CRISPR-Generated Nrf2a Loss- and Gain-of-Function Mutants Facilitate Mechanistic Analysis of Chemical Oxidative Stress-Mediated Toxicity in Zebrafish.

The transcription factor Nrf2a induces a cellular antioxidant response and provides protection against chemical-induced oxidative stress, as well as playing a critical role in development and disease. Zebrafish are a powerful model to study the role of Nrf2a in these processes but have been limited by reliance on transient gene knockdown techniques or mutants with only partial functional alteration. We developed several lines of zebrafish carrying different null (loss of function, LOF) or hyperactive (gain of function, GOF) mutations to facilitate our understanding of the Nrf2a pathway in protecting against oxidative stress. The mutants confirmed Nrf2a dependence for induction of the antioxidant genes gclc, gstp, prdx1, and gpx1a and identified a role for Nrf2a in the baseline expression of these genes, as well as for sod1. Specifically, the 4-fold induction of gstp by tert-butyl hydroperoxide (tBHP) in wild type fish was abolished in LOF mutants. In addition, baseline gstp expression in GOF mutants increased by 12.6-fold and in LOF mutants was 0.8-fold relative to wild type. Nrf2a LOF mutants showed increased sensitivity to the acute toxicity of cumene hydroperoxide (CHP) and tBHP throughout the first 4 days of development. Conversely, GOF mutants were less sensitive to CHP toxicity during the first 4 days of development and were protected against the toxicity of both hydroperoxides after 4 dpf. Neither gain nor loss of Nrf2a modulated the toxicity of R-(-)-carvone (CAR), despite the ability of this compound to potently induce Nrf2a-dependent antioxidant genes. Similar to other species, GOF zebrafish mutants exhibited significant growth and survival defects. In summary, these new genetic tools can be used to facilitate the identification of downstream gene targets of Nrf2a, better define the role of Nrf2a in the toxicity of environmental chemicals, and further the study of diseases involving altered Nrf2a function.

Determining protein complex structures based on a Bayesian model of in vivo Förster resonance energy transfer (FRET) data.

The use of in vivo Förster resonance energy transfer (FRET) data to determine the molecular architecture of a protein complex in living cells is challenging due to data sparseness, sample heterogeneity, signal contributions from multiple donors and acceptors, unequal fluorophore brightness, photobleaching, flexibility of the linker connecting the fluorophore to the tagged protein, and spectral cross-talk. We addressed these challenges by using a Bayesian approach that produces the posterior probability of a model, given the input data. The posterior probability is defined as a function of the dependence of our FRET metric FRETR on a structure (forward model), a model of noise in the data, as well as prior information about the structure, relative populations of distinct states in the sample, forward model parameters, and data noise. The forward model was validated against kinetic Monte Carlo simulations and in vivo experimental data collected on nine systems of known structure. In addition, our Bayesian approach was validated by a benchmark of 16 protein complexes of known structure. Given the structures of each subunit of the complexes, models were computed from synthetic FRETR data with a distance root-mean-squared deviation error of 14 to 17 Å. The approach is implemented in the open-source Integrative Modeling Platform, allowing us to determine macromolecular structures through a combination of in vivo FRETR data and data from other sources, such as electron microscopy and chemical cross-linking.

Vestibular schwannomas occur in schwannomatosis and should not be considered an exclusion criterion for clinical diagnosis.

Schwannomatosis is a recently delineated inherited condition that has clinical overlap with neurofibromatosis type 2 (NF2). Diagnostic criteria have been developed to distinguish schwannomatosis from NF2, but the existence of mosaic NF2, which may closely mimic schwannomatosis, makes even these criteria problematic. In particular, it is not clear why there is a relative sparing of the cranial nerves from schwannomas in schwannomatosis. We have identified two individuals with schwannomatosis and a unilateral vestibular schwannoma (VS), where a diagnosis of NF2 has been excluded. A third case with an identified SMARCB1 mutation was reported by two radiologists to have a VS, but this was later confirmed as a jugular schwannoma. These cases question whether the current exclusion of a VS from the clinical diagnosis of schwannomatosis is justified.

Cranial meningiomas in 411 neurofibromatosis type 2 (NF2) patients with proven gene mutations: clear positional effect of mutations, but absence of female severity effect on age at onset.

BACKGROUND: Meningiomas have been reported to occur in approximately 50% of neurofibromatosis type 2 (NF2) patients. The NF2 gene is commonly biallelically inactivated in both schwannomas and meningiomas. The spectrum of NF2 mutations consists mainly of truncating (nonsense and frameshift) mutations. A smaller number of patients have missense mutations, which are associated with a milder disease phenotype. METHODS: This study analysed the cumulative incidence and gender effects as well as the genotype-phenotype correlation between the position of the NF2 mutation and the occurrence of cranial meningiomas in a cohort of 411 NF2 patients with proven NF2 mutations. RESULTS AND CONCLUSION: Patients with mutations in exon 14 or 15 were least likely to develop meningiomas. Cumulative risk of cranial meningioma to age 50 years was 70% for exons 1-3, 81% for exons 4-6, 49% for exons 7-9, 56% for exons 10-13, and 28% for exons 14-15. In the cohort of 411 patients, no overall gender bias was found for occurrence of meningioma in NF2 disease. Cumulative incidence of meningioma was close to 80% by 70 years of age for both males and females, but incidence by age 20 years was slightly increased in males (male 25%, female 18%; p=0.023). Conversely, an increased risk of meningiomas in women with mosaic NF2 disease was also found.

An intein with genetically selectable markers provides a new approach to internally label proteins with GFP.

BACKGROUND: Inteins are proteins that catalyze their own removal from within larger precursor proteins. In the process they splice the flanking protein sequences, termed the N-and C-terminal exteins. Large inteins frequently have a homing endonuclease that is involved in maintaining the intein in the host. Splicing and nuclease activity are independent and distinct domains in the folded structure. We show here that other biochemical activities can be incorporated into an intein in place of the endonuclease without affecting splicing and that these activities can provide genetic selection for the intein. We have coupled such a genetically marked intein with GFP as the N-terminal extein to create a cassette to introduce GFP within the interior of a targeted protein. RESULTS: The Pch PRP8 mini-intein of Penicillium chrysogenum was modified to include: 1) aminoglycoside phosphotransferase; 2) imidazoleglycerol-phosphate dehydratase, His5 from S. pombe ; 3) hygromycin B phosphotransferase; and 4) the transcriptional activator LexA-VP16. The proteins were inserted at the site of the lost endonuclease. When expressed in E. coli, all of the modified inteins spliced at high efficiency. Splicing efficiency was also greater than 96% when expressed from a plasmid in S. cerevisiae. In addition the inteins conferred either G418 or hygromycin resistance, or histidine or leucine prototropy, depending on the inserted marker and the yeast genetic background. DNA encoding the marked inteins coupled to GFP as the N-terminal extein was PCR amplified with ends homologous to an internal site in the yeast calmodulin gene CMD1. The DNA was transformed into yeast and integrants obtained by direct selection for the intein's marker. The His5-marked intein yielded a fully functional calmodulin that was tagged with GFP within its central linker. CONCLUSIONS: Inteins continue to show their flexibility as tools in molecular biology. The Pch PRP8 intein can successfully tolerate a variety of genetic markers and still retain high splicing efficiency. We have shown that a genetically marked intein can be used to insert GFP in one-step within a target protein in vivo.

Empirical development of improved diagnostic criteria for neurofibromatosis 2.

PURPOSE: Four sets of clinical diagnostic criteria have been proposed for neurofibromatosis 2, but all have low sensitivity at the time of initial clinical assessment for the disease among patients with a negative family history who do not present with bilateral vestibular schwannomas. We have empirically developed and tested an improved set of diagnostic criteria that uses current understanding of the natural history and genetic characteristics of neurofibromatosis 2 to increase sensitivity while maintaining very high specificity. METHODS: We used data from the UK Neurofibromatosis 2 Registry and Kaplan-Meier curves to estimate frequencies of clinical features at various ages among patients with or without unequivocal neurofibromatosis 2. On the basis of this analysis, we developed the Baser criteria, a new diagnostic system that incorporates genetic testing and gives more weight to the most characteristic features and to those that occur before 30 years of age. RESULTS: In an independent validation subset of patients with unequivocal neurofibromatosis 2, the Baser criteria increased diagnostic sensitivity to 79% (9-15% greater than previous sets of criteria) while maintaining 100% specificity at the age at onset of the first characteristic sign of neurofibromatosis 2. CONCLUSION: The Baser criteria permit early diagnosis in a greater proportion of patients with neurofibromatosis 2 than previous sets of diagnostic criteria.

Brain stem responses evoked by stimulation of the mature cochlear nucleus with an auditory brain stem implant.

OBJECTIVES: The Nucleus auditory brain stem implant (ABI) has been used in the hearing rehabilitation of totally deaf individuals for whom a cochlear implant is not an option such as in the case of neurofibromatosis type 2 (NF2). Intraoperative electrically evoked auditory brain stem responses (EABRs) are recorded to assist in the placement of the electrode array over the dorsal and ventral cochlear nuclei in the lateral recess of the IVth ventricle of the brain stem. This study had four objectives: (1) to characterize EABRs evoked by stimulation with an ABI in adolescents and adults with NF2, (2) to evaluate how the EABR morphology relates to auditory sensations elicited from stimulation by an ABI, (3) to establish whether there is evidence of morphology changes in the EABR with site of stimulation by the ABI, and (4) to investigate how the threshold of the EABR relates to behavioral threshold and comfortably loud sensations measured at initial device activation. DESIGN: Intraoperative EABRs were recorded from 34 subjects with ABIs: 19 male and 15 female, mean age 27 yrs (range 12 to 52 yrs). ABI stimulation was applied at seven different sites using either wide bipolar stimulation across the array or in subsections of the array from medial to lateral and inferior to superior. The EABRs were analyzed with respect to morphology, peak latency, and changes in these characteristics with the site of stimulation. In a subset of eight subjects, additional narrow bipolar sites were stimulated to compare the intraoperative EABR threshold levels with the behavioral threshold (T) and comfortably loud (C) levels of stimulation required at initial device activation. RESULTS: EABRs were elicited from 91% of subjects. Morphology varied from one to four vertex-positive peaks with mean latencies of 0.76, 1.53, 2.51, and 3.64 msecs, respectively. The presence of an EABR from stimulation by electrodes across the whole array had a high predictive value for the presence of auditory electrodes at initial device activation. When examining subsections of the array, the absence of an EABR was a poor predictor for the absence of auditory electrodes. The morphology of the EABRs varied with site of stimulation in 16 cases, but there was no consistent pattern of change with stimulation site. There was a trend for more auditory electrodes to be present in stimulation sites that evoked EABRs with a higher number of peaks in the waveform. The EABR threshold was closer to the behavioral C level than the T level, but there was no overall correlation between the intraoperative EABR threshold level and the behavioral T and C levels. CONCLUSIONS: The presence of an intraoperative EABR corresponded well to the presence of auditory electrodes. The absence of an EABR from stimulating subsections of the array was not; however, a good indicator for the absence of auditory electrodes and the EABR from such stimulation would not be of assistance in identifying the nonauditory sections of the array to exclude in behavioral fitting of the device. The morphology of the EABR did not relate to site of stimulation. More peaks in the EABR was associated with a greater number of electrodes with auditory sensations, suggesting that correct positioning of the ABI activated more auditory subsystems within the cochlear nucleus. The intraoperative EABR thresholds did not correlate with the behavioral T and C levels and could not be used to assist in device fitting.

Brain stem responses evoked by stimulation with an auditory brain stem implant in children with cochlear nerve aplasia or hypoplasia.

OBJECTIVES: The inclusion criteria for an auditory brain stem implant (ABI) have been extended beyond the traditional, postlingually deafened adult with Neurofibromatosis type 2, to include children who are born deaf due to cochlear nerve aplasia or hypoplasia and for whom a cochlear implant is not an option. Fitting the ABI for these new candidates presents a challenge, and intraoperative electrically evoked auditory brain stem responses (EABRs) may assist in the surgical placement of the electrode array over the dorsal and ventral cochlear nucleus in the brain stem and in the postoperative programming of the device. This study had four objectives: (1) to characterize the EABR by stimulation of the cochlear nucleus in children, (2) to establish whether there are any changes between the EABR recorded intraoperatively and again just before initial behavioral testing with the device, (3) to establish whether there is evidence of morphology changes in the EABR depending on the site of stimulation with the ABI, and (4) to investigate how the EABR relates to behavioral measurements and the presence of auditory and nonauditory sensations perceived with the ABI at initial device activation. DESIGN: Intra- and postoperative EABRs were recorded from six congenitally deaf children with ABIs, four boys and two girls, mean age 4.2 yrs (range 3.2 to 5.0 yrs). The ABI was stimulated at nine different bipolar sites on the array, and the EABRs recorded were analyzed with respect to the morphology and peak latency with site of stimulation for each recording session. The relationship between the EABR waveforms and the presence or absence of auditory electrodes at initial device activation was investigated. The EABR threshold levels were compared with the behavioral threshold (T) and comfortably loud (C) levels of stimulation required at initial device activation. RESULTS: EABRs were elicited from all children on both test occasions. Responses contained a possible combination of one to three peaks from a total of four identifiable peaks with mean latencies of 1.04, 1.81, 2.61, and 3.58 msecs, respectively. The presence of an EABR was a good predictor of an auditory response; however, the absence of the EABR was poor at predicting a site with no auditory response. The morphology of EABRs often varied with site of stimulation and between EABR test occasions. Postoperatively, there was a trend for P1, P3, and P4 to be present at the lateral end of the array and P2 at the medial end of the array. Behavioral T and C levels showed a good correlation with postoperative EABR thresholds but a poor correlation with intraoperative EABR thresholds. CONCLUSIONS: The presence of an intraoperative EABR was a good indicator for the location of electrodes on the ABI array that provided auditory sensations. The morphology of the EABR was often variable within and between test sessions. The postoperative EABR thresholds did correlate with the behavioral T and C levels and could be used to assist with initial device fitting.

Outcome from surgery for vestibular schwannomas in children.

OBJECT: A review of sporadic and NF2-related vestibular schwannoma surgery in children (under 18 years of age) with a specific interest in resection rates, recurrence, facial nerve outcomes, hearing preservation, hearing rehabilitation and genetic analysis. METHODS: A retrospective analysis of prospectively collected data of 35 consecutively operated vestibular schwannomas in 29 paediatric patients that underwent 38 operations between 1992 and 2007. Pre- and post-operative radiology, facial nerve function, pure tone audiogram and speech discrimination tests were performed with a mean follow-up of 4.5 years. Tumour and blood mutations were analysed in 86% of patients. RESULTS: Total resection was achieved in all sporadic cases and 68% of NF2 cases. Near total resection led to tumour recurrence in 5 out of 10 cases. The facial nerve was anatomically preserved in 92%. Facial nerve function was excellent to good (Grades 1-3) in 88% with outcome related to tumour size. Hearing preservation was successful in 3 of 11 cases. CONCLUSIONS: Surgery with complete resection results in excellent tumour control, but it is more difficult to attain total resection in NF2 with a relatively high recurrence rate of persistently growing tumours. A better facial outcome is associated with smaller tumours, near-total resection and first time surgery. Hearing preservation is possible in a minority. Hearing rehabilitation can be successful by utilising cochlear implants and auditory brain stem implants (ABI) as appropriate. Overall there is a low complication rate and results are comparable with adult series.

What are the implications in individuals with unilateral vestibular schwannoma and other neurogenic tumors?

OBJECTIVES: Individuals who develop a unilateral vestibular schwannoma (VS) and other neurogenic tumors are at high risk of having the inherited condition neurofibromatosis Type 2 (NF2). The risk of bilateral disease and transmission risk to offspring are important in surgical planning and counseling. The authors have attempted to resolve these risks. METHODS: A large NF2 dataset was interrogated for individuals who had initially presented with a unilateral VS and other tumors before developing bilateral disease, to assess the contralateral and offspring risks. RESULTS: Ninety-six patients with a unilateral VS and additional neurogenic tumors had a bilaterality rate of 48% at 20 years in those initially diagnosed when > 18 years of age and 82% if presenting earlier. Constitutional NF2 mutations were found in blood in 25 (27%) of 92, but 13 (76%) of 17 patients presenting with unilateral VS at < or = 18 years of age. Tumor analysis suggests that the vast majority of the remainder are mosaic for an NF2 mutation. CONCLUSIONS: Patients with unilateral VS and other NF2-related tumors who fulfill Manchester criteria have a high risk of developing a contralateral tumor, especially if presenting in childhood. Transmission risks are reduced for offspring, particularly in the older patients who are likely to be mosaic.

Cochlear implantation in the neurofibromatosis type 2 patient: long-term follow-up.

OBJECTIVE: To evaluate the long-term hearing outcomes of neurofibromatosis type 2 (NF2) patients with cochlear implants. METHODS: Retrospective analysis of cochlear implant performance in NF2 patients using open- and closed-set speech perception testing. RESULTS: Patients with NF2-associated bilateral vestibular schwannomas frequently become profoundly deaf. The aim of surgical resection should be to preserve serviceable hearing in at least one ear; however, this goal can be difficult to achieve. Frequently, tumor size or poor preoperative hearing status can require a surgical approach that leaves the patient with a profound, bilateral sensorineural hearing loss. If the cochlear nerve is preserved anatomically after vestibular schwannoma surgery, and if promontory stimulation confirms the functionality of the cochlear nerve, then cochlear implantation is an excellent option to restore hearing. We present six cochlear implant patients with NF2 who attained a significant improvement in open- and closed-set speech understanding with a mean follow-up of 7.9 (range: 5-13) years after surgery. In all but one case, the hearing results did not deteriorate over the follow-up period. CONCLUSION: Early surgical intervention for vestibular schwannomas in NF2 patients when the cochlear nerve can be spared is an important consideration to allow for possible cochlear implantation. A 6- to 8-week recovery period for the anatomically intact cochlear nerve may be necessary to obtain a positive promontory stimulation response following tumor resection and should be performed prior to cochlear implantation.

Primary cultures of human vestibular schwannoma: selective growth of schwannoma cells.

OBJECTIVE: To establish primary vestibular schwannoma (VS) cultures that selectively favor the growth of schwannoma cells. BACKGROUND: The lack of a suitable in vitro model of human VS cells has directly limited the progress of research on tumorigenesis and therapy. The problems of establishing pure VS culture include control of fibroblast proliferation. Current efforts to extend VS cell life span using viral oncogenes, by conferring the ability to proliferate in vitro, will yield cells intrinsically different from in vivo VS tumors. Much more desirable is the ability to culture VS cells without cellular transformation. METHODS: Tumor specimens from 17 patients were processed for cell culture and grown at 37 degrees C with 5% CO2 and 100% humidity. Key modifications limiting fibroblast proliferation included using selective medium without L-valine, supplemented by Nu-Serum for at least a week; the use of cytosine arabinoside to kill contaminating fibroblasts; and using the Dulbecco modified medium, supplemented with brain-derived neurotrophic factor and 10% fetal calf serum after the initial serum-free period. RESULTS: Twelve of 17 VS were successfully cultured. The presence of schwannoma cells and the absence of fibroblasts were confirmed immunohistochemically using S100 and CD90 markers, respectively. Scanning and transmission electron microscopy demonstrated typical spindle-shaped cells and the presence of "fibrous long-spacing collagen." CONCLUSION: We describe a method for obtaining short-term, essentially fibroblast-free, primary VS cultures. Such pure VS cultures, retaining in vivo characteristics, are extremely useful as an in vitro model to study the pathobiology of schwannoma cells.

Revision surgery for residual or recurrent vestibular schwannoma.

OBJECTIVE: Assess the requirement for and describe the complication rates of revision surgery for vestibular schwannoma. STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral center. PATIENTS: Patients undergoing surgery for vestibular schwannoma by the Manchester Neurotology Service between 1978 and 2004. INTERVENTION: Surgery. MAIN OUTCOME MEASURE(S): The presence of recurrent or residual tumor; necessity for further treatment; complications from revision surgery. RESULTS: Primary surgery was undertaken on 1,037 tumors, with 866 total (19 recurred), 128 near-total, and 43 subtotal removals. Further treatment was performed for 4 recurrent, 2 near-total, and 11 subtotal excised tumors. Thirty-five revision operations resulted in 14 total (1 recurred), 8 near-total, and 13 subtotal removals. Further treatment was required for 3 near-total and 6 subtotal excisions. Poor preoperative facial function (House-Brackmann Grades 4-6) was present in 9 of the 35 patients. A further 10 deteriorated by at least 3 grades by 1 year postoperatively. Other complications of revision surgery included 3 patients with cerebrospinal fluid leaks, a postoperative hematoma requiring evacuation, 2 cerebrovascular accidents, and 2 patients with new cranial nerve deficits. CONCLUSION: Most residual tumors after primary surgery are successfully managed with watch and rescan. Tumor fragment size is the greatest determinant of revision treatment. After revision surgery, tumor regrowth is much less predictable. Revision surgery is usually considerably more difficult than primary surgery, with a higher complication rate, particularly with regard to the facial nerve. Changing the approach for revision surgery may confer an advantage to facial nerve function.

Intracranial/Extracranial meningioma arising in the hypoglossal canal: case report.

A case of a patient with a posterior fossa meningioma extending through the hypoglossal canal to the cervical region as described in this article has not been previously described in the literature. Investigations and surgical management are outlined and pathological classifications are discussed. A literature review including recent reports of extracranial meningiomas is presented. Extracranial meningiomas are exceedingly rare and a high index of suspicion is necessary to make the diagnosis.

Does Meningitis Stop CSF Rhinorrhea following Lateral Skull Base Surgery?

In cases of cerebrospinal fluid (CSF) rhinorrhea following lateral skull base surgery, fibrosis and fibrin formation resulting from meningitis has been postulated as a mechanism of spontaneously resolving the CSF leak. This study was undertaken to explore any possible relationship between the cessation of CSF leak and meningitis. A retrospective study at a tertiary referral center of 232 consecutive patients was performed. Out of a total of 232 procedures, 29 patients developed CSF rhinorrhea, of whom 7 subsequently developed meningitis. Bacteria were isolated in CSF obtained at lumbar puncture in 5 cases, with the CSF analysis in the remaining 2 cases suggesting aseptic meningitis. Conservative treatment failed to stop the CSF rhinorrhea in 6 of 7 cases. In this study, the development of meningitis did not appear to aid in the resolution of the CSF rhinorrhea. We conclude that surgical intervention should not be delayed in the expectation that meningitis and conservative interventions may promote CSF leak resolution.

Long-term Quality of Life Following Vestibular Schwannoma Excision Via the Translabyrinthine Approach.

OBJECTIVE: To assess postoperative quality of life (QOL) and other patient-reported outcomes following surgery for vestibular schwannoma. STUDY DESIGN: Cross-sectional retrospective case review using postal questionnaires. SETTING: Tertiary referral center. PATIENTS: Five hundred consecutive patients undergoing surgery for vestibular schwannoma. INTERVENTION(S): Patients undergoing surgery via the translabyrinthine approach (excluding neurofibromatosis type 2) under the senior author, with a minimum of 5 years follow-up, were included. MAIN OUTCOME MEASURE(S): QOL was assessed using the Short Form 36 (SF-36) questionnaire and a disease-specific survey to assess patients' subjective outcomes. RESULTS: The SF-36 scores in this group were significantly lower than the general UK population, though 24% of respondents reported a subjective improvement in overall QOL. Tumors larger than 4 cm were related to a reduced SF-36 total mental component score (p = 0.037). Increased age at time of surgery correlated with a reduced physical component of QOL (correlation coefficient = -0.26) and an improved mental component (correlation coefficient = 0.26). Subjective reports of postoperative symptoms and return to work, driving and social activities were similar to other published studies. 35% of patients reported vivid dreams or nightmares following surgery; the first reported incidence of this phenomenon in a large group of vestibular schwannoma patients. CONCLUSIONS: Generic measures of QOL in patients following translabyrinthine surgery for vestibular schwannoma do not always match subjective reports, reflecting the complexity of QOL assessment and the range of outcomes in this group. Increased time since surgery appears to be associated with an improvement in mental health.

Alternatively spliced isoforms of the human constitutive androstane receptor.

The nuclear receptor CAR (NR1I3) regulates transcription of genes encoding xenobiotic- and steroid-metabolizing enzymes. Regulatory processes that are mediated by CAR are modulated by a structurally diverse array of chemicals including common pharmaceutical and environmental agents. Here we describe four in-frame splice variants of the human CAR receptor gene. The variant mRNA splice transcripts were expressed in all human livers evaluated. Molecular modeling of the splice variant proteins predicts that the structural effects are localized within the receptor's ligand-binding domain. Assays to assess function indicate that the variant proteins, when compared with the reference protein isoform, exhibit compromised activities with respect to DNA binding, transcriptional activation and coactivator recruitment.

Residual hearing conservation and electroacoustic stimulation with the nucleus 24 contour advance cochlear implant.

OBJECTIVE: To assess the conservation of residual hearing in recipients of the Nucleus 24 Contour Advance cochlear implant (CI) and the benefits of combined electrical and acoustic stimulation. STUDY DESIGN: Prospective multicenter study. SETTING: CI clinics in Western Europe. PATIENTS: Adult candidates for conventional cochlear implantation with a minimum preoperative word recognition score of 10% in the ear to be implanted. INTERVENTION: "Soft-surgery" protocol, including a 1- to 1.2-mm anterior and inferior cochleostomy hole with the electrode array, inserted 17 mm using the "advance-off-stylet" technique. Patients with postoperative pure-tone hearing threshold levels (HTLs) of 80 dB hearing loss or less at 125 and 250 Hz and 90 dB hearing loss or less at 500 Hz were refitted with an in-the-ear hearing aid for combined ipsilateral electrical and acoustic (El-Ac) stimulation. MAIN OUTCOME MEASURES: A questionnaire to collect information regarding surgery. Pure-tone HTLs measured at intervals. Word recognition tested in quiet and sentence recognition tested in noise at 10 and 5 dB signal-to-noise ratio (SNR). HEARING CONSERVATION RESULTS: HTL data were available for 27 patients. HTLs were conserved within 20 dB of preoperative levels for 33, 26, and 19% of patients for 125, 250, and 500 Hz, respectively. However, the recommended soft-surgery protocol was strictly followed in only 12 of 27 patients. For these 12 patients, hearing thresholds were conserved within 20 dB for 50, 50, and 33% of patients. Median threshold increases were 40 dB (range, 250-500 Hz) for the whole group and 23 dB for the strict surgery group. Ten patients retained sufficient HTLs to enter the El-Ac user group. SPEECH RECOGNITION RESULTS: Group mean recognition scores for nine El-Ac users for words presented at 65 dB sound pressure level were 45% for CI alone and 55% for CI + ipsilateral hearing aid (p < 0.05, paired t). For sentences presented in noise at 5 dB SNR, mean word scores were 46% CI alone and 56% CI + ipsilateral hearing aid (p < 0.01, paired t). CONCLUSION: Hearing was conserved for conventional candidates for cochlear implantation where the recommended soft-surgery protocol was strictly adhered. Combined ipsilateral electrical and acoustic stimulation provided considerable benefits for speech recognition in noise, equivalent to between 3 and 5 dB SNR, compared with CI alone.

Dual NRF2 paralogs in Coho salmon and their antioxidant response element targets.

The transcription factor NFE2L2 (Nuclear Factor, Erythroid 2-Like 2, or NRF2) plays a key role in maintaining the redox state within cells. Characterization of this pathway has extended to fish, most notably zebrafish (Danio rerio), in which two paralogs of the transcription factor exist: Nrf2a, an activator, and Nrf2b, a negative regulator during embryogenesis. Only one ARE target has been thoroughly delineated in zebrafish, and this deviated from the canonical sequence derived from studies in mammals. In general, the mechanistic pathway has not been characterized in non-model aquatic organisms that are commonly exposed to environmental pollutants. The current study compares the zebrafish paralogs to those found in a non-model teleost, the ecologically important salmonid, Oncorhnychus kisutch (coho salmon). Two salmon paralogs, Nrf2A and -2B, described here were found to possess only slightly greater identity between one another (84% of amino acids) than to the singleton ortholog of the esocid Esox lucius (80-82%), the nearest non-salmonid outgroup. Unlike one of the zebrafish forms, each is a strong activating factor based on sequence homology and in vitro testing. To uncover functional target AREs in coho, promoter flanking sequences were isolated for five genes that protect cells against oxidative stress: heme oxygenase 1, peroxiredoxin 1, glutamate-cysteine ligase, and the glutathione S-transferases pi and rho (hmox1, prdx1, gclc, gstp, and gstr). All except gstr had functional elements and all fit the standard mammalian-derived canonical sequence, unlike the motif found in zebrafish gstp. Expression studies demonstrate the presence of both Nrf2 paralogs in multiple organs, although in differing ratios. Collectively, our findings extend the conservation of Nrf2 and the ARE to salmonids, and should help inform future work in teleosts on mechanisms of redox control, as well as responsiveness of this pathway and its downstream antioxidant gene targets to chemical exposures in the environment.

Auditory Brainstem Implantation in Neurofibromatosis Type 2: Experience From the Manchester Programme.

OBJECTIVE: To describe the experience of auditory brainstem implantation (ABI) in patients with Neurofibromatosis type 2 (NF2). STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral centre. PATIENTS: Implanted with a Cochlear ABI22 or ABI24M between 1994 and 2009 because of NF2 disease. INTERVENTION(S): Rehabilitative. MAIN OUTCOME MEASURE(S): Surgical complication rate; audiological outcomes. RESULTS: There were 50 primary ABI insertions in 49 patients, including 16 inserted at the time of first side tumor removal as a sleeper, and two revision repositionings which failed to improve outcome. Postoperatively three patients had cerebrospinal fluid leaks which did not require reoperation, one patient had meningitis, and eleven patients suffered either temporary or permanent lower cranial nerve dysfunction. Twenty-nine patients became full time users; a further 12 patients became non-users. Three patients died while their device was inactive. Five patients retain serviceable contralateral hearing. Audiological open set testing of users showed means of: environmental sounds discrimination 51%; phoneme discrimination: with ABI alone 22%/lip reading (LR) 45%/ABI with LR 65%; sentence testing: with ABI alone 13%/LR 19%/ABI with LR 54%. CONCLUSIONS: The majority of patients with NF2 implanted with an ABI find the device a useful aid to communication in conjunction with LR and in recognizing common environmental sounds. A small proportion gain open set discrimination. Almost a third of patients may end up as non-users. There is probably an increased risk of postoperative lower cranial nerve dysfunction so careful preoperative assessment is advised.