHz band over ~12 trial pairs. After a probe refit, an additional trial pair was collected for the two early-stopping signal-to-noise ratio criteria (15 and 20 dB). They were evaluated for single-trial reliability and test time. Nineteen adult participants with normal hearing (38 ears) completed the study. The TEOAE clicks and contralateral elicitor noise were calibrated in situ using FPL and delivered with automated timing. RESULTS: MOCR% for linear-mode clicks was distinguishable from measurement variability in 98% to 100% of participants' ears (both experiments), compared with only 73% for the nonlinear-mode chirp (experiment 1). MOCR detectability was assessed using the MOCR% across-subject/within-subject variance ratio. The ratio in experiment 1 for linear-mode clicks was higher (8.0) than for nonlinear-mode chirps (6.4). The ratio for linear-mode clicks (8.9) in experiment 2 was slightly higher than for the comparable linear-mode stimulus (8.0) in experiment 1. TEOAEs showed excellent reliability with high signal-to-noise ratios in both experiments, but reliability was higher for linear-mode clicks than nonlinear-mode chirps. MOCR reliability for the two stimuli was comparable. The FPL pressure response retest reliability derived from the SPL at the microphone was higher than the SPL retest reliability across 0.4 to 8 kHz. Stable results required 2 to 3 trial pairs for the linear-mode click (experiments 1 and 2) and three for the nonlinear-mode chirp (experiment 1), taking around 2 min on average. CONCLUSIONS: The linear-mode click assay produced measurable, reliable, and stable TEOAE and MOCR results on both hardware platforms in around 2 min per ear. The stimulus design and response window ensured that any stimulus artifact in linear mode was unlikely to confound the results. The refined assay is ready to produce high-quality data quickly for clinical and field studies to develop population norms, recognize diagnostic patterns, and determine risk profiles.

Serial Monitoring of Otoacoustic Emissions in Clinical Trials.

The purpose of this report is to provide guidance on the use of otoacoustic emissions (OAEs) as a clinical trial outcome measure for pharmaceutical interventions developed to prevent acquired hearing loss secondary to cochlear insult. OAEs are a rapid, noninvasive measure that can be used to monitor cochlear outer hair cell function. Serial monitoring of OAEs is most clearly established for use in hearing conservation and ototoxicity monitoring programs in which they exhibit more frequent and earlier changes compared with pure-tone audiometry. They also show promise in recent human trials of otoprotectants. Questions remain, however, concerning the most appropriate OAE protocols to use and what constitutes a "significant" OAE response change. Measurement system capabilities are expanding and test efficacy will vary across locations and patient populations. Yet, standardizing minimal measurement criteria and reporting of results is needed including documentation of test-retest variability so that useful comparisons can be made across trials. It is also clear that protocols must be theoretically sound based on known patterns of damage, generate valid results in most individuals tested, be accurate, repeatable, and involve minimal time. Based on the potential value added, OAEs should be included in clinical trials when measurement conditions and time permit.

Air toxics and the risk of autism spectrum disorder: the results of a population based case-control study in southwestern Pennsylvania.

BACKGROUND: Autism spectrum disorders (ASD) constitute a major public health problem affecting one in 68 children. There is little understanding of the causes of ASD despite its serious social impact. Air pollution contains many toxicants known to have adverse effects on the fetus. We conducted a population based case-control study in southwestern Pennsylvania to estimate the association between ASD and 2005 US EPA modeled NATA (National Air Toxics Assessment) levels for 30 neurotoxicants. METHODS: A total of 217 ASD cases born between 2005 and 2009 were recruited from local ASD diagnostic and treatment centers. There were two different control groups: 1) interviewed controls (N = 224) frequency matched by child's year of birth, sex and race with complete residential histories from prior to pregnancy through the child's second birthday, and 2) 5,007 controls generated from a random sample of birth certificates (BC controls) using residence at birth. We used logistic regression analysis comparing higher to first quartile of exposure to estimate odds ratios (ORs) and 95% confidence intervals (CI), adjusting for mother's age, education, race, smoking status, child's year of birth and sex. RESULTS: Comparing fourth to first quartile exposures for all births, the adjusted OR for styrene was 2.04 (95% CI = 1.17-3.58, p = 0.013) for the interviewed case-control analysis and 1.61 (95% CI = 1.08-2.40, p = 0.018) for the BC analysis. In the BC comparison, chromium also exhibited an elevated OR of 1.60 (95% CI = 1.08-2.38, p = 0.020), which was similarly elevated in the interviewed analysis (OR = 1.52, 95% CI = 0.87-2.66). There were borderline significant ORs for the BC comparison for methylene chloride (OR = 1.41, 95% CI = 0.96-2.07, p = 0.082) and PAHs (OR = 1.44, 95% CI = 0.98-2.11, p = 0.064). CONCLUSIONS: Living in areas with higher levels of styrene and chromium during pregnancy was associated with increased risk of ASD, with borderline effects for PAHs and methylene chloride. These results are consistent with other studies. It is unclear, however, whether these chemicals are risk factors themselves or if they reflect the effect of a mixture of pollutants. Future work should include improved spatiotemporal estimates of exposure to air toxics, taking into account the dynamic movement of individuals during daily life.

Effective identification of functional hearing loss using behavioral threshold measures.

PURPOSE: Four functional hearing loss protocols were evaluated. METHOD: For each protocol, 30 participants feigned a hearing loss first on an audiogram and then for a screening test that began a threshold search from extreme levels (-10 or 90 dB HL). Two-tone and 3-tone protocols compared thresholds for ascending and descending tones for 2 (0.5 and 1.0 kHz) and 3 (0.5, 1.0, and 2.0 kHz) frequencies, respectively. A noise-band protocol compared an ascending noise-band threshold with that for 2 descending tones (0.5 and 1.0 kHz). A spondee protocol compared an ascending spondee threshold with that for 2 descending tones (0.5 and 1.0 kHz). These measures were repeated without the participants feigning losses. RESULTS: With nonfeigning participants, ascending and descending threshold differences were minimal for all protocols. When the participants feigned a loss, the spondee protocol produced the largest average threshold difference (30.8 dB), whereas the other protocols produced smaller differences (19.6-22.2 dB). CONCLUSIONS: Using both the screening test and a comparison of the initial audiogram with the screening test, the spondee and 3-tone protocols resulted in 100% true positives and 0% false positives for functional hearing loss. Either of these protocols could be used clinically or in occupational hearing conservation programs.

The association of respiratory hospitalization rates in WV counties, total, underground, and surface coal production and sociodemographic covariates.

BACKGROUND: Exposures associated with coal mining have long been linked to occupational disease. More recently, investigators have suggested that this industry may affect community health. METHODS: We explored associations between age-adjusted, county-level respiratory disease hospitalization rates (RHRs) in West Virginia and total, surface, and underground coal production, taking into account relevant sociodemographic and behavioral covariates. RHRs were calculated for 2005 to 2009, and analyses were performed to assess the effect of coal production after adjusting for sociodemographic factors. RESULTS: After controlling for percent below poverty, percent urban, and smoking, neither total nor underground tonnage was associated with RHR. Surface coal production, however, was significantly related with RHR (P < 0.05). CONCLUSIONS: Surface coal production makes a small but significant contribution to RHR in West Virginia after accounting for other important sociodemographic and behavioral determinants of health.

Otoacoustic-emission-based medial-olivocochlear reflex assays for humans.

Otoacoustic emission (OAE) tests of the medial-olivocochlear reflex (MOCR) in humans were assessed for viability as clinical assays. Two reflection-source OAEs [TEOAEs: transient-evoked otoacoustic emissions evoked by a 47 dB sound pressure level (SPL) chirp; and discrete-tone SFOAEs: stimulus-frequency otoacoustic emissions evoked by 40 dB SPL tones, and assessed with a 60 dB SPL suppressor] were compared in 27 normal-hearing adults. The MOCR elicitor was a 60 dB SPL contralateral broadband noise. An estimate of MOCR strength, MOCR%, was defined as the vector difference between OAEs measured with and without the elicitor, normalized by OAE magnitude (without elicitor). An MOCR was reliably detected in most ears. Within subjects, MOCR strength was correlated across frequency bands and across OAE type. The ratio of across-subject variability to within-subject variability ranged from 2 to 15, with wideband TEOAEs and averaged SFOAEs giving the highest ratios. MOCR strength in individual ears was reliably classified into low, normal, and high groups. SFOAEs using 1.5 to 2 kHz tones and TEOAEs in the 0.5 to 2.5 kHz band gave the best statistical results. TEOAEs had more clinical advantages. Both assays could be made faster for clinical applications, such as screening for individual susceptibility to acoustic trauma in a hearing-conservation program.

Attenuation as a function of the canal length of custom-molded earplugs: a pilot study.

Custom-molded earplugs (CMEPs) whose canal segments extend beyond the second bend of the ear canal can provide excellent attenuation but can sometimes be uncomfortable. Attenuation was measured for CMEPs whose canal segments were shortened in 2-mm increments. The within-subjects design permitted illustration of the form of the function relating attenuation to canal segment length for individuals. Reduction of attenuation due to canal segment shortening was generally more pronounced for frequencies ≤1000 Hz. Some regions of the canal segments were more critical than others in maintaining attenuation. The relationship between comfort and canal segment length was not straightforward.

Assessment and longitudinal analysis of health impacts and stressors perceived to result from unconventional shale gas development in the Marcellus Shale region.

INTRODUCTION: Concerns for health and social impacts have arisen as a result of Marcellus Shale unconventional natural gas development. Our goal was to document the self-reported health impacts and mental and physical health stressors perceived to result from Marcellus Shale development. METHODS: Two sets of interviews were conducted with a convenience sample of community members living proximal to Marcellus Shale development, session 1 March-September 2010 (n = 33) and session 2 January-April 2012 (n = 20). Symptoms of health impacts and sources of psychological stress were coded. Symptom and stressor counts were quantified for each interview. The counts for each participant were compared longitudinally. RESULTS: Participants attributed 59 unique health impacts and 13 stressors to Marcellus Shale development. Stress was the most frequently-reported symptom. Over time, perceived health impacts increased (P = 0·042), while stressors remained constant (P = 0·855). DISCUSSION: Exposure-based epidemiological studies are needed to address identified health impacts and those that may develop as unconventional natural gas extraction continues. Many of the stressors can be addressed immediately.

Measurements of earplug attenuation under supra-aural and circumaural headphones.

OBJECTIVE: Supra-aural audiometric headphones are generally not recommended for use in measuring the attenuation of earplugs, because contact between the headphone and pinna and/or earplug could alter the attenuation obtained, and because of concerns of non-comparability between modes of excitation from supra-aural headphones and the sound-field procedure required by the standardized method. In this study, we compared measurements of earplug attenuation obtained under Telephonics TDH-50P supra-aural headphones with measurements obtained under circumaural headphones designed expressly for such testing. DESIGN: The attenuation of three types of earplugs (foam, premolded quadruple-flange, and custom-molded) was measured in a repeated-measures design. STUDY SAMPLE: The study sample comprised 42 normal-hearing adults (21 females, 21 males). RESULTS: With the foam earplugs, nearly all of the attenuation measurements under the supra-aural headphones fell within 10 dB of the measurements under the circumaural headphones. With the flange and custom earplugs, approximately 10% of individuals obtained spuriously high attenuation under the supra-aural headphones. CONCLUSIONS: We conclude that standard supra-aural audiometric headphones are suitable for measuring the attenuation provided by foam earplugs. However, supra-aural headphones should not be used to measure the attenuation of flange or custom-molded earplugs. The potential exists for substantial over-estimation of attenuation, especially of custom plugs.

Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila.

The nutrient/target-of-rapamycin (TOR) pathway has emerged as a key regulator of tissue and organismal growth in metazoans. The signalling components of the nutrient/TOR pathway are well defined; however, the downstream effectors are less understood. Here, we show that the control of RNA polymerase (Pol) III-dependent transcription is an essential target of TOR in Drosophila. We find that TOR activity controls Pol III in growing larvae via inhibition of the repressor Maf1 and, in part, via the transcription factor Drosophila Myc (dMyc). Moreover, we show that loss of the Pol III factor, Brf, leads to reduced tissue and organismal growth and prevents TOR-induced cellular growth. TOR activity in the larval fat body, a tissue equivalent to vertebrate fat or liver, couples nutrition to insulin release from the brain. Accordingly, we find that fat-specific loss of Brf phenocopies nutrient limitation and TOR inhibition, leading to decreased systemic insulin signalling and reduced organismal growth. Thus, stimulation of Pol III is a key downstream effector of TOR in the control of cellular and systemic growth.

Drosophila RNA polymerase III repressor Maf1 controls body size and developmental timing by modulating tRNAiMet synthesis and systemic insulin signaling.

The target-of-rapamycin pathway couples nutrient availability with tissue and organismal growth in metazoans. The key effectors underlying this growth are, however, unclear. Here we show that Maf1, a repressor of RNA polymerase III-dependent tRNA transcription, is an important mediator of nutrient-dependent growth in Drosophila. We find nutrients promote tRNA synthesis during larval development by inhibiting Maf1. Genetic inhibition of Maf1 accelerates development and increases body size. These phenotypes are due to a non-cell-autonomous effect of Maf1 inhibition in the fat body, the main larval endocrine organ. Inhibiting Maf1 in the fat body increases growth by promoting the expression of brain-derived insulin-like peptides and consequently enhanced systemic insulin signaling. Remarkably, the effects of Maf1 inhibition are reproduced in flies carrying one extra copy of the initiator methionine tRNA, tRNA(i)(Met). These findings suggest the stimulation of tRNA(i)(Met) synthesis via inhibition of dMaf1 is limiting for nutrition-dependent growth during development.

Regulation of TFIIIB during F9 cell differentiation.

BACKGROUND: Differentiation of F9 embryonal carcinoma (EC) cells into parietal endoderm (PE) provides a tractable model system for studying molecular events during early and inaccessible stages of murine development. PE formation is accompanied by extensive changes in gene expression both in vivo and in culture. One of the most dramatic is the ~10-fold decrease in transcriptional output by RNA polymerase (pol) III. This has been attributed to changes in activity of TFIIIB, a factor that is necessary and sufficient to recruit pol III to promoters. The goal of this study was to identify molecular changes that can account for the low activity of TFIIIB following F9 cell differentiation. RESULTS: Three essential subunits of TFIIIB decrease in abundance as F9 cells differentiate; these are Brf1 and Bdp1, which are pol III-specific, and TBP, which is also used by pols I and II. The decreased levels of Brf1 and Bdp1 proteins can be explained by reduced expression of the corresponding mRNAs. However, this is not the case for TBP, which is regulated post-transcriptionally. In proliferating cells, pol III transcription is stimulated by the proto-oncogene product c-Myc and the mitogen-activated protein kinase Erk, both of which bind to TFIIIB. However, c-Myc levels fall during differentiation and Erk becomes inactive through dephosphorylation. The diminished abundance of TFIIIB is therefore likely to be compounded by changes to these positive regulators that are required for its full activity. In addition, PE cells have elevated levels of the retinoblastoma protein RB, which is known to bind and repress TFIIIB. CONCLUSION: The low activity of TFIIIB in PE can be attributed to a combination of changes, any one of which could be sufficient to inhibit pol III transcription. Declining levels of essential TFIIIB subunits and of activators that are required for maximal TFIIIB activity are accompanied by an increase in a potent repressor of TFIIIB. These events provide fail-safe guarantees to ensure that pol III output is appropriate to the diminished metabolic requirements of terminally differentiated cells.

Estimation of equivalent noise exposure level using hearing threshold levels of a population.

OBJECTIVES: Noise-induced hearing loss (NIHL) is costly in both human and economic terms. One means of reducing NIHL is to apply engineering controls to hazardous noise sources. To trade off the cost of engineering controls against the total direct monetary costs incurred by NIHL, a means of predicting the amount of NIHL that will be incurred over the life-cycle of a hazardous noise source is necessary. A widely known algorithm for the prediction of NIHL is published in ANSI S3.44-1996. However, the algorithm inputs, noise exposure level and duration, may be difficult to determine in some cases. This paper describes the conceptual basis of an approach for using ANSI S3.44-1996 to predict hearing thresholds in a population even when noise exposure levels and durations are not precisely known, and demonstrates the initial application of this approach to a single military population. DESIGN: Retrospective data were obtained on the hearing-threshold levels, demographic characteristics, and noise exposure history of 250 male U.S. Navy machinists' mates. A maximum-likelihood fitting procedure was developed in which the noise level input to the algorithm was varied in order to determine the noise level that best accounted for all of the data. RESULTS: The maximum likelihood fitting produced a value for the noise level input of approximately 93 dBA, with a standard error of approximately 0.3. The low standard error virtually eliminates any estimate above 94 or below 92 dBA, and indicates that a good fit to the data was achieved. CONCLUSIONS: This research demonstrates the feasibility of calibrating the algorithm to an individual population, even when noise exposure level or duration is not precisely known. Future work will focus on validating and generalizing this approach so that it may be used to predict hearing-threshold levels in various populations. Such an approach may be used in calculating potential cost savings in compensable hearing loss due to the application of noise control solutions.

Detecting incipient inner-ear damage from impulse noise with otoacoustic emissions.

Audiometric thresholds and otoacoustic emissions (OAEs) were measured in 285 U.S. Marine Corps recruits before and three weeks after exposure to impulse-noise sources from weapons' fire and simulated artillery, and in 32 non-noise-exposed controls. At pre-test, audiometric thresholds for all ears were

Non-coding RNA production by RNA polymerase III is implicated in cancer.

RNA polymerase III (Pol III) makes a variety of small non-coding RNAs, such as tRNA and 5S ribosomal RNA. Increased expression of pol III products is often observed in transformed cells. Much progress has been made in determining how Pol III-dependent transcription is regulated and how it increases in cancers, but the importance of this increase has not been clearly established. New evidence suggests that Pol III output can substantially affect transformation.

Elevated RNA polymerase III transcription drives proliferation and oncogenic transformation.

RNA polymerase (Pol) III is the largest of the RNA polymerases with seventeen subunits. It is responsible for the production of short, untranslated RNAs that play important roles in determining the biosynthetic capacity of the cell. Pol III transcription is generally elevated in transformed cells and tumours; however, it has remained a matter for conjecture as to whether this activation is a cause or consequence of the transformation process.

Recruitment of RNA polymerase III in vivo.

RNA polymerase (pol) III contains a dissociable subcomplex that is required for initiation, but not for elongation or termination of transcription. This subcomplex is composed of subunits RPC3, RPC6 and RPC7, and interacts with TFIIIB, a factor that is necessary and sufficient to support accurate pol III transcription in vitro. Direct binding of TFIIIB to RPC6 is believed to recruit pol III to its genetic templates. However, this has never been tested in vivo. Here we combine chromatin immunoprecipitation with RNA interference to demonstrate that the RPC3/6/7 subcomplex is required for pol III recruitment in mammalian cells. Specific knockdown of RPC6 by RNAi results in post-transcriptional depletion of the other components of the subcomplex, RPC3 and RPC7, without destabilizing core pol III subunits or TFIIIB. The resultant core enzyme is defective in associating with TFIIIB and target genes in vivo. Promoter occupancy by pol II is unaffected, despite sharing five subunits with the pol III core. These observations provide evidence for the validity in vivo of the model for pol III recruitment that was built on biochemical data.

Regulation of RNA polymerase III transcription by Maf1 in mammalian cells.

RNA polymerase (pol) III produces essential components of the biosynthetic machinery; therefore, its output is tightly coupled with the rate of cell growth and proliferation. In Saccharomyces cerevisiae, Maf1 is an essential mediator of pol III repression in response to starvation. We demonstrate that a Maf1 ortholog is also used to restrain pol III activity in mouse and human cells. Mammalian Maf1 represses pol III transcription in vitro and in transfected fibroblasts. Furthermore, genetic deletion of Maf1 elevates pol III transcript expression, thus confirming the role of endogenous Maf1 as an inhibitor of mammalian pol III output. Maf1 is detected at chromosomal pol III templates in rodent and human cells. It interacts with pol III as well as its associated initiation factor TFIIIB and is phosphorylated in a serum-sensitive manner in vivo. These aspects of Maf1 function have been conserved between yeast and mammals and are therefore likely to be of fundamental importance in controlling pol III transcriptional activity.

Elevated tRNA(iMet) synthesis can drive cell proliferation and oncogenic transformation.

Characteristics of transformed and tumor cells include increased levels of protein synthesis and elevated expression of RNA polymerase (pol) III products, such as tRNAs and 5S rRNA. However, whether deregulated pol III transcription contributes to transformation has been unclear. Generating cell lines expressing an inducible pol III-specific transcription factor, Brf1, allowed us to raise tRNA and 5S rRNA levels specifically. Brf1 induction caused an increase in cell proliferation and oncogenic transformation, whereas depletion of Brf1 impeded transformation. Among the gene products induced by Brf1 is the tRNA(iMet) that initiates polypeptide synthesis. Overexpression of tRNA(iMet) is sufficient to stimulate cell proliferation and allow immortalized fibroblasts to form foci in culture and tumors in mice. The data indicate that elevated tRNA synthesis can promote cellular transformation.

TRRAP and GCN5 are used by c-Myc to activate RNA polymerase III transcription.

Activation of RNA polymerase (pol) II transcription by c-Myc generally involves recruitment of histone acetyltransferases and acetylation of histones H3 and H4. Here, we describe the mechanism used by c-Myc to activate pol III transcription of tRNA and 5S rRNA genes. Within 2 h of its induction, c-Myc appears at these genes along with the histone acetyltransferase GCN5 and the cofactor TRRAP. At the same time, occupancy of the pol III-specific factor TFIIIB increases and histone H3 becomes hyperacetylated, but increased histone H4 acetylation is not detected at these genes. The rapid acetylation of histone H3 and promoter assembly of TFIIIB, c-Myc, GCN5, and TRRAP are followed by recruitment of pol III and transcriptional induction. The selective acetylation of histone H3 distinguishes pol III activation by c-Myc from mechanisms observed in other systems.