Combined clarification and affinity capture using magnetic resin enables efficient separation of rAAV5 from cell lysate.

Recombinant adeno-associated virus (rAAV) vectors have displayed enormous potential as a platform for delivery of gene therapies. Purification of rAAV at industrial scale involves a series of elaborate, material, and time-consuming midstream steps, such as clarification by depth filtration and concentration/buffer exchange by tangential flow filtration. In this study, we developed a filter-less flow capture method for purification of rAAV serotype 5, using a high-gradient magnetic separator and magnetic Mag Sepharose beads coupled to an AVB affinity ligand. In under 2 h, we captured and eluted rAAV5 directly from ∼5 L of cell lysate with a recovery yield of 63% (±5%, n = 3). Compared to cell lysate, the eluate showed a 3-log reduction of host cell DNA and host cell proteins. The process developed eliminates the need for filtration and column chromatography in the early steps of industrial rAAV purification. This will be of high value for industrial-scale manufacturing of rAAVs by reducing time and material in the purification process, without compromising product recovery and purity.

Electrophysiological characteristics in children with listening difficulties, with or without auditory processing disorder.

Objective: To determine if the auditory middle latency responses (AMLR), auditory late latency response (ALLR) and auditory P300 were sensitive to auditory processing disorder (APD) and listening difficulties in children, and further to elucidate mechanisms regarding level of neurobiological problems in the central auditory nervous system. Design: Three-group, repeated measure design. Study sample: Forty-six children aged 8-14 years were divided into three groups: children with reported listening difficulties fulfilling APD diagnostic criteria, children with reported listening difficulties not fulfilling APD diagnostic criteria and normally hearing children. Results: AMLR Na latency and P300 latency and amplitude were sensitive to listening difficulties. No other auditory evoked potential (AEP) measures were sensitive to listening difficulties, and no AEP measures were sensitive to APD only. Moderate correlations were observed between P300 latency and amplitude and the behavioural AP measures of competing words, frequency patterns, duration patterns and dichotic digits. Conclusions: Impaired thalamo-cortical (bottom up) and neurocognitive function (top-down) may contribute to difficulties discriminating speech and non-speech sounds. Cognitive processes involved in conscious recognition, attention and discrimination of the acoustic characteristics of the stimuli could contribute to listening difficulties in general, and to APD in particular.

Contralateral suppression of otoacoustic emissions in a clinical sample of children with auditory processing disorder.

OBJECTIVE: The suppression of evoked otoacoustic emissions (EOAE) may serve as a clinical tool to evaluate the medial olivocochlear (MOC) reflex, which is thought to aid speech discrimination (particularly in noise) by selectively inhibiting cochlear amplification. The present study aimed to determine if contralateral transient evoked otoacoustic emission (TEOAE) suppression was present in a clinical sample of children with listening difficulties with and without auditory processing disorder (APD). DESIGN: A three-group, repeated measure design was used. STUDY SAMPLE: Forty three children aged 8-14 years underwent an auditory processing assessment and were divided into three groups: children with reported listening difficulties with APD, children with reported listening difficulties without APD, and children with normal hearing. APD was defined as per British Society of Audiology. RESULTS: TEOAE suppression was present in all three participant groups. No significant group, age or ear effects were observed for TEOAE suppression in dB or as a normalised index. CONCLUSION: Contralateral TEOAE suppression method could not be used as a clinical tool to identify APD in this study's participating children and did not support the hypothesised link between reduced MOC function and general listening difficulties in background noise in children with or without APD.

One-step integrated clarification and purification of a monoclonal antibody using Protein A Mag Sepharose beads and a cGMP-compliant high-gradient magnetic separator.

Monoclonal antibodies are a dominant component of today's biopharmaceutical market and are typically purified by classical platform processes. However, high costs and rising demands are drivers for the development of new, efficient and flexible integrated purification processes. Currently, high-gradient magnetic separation as a direct capturing tool for protein purification suffers from the lack of suitable GMP-compliant separation equipment for industrial scale. As a solution for this bottleneck, we present a purification process for a monoclonal antibody directly from CHO cell culture by use of protein A-functionalized magnetic particles together with the first pilot-scale GMP-compliant 'rotor-stator' high-gradient magnetic separator. Five consecutive purification cycles were performed, achieving consistent yields of over 85% and purities of over 95%. Stable cell viabilities during the magnetic separation process enable integration of the device as an in situ product removal tool. A comparison with state-of-the-art protein A column-based purification processes reveals a 3-times higher process productivity per mL of applied resin and demonstrates the great potential of magnetic separation in downstream processing.

Normative data for diagnosing auditory processing disorder in Norwegian children aged 7-12 years.

OBJECTIVE: The main purpose of this research was to obtain normative data for auditory processing tests for Norwegian speaking children. DESIGN: Participants were administered routine audiological tests and an auditory processing test-battery consisting of Filtered Words, Competing Words, Dichotic Digits, Gaps In Noise, Duration- and Frequency Pattern, Binaural Masking Level Difference and HIST Speech in Noise test. A group of 10-year-old children were retested after two weeks. The effects ear, age and gender and the test-retest reliability were investigated. STUDY SAMPLE: There were 268 normal hearing children aged 7-12 years who participated in the study. RESULTS: Results revealed no differences between genders. The children showed improving performance by age on all tests, except from the Gaps In Noise and Binaural Masking Level Difference. As expected, the children showed a right ear advantage on dichotic speech tests that decreased with age. The test-retest reliability for the tests was good, with a small learning effect on the Filtered Words test. CONCLUSION: Normative data were established and the preferred tests for diagnosing Auditory Processing Disorder were suggested for Norwegian children aged 7-12 years.

Hearing loss in the Royal Norwegian Navy: A longitudinal study.

The aims of this longitudinal study were to investigate a significant threshold shift (STS) among personnel working on board the Royal Norwegian Navy's (RNoN) vessels between 2012 and 2014 and to identify possible determinants of STS. Hearing thresholds were measured by pure tone audiometry in two consecutive examinations (n = 226). STS was defined as an average change in hearing thresholds ≥ + 10 dB at 2,000 Hz, 3,000 Hz, and 4,000 Hz in either ear. Determinants of STS were assessed through a questionnaire. The incidence of STS was 23.0%. Significant determinants of STS were the number of episodes of temporary threshold shifts (TTS) in the Navy, exposure to continuous loud noise during work on board, and the number of gun shots (in the Navy, hunting, and sports). This study indicated a significant association between noise exposure on board Navy vessels and development of STS.

High myopia-excavated optic disc anomaly associated with a frameshift mutation in the MYC-binding protein 2 gene (MYCBP2).

PURPOSE: To investigate the ocular and neurologic manifestations, and to identify the causative mutation in a family with an excavated optic disc anomaly, high myopia, enlarged axial lengths, and abnormal visual evoked response (VER). DESIGN: Prospective observational case series with whole exome sequencing. METHODS: Institutional study of 8 family members from 3 generations. Clinical examination included visual field examination, optical coherence tomography, axial length measurement, audiometry, visual evoked response (VER), orbital and cerebral magnetic resonance imaging (MRI), and renal ultrasound. DNA was analyzed by whole exome sequencing and Sanger sequencing. Main outcome measures were clinical and radiological findings, and DNA sequence data. RESULTS: Three affected family members, a father and his 2 daughters, were examined. The parents and siblings of the father were healthy. Affected individuals presented with excavated optic discs, high myopia (-1.00 to -16.00 diopters), and increased axial lengths. Reduced visual acuity (0.05-0.8) and decreased sensitivity on visual field examination were observed. VER revealed prolonged latency times. Affected eyes appeared ovoid on MRI and the father had thin optic nerves. Exome sequencing revealed that the father was heterozygous for a de novo 5 bp deletion in MYCBP2, c.5906_5910del; p.Glu1969Valfs*26. The same mutation was found in his 2 affected daughters, but not in his parents or siblings, or in public databases. CONCLUSION: We describe a distinct excavated optic disc anomaly associated with high myopia and increased axial length. The condition appears to follow an autosomal dominant pattern and segregate with a deletion in MYCBP2. We suggest naming this entity high myopia-excavated optic disc anomaly.

Hearing loss in the royal Norwegian Navy: a cross-sectional study.

OBJECTIVES: Prior studies have indicated a high prevalence of noise-induced hearing loss (NIHL) among Navy personnel; however, it is not clear whether this is caused by work on board. The present study aimed to assess the prevalence of hearing loss among Navy personnel in the Royal Norwegian Navy (RNoN), and to investigate whether there is an association between work on board RNoN vessels and occurrence of hearing loss. METHODS: Navy personnel currently working on board RNoN vessels were recruited to complete a questionnaire on noise exposure and health followed by pure tone audiometry. Hearing loss was defined as hearing threshold levels ≥25 dB in either ear at the frequencies 3,000, 4,000 or 6,000 Hz. Hearing thresholds were adjusted for age and gender using ISO 7029. RESULTS: The prevalence of hearing loss among Navy personnel was 31.4 %. The work exposure variables: years of work in the Navy, years on vessel(s) in the Navy and years of sailing in the Navy were associated with reduced hearing after adjusting for age, gender and otitis as an adult. Among the work exposure variables, years of sailing in the Navy was the strongest predictor of reduced hearing, and significantly reduced hearing was found at the frequencies 1,000, 3,000 and 4,000 Hz. CONCLUSIONS: Our results indicate that time spent on board vessels in the RNoN is a predictor of reduced hearing.

Frequency discrimination in ears with and without contralateral cochlear dead regions.

OBJECTIVE: The purpose of this study was to test the ability to discriminate low-frequency pure-tone stimuli for ears with and without contralateral dead regions, in subjects with bilateral high-frequency hearing loss; we examined associations between hearing loss characteristics and frequency discrimination of low-frequency stimuli in subjects with high-frequency hearing loss. DESIGN: Cochlear dead regions were diagnosed using the TEN-HL test. A frequency discrimination test utilizing an adaptive three-alternative forced choice method provided difference limens for reference frequencies 0.25 kHz and 0.5 kHz. STUDY SAMPLE: Among 105 subjects with bilateral high-frequency hearing loss, unilateral dead regions were found in 15 subjects. These, and an additional 15 matched control subjects without dead regions, were included in the study. RESULTS: Ears with dead regions performed best at the frequency discrimination test. Ears with a contralateral dead region performed significantly better than ears without a contralateral dead region at 0.5 kHz, the reference frequency closest to the mean audiogram cut-off, while the opposite result was obtained at 0.25 kHz. CONCLUSIONS: Results may be seen as sign of a contralateral effect of unilateral dead regions on the discrimination of stimuli with frequencies well below the audiogram cut-off in adult subjects with bilateral high-frequency hearing loss.

Forte Fortissimo for amateur musicians: no effect on otoacoustic emissions.

Many amateur musicians are likely to be exposed to high sound levels during rehearsals. Measuring otoacoustic emissions (OAE) is an objective technique to assess hearing ability and can be used as an early predictor of hearing loss. This study aimed to record sound levels at an amateur brass band rehearsal and to examine the cochlear function of the musicians by evaluating distortion product (DP) OAE amplitudes before and after sound exposure. Twenty-four brass band musicians and 10 office workers were examined with personal noise measurements and DPOAE tests before and after a music rehearsal or one office workday, respectively. The sound levels in the brass band ranged from 92 to 100 dBA, LaEQ 96 dBA. However, the amateur brass band members had no change in DPOAEs after this exposure compared to pre-exposure levels and compared to office workers.

Strategies for selecting optimal sampling and work-up procedures for analysing alkylphenol polyethoxylates in effluents from non-activated sludge biofilm reactors.

Trace-level analysis of alkylphenol polyethoxylates (APEOs) in wastewater containing sludge requires the prior removal of contaminants and preconcentration. In this study, the effects on optimal work-up procedures of the types of alkylphenols present, their degree of ethoxylation, the biofilm wastewater treatment and the sample matrix were investigated for these purposes. The sampling spot for APEO-containing specimens from an industrial wastewater treatment plant was optimized, including a box that surrounded the tubing outlet carrying the wastewater, to prevent sedimented sludge contaminating the collected samples. Following these changes, the sampling precision (in terms of dry matter content) at a point just under the tubing leading from the biofilm reactors was 0.7% RSD. The findings were applied to develop a work-up procedure for use prior to a high-performance liquid chromatography-fluorescence detection analysis method capable of quantifying nonylphenol polyethoxylates (NPEOs) and poorly investigated dinonylphenol polyethoxylates (DNPEOs) at low microg L(-1) concentrations in effluents from non-activated sludge biofilm reactors. The selected multi-step work-up procedure includes lyophilization and pressurized fluid extraction (PFE) followed by strong ion exchange solid phase extraction (SPE). The yields of the combined procedure, according to tests with NP10EO-spiked effluent from a wastewater treatment plant, were in the 62-78% range.

Psychological well-being among cochlear implant users: a comparison with the general population.

OBJECTIVES: In this study, we describe the psychological well-being experienced by cochlear implant users, and compare it to that of the general population in Norway. METHODS: We conducted a questionnaire-based cross-sectional multicentre study, in which 53 of 73 (73%) unilateral cochlear implant users and 177 of 318 (56%) matched reference subjects from the general population participated. Psychological well-being was measured by the Psychological General Well-being Index (PGWB). The indexes of the cochlear implant users and the general population sample were compared using ordinary linear regression, adjusting for the matching variables. RESULTS: Mean age of the cochlear implant users was 56 (SD 16) years, 66% of them were females, and they had used their implants for a mean of 18 (SD 6) months. The distribution of socio-demographic variables was similar to that of the general population sample. We found no difference in mean PGWB index between the cochlear implant users and the general population sample with indexes of 85.5 and 83.4, respectively. The 95% confidence interval for the adjusted difference was -3.0 to 6.9. The cochlear implant users had slightly better scores in the dimensions general health and vitality. DISCUSSION: The cochlear implant users experienced a psychological well-being similar to that of the general population.

Dichotic listening and otoacoustic emissions: shared variance between cochlear function and dichotic listening performance in adults with normal hearing.

The present study investigated a possible connection between speech processing and cochlear function. Twenty-two subjects with age range from 18 to 39, balanced for gender with normal hearing and without any known neurological condition, were tested with the dichotic listening (DL) test, in which listeners were asked to identify CV-syllables in a nonforced, and also attention-right, and attention-left condition. Transient evoked otoacoustic emissions (TEOAEs) were recorded for both ears, with and without the presentation of contralateral broadband noise. The main finding was a strong negative correlation between language laterality as measured with the dichotic listening task and of the TEOAE responses. The findings support a hypothesis of shared variance between central and peripheral auditory lateralities, and contribute to the attentional theory of auditory lateralization. The results have implications for the understanding of the cortico-fugal efferent control of cochlear activity.

Development of a high-performance liquid chromatography-fluorescence detection method for analyzing nonylphenol/dinonylphenol-polyethoxylate-based phosphate esters.

A novel reversed phase HPLC method for the simultaneous analysis of surfactants containing nonylphenol/dinonylphenol-polyethoxylates and their o-phosphate esters is reported, in which eluting substances are detected fluorescently. Their chemical structures were elucidated by direct infusion electrospray-mass spectrometry in positive mode. The limits of quantification and range of the method were determined to be 0.1mg and 0.1-100 mg surfactant L(-1), respectively, with a reproducibility (RSD) at a concentration of 38 mg surfactant L(-1) of 5.6%. The accuracy was determined by spiking selected process water samples with known amounts of surfactant, and recoveries were typically in the 82-102% range.

Effects of cochlear implants: a qualitative study.

This qualitative study aimed to gain a deeper insight into the effects of cochlear implants on recipients' lives, as perceived by the recipients themselves. To obtain this insight, we used four open-ended questions. We invited 107 adult patients from two Norwegian implant centres. Of these, 74 returned completed questionnaires (69%). Recipients perceived that they had got 'A new life' with the implant. Contributing to this global category of effects were four other categories. These concerned subjects' interactions with the world around them, experience of themselves, ability to hear the world around them and finally certain device-related issues. Overall, psychological well-being was improved. The questionnaire described might prove a useful tool in the follow-up of patients.

Preparation and characterization of prototypes for multi-modal separation media aimed for capture of negatively charged biomolecules at high salt conditions.

Several prototypes of multi-modal ligands suitable for the capture of negatively charged proteins from high conductivity (28 mS/cm) mobile phases were coupled to Sepharose 6 Fast Flow. These new prototypes of multi-modal anion-exchangers were found by screening a diverse library of multi-modal ligands and selecting anion-exchangers resulting in elution of test proteins at high ionic strength. Candidates were then tested with respect to breakthrough capacity of BSA in a buffer adjusted to a high conductivity (20 mM Piperazine and 0.25 M NaCl, pH 6.0). The recovery of BSA was also tested with a salt step (from 0.25 to 2.0 M NaCl using 20 mM Piperazine as buffer, pH 6.0) or with a pH-step to pH 4.0. We have found that non-aromatic multi-modal anion-exchange ligands based on primary or secondary amines (or both) are optimal for the capture of proteins at high salt conditions. Furthermore, these new multi-modal anion-exchange ligands have been designed to take advantage not only of electrostatic but also hydrogen bond interactions. This has been accomplished through modification of the ligands by the introduction of hydroxyl groups in the proximity of the ionic group. Experimental evidence on the importance of the relative position of the hydroxyl groups on the ligand in order to improve the breakthrough capacity of BSA has been found. Compared to strong anion-exchangers such as Q Sepharose Fast Flow the new multi-modal weak anion-exchangers have breakthrough capacities of BSA at mobile phases of 28 mS/cm and pH 6.0 that are 20-30 times higher. The new multi-modal anion-exchangers can also be used at normal anion-exchange conditions and with either a salt step or a pH-step to acidic pH can accomplish the elution of proteins. In addition, the functional performance of the new anion-exchangers was found to be intact after treatment in 1.0 M sodium hydroxide solution for 1 week. A number of multi-modal anion-exchange ligands based on aromatic amines exhibiting high breakthrough capacity of BSA have been found. With these ligands recovery was often found to be low due to strong non-electrostatic interactions. However, for phenol derived anion-exchange media the recovery can be improved by desorption at high pH.

Preparation and characterization of prototypes for multi-modal separation aimed for capture of positively charged biomolecules at high-salt conditions.

Several prototypes of aromatic (Ar) and non-aromatic (NoAr) cation-exchange ligands suitable for capture of proteins from high conductivity (ca. 30 mS/cm) mobile phases were coupled to Sepharose 6 Fast Flow. These new prototypes of multi-modal cation-exchangers were found by screening a diverse library of multi-modal ligands and selecting cation-exchangers resulting in elution of test proteins at high ionic-strength. Candidates were then tested with respect to breakthrough capacity of bovine serum albumin (BSA), human IgG and lysozyme in buffers adjusted to a high conductivity. By applying a salt-step or a pH-step the recoveries were also tested. We have found that aromatic multi-modal cation-exchanger ligands based on carboxylic acids seem to be optimal for the capture of proteins at high-salt conditions. Experimental evidence on the importance of the relative position of the aromatic group in order to improve the breakthrough capacity at high-salt conditions has been found. It was also found that an amide group on the alpha-carbon was essential for capture of proteins at high-salt conditions. Compared to a strong cation-exchanger such as SP Sepharose Fast Flow the best new multi-modal weak cation-exchangers have breakthrough capacities of BSA, human IgG and lysozyme that are 10-30 times higher at high-salt conditions. The new multi-modal cation-exchangers can also be used at normal cation-exchange conditions and with either a salt-step or a pH-step (to pH-values where the proteins are negatively charged) to accomplish elution of proteins. In addition, the functional performance of the new cation-exchangers was found to be intact after treatment in 1.0 M sodium hydroxide solution for 10 days. For BSA it was also possible to design cation-exchangers based on non-aromatic carboxyl acid ligands with high capacities at high-salt conditions. A common feature of these ligands is that they contain hydrogen acceptor groups close to the carboxylic group. Furthermore, it was also possible to obtain high breakthrough capacities for lysozyme and BSA of a strong cation-exchanger (SP Sepharose Fast Flow) if phenyl groups were attached to the beads. Varying the ligand ratio (SP/Phenyl) could be used for optimizing the function of mixed-ligand ion-exchange media.

Acute mastoiditis in a Norwegian population: a 20 year retrospective study.

We have retrospectively examined the nature of acute mastioditis (in western Norway) during a 20 year period (1980-2000). Sixty-one cases of AM were identified in 57 patients with a mean age of 3.6 years. We found no significant change in the incidence of AM during the last 20 years. Seven patients were treated solely with intravenous antibiotics and myringotomies. Fifty patients also underwent cortical mastoidectomy, four cases with bilateral surgery. Antibiotic treatment was given to 31 of the patients before admission to hospital and this group had a significant longer duration of symptoms (12.4 days) compared to untreated patients (7.3 days). Streptococcus pneumoniae was the most common organism recovered from patient cultures. Surgery was found to correlate to patients with retroauricular fluctuation or to children with at least two of the three clinical signs: protrusion of the ear, retroauricular oedema and swelling of the ear canal. Our data show that clinical examination only reveal 50% of the cases with surgically proven retroauricular subperiostal abscess. We therefore recommend a CT scan of patients treated conservatively.