Sound localization with bilateral bone conduction devices.

PURPOSE: To investigate sound localization in patients bilaterally fitted with bone conduction devices (BCDs). Additionally, clinically applicable methods to improve localization accuracy were explored. METHODS: Fifteen adults with bilaterally fitted percutaneous BCDs were included. At baseline, sound localization, (un)aided pure-tone thresholds, device use, speech, spatial and qualities of hearing scale (SSQ) and York hearing-related quality of life (YHRQL) questionnaire were measured. Settings to optimize sound localizing were added to the BCDs. At 1 month, sound localization was assessed again and localization was practiced with a series of sounds with visual feedback. At 3 months¸ localization performance, device use and questionnaire scores were determined again. RESULTS: At baseline, one patient with congenital hearing loss demonstrated near excellent localization performance and four other patients (three with congenital hearing loss) localized sounds (quite) accurately. Seven patients with acquired hearing loss were able to lateralize sounds, i.e. identify whether sounds were coming from the left or right side, but could not localize sounds accurately. Three patients (one with congenital hearing loss) could not even lateralize sounds correctly. SSQ scores were significantly higher at 3 months. Localization performance, device use and YHRQL scores were not significantly different between visits. CONCLUSION: In this study, the majority of experienced bilateral BCD users could lateralize sounds and one third was able to localize sounds (quite) accurately. The localization performance was robust and stable over time. Although SSQ scores were increased at the last visit, optimizing device settings and a short practice session did not improve sound localization.

A mobile sound localization setup.

In this paper, a mobile sound localization setup is described that can be used to measure a persons' localization performance in a sophisticated way. With this mobile setup, researchers can travel to subjects, and studies are not limited by the willingness of participants to visit the clinic. In the setup, sounds are presented within a partial sphere in both the horizontal (-70° to 70° azimuth) and vertical (-35° to 40° elevation) plane. Participants are asked to indicate the perceived sound origin by pointing with a head-mounted LED. Head movements are recorded and instantly visualized (i.e. online target response plots). Depending on the research question, the setup can be adjusted for more advanced or simplified measurements, making the setup suitable for a wide range of research questions. The rationale for building this mobile setup was to test horizontal sound localization abilities (binaural hearing) and vertical sound localization abilities (monaural hearing) of children and patients who were otherwise not accessible for testing. In this setup loudspeakers are not visible and subjects are asked to indicate the perceived sound direction by a natural head-pointing response towards the perceived location. An advantage of the implemented pointing-method is the playful manner in which children are tested. They are 'shooting' at the perceived sound target location with a head-mounted LED and have fun while performing the test. •We present a mobile sound localization setup suitable for measuring horizontal and vertical sound localization in children and adult patients in the convenience of their own environment.

Improved directional hearing of children with congenital unilateral conductive hearing loss implanted with an active bone-conduction implant or an active middle ear implant.

Different amplification options are available for listeners with congenital unilateral conductive hearing loss (UCHL). For example, bone-conduction devices (BCDs) and middle ear implants. The present study investigated whether intervention with an active BCD, the Bonebridge, or a middle ear implant, the Vibrant Soundbridge (VSB), affected sound-localization performance of listeners with congenital UCHL. Listening with a Bonebridge or VSB might provide access to binaural cues. However, when fitted with the Bonebridge, but not with a VSB, binaural processing might be affected through cross stimulation of the contralateral normal hearing ear, and could interfere with processing of binaural cues. In the present study twenty-three listeners with congenital UCHL were included. To assess processing of binaural cues, we investigated localization abilities of broadband (BB, 0.5-20 kHz) filtered noise presented at varying sound levels. Sound localization abilities were analyzed separately for stimuli presented at the side of the normal-hearing ear, and for stimuli presented at the side of the hearing-impaired ear. Twenty-six normal hearing children and young adults were tested as control listeners. Sound localization abilities were measured under open-loop conditions by recording head-movement responses. We demonstrate improved sound localization abilities of children with congenital UCHL, when listening with a Bonebridge or VSB, predominantly for stimuli presented at the impaired (aided) side. Our results suggest that the improvement is not related to accurate processing of binaural cues. When listening with the Bonebridge, despite cross stimulation of the contralateral cochlea, localization performance was not deteriorated compared to listening with a VSB.

Hearing aid fitting for visual and hearing impaired patients with Usher syndrome type IIa.

OBJECTIVES: Usher syndrome is the leading cause of hereditary deaf-blindness. Most patients with Usher syndrome type IIa start using hearing aids from a young age. A serious complaint refers to interference between sound localisation abilities and adaptive sound processing (compression), as present in today's hearing aids. The aim of this study was to investigate the effect of advanced signal processing on binaural hearing, including sound localisation. DESIGN AND PARTICIPANTS: In this prospective study, patients were fitted with hearing aids with a nonlinear (compression) and linear amplification programs. Data logging was used to objectively evaluate the use of either program. Performance was evaluated with a speech-in-noise test, a sound localisation test and two questionnaires focussing on self-reported benefit. RESULTS: Data logging confirmed that the reported use of hearing aids was high. The linear program was used significantly more often (average use: 77%) than the nonlinear program (average use: 17%). The results for speech intelligibility in noise and sound localisation did not show a significant difference between type of amplification. However, the self-reported outcomes showed higher scores on 'ease of communication' and overall benefit, and significant lower scores on disability for the new hearing aids when compared to their previous hearing aids with compression amplification. CONCLUSIONS: Patients with Usher syndrome type IIa prefer a linear amplification over nonlinear amplification when fitted with novel hearing aids. Apart from a significantly higher logged use, no difference in speech in noise and sound localisation was observed between linear and nonlinear amplification with the currently used tests. Further research is needed to evaluate the reasons behind the preference for the linear settings.

Deletion of two exons from the Lymnaea stagnalis beta1-->4-N-acetylglucosaminyltransferase gene elevates the kinetic efficiency of the encoded enzyme for both UDP-sugar donor and acceptor substrates.

Lymnaea stagnalis UDP-GlcNAc:GlcNAcbeta-R beta1-->4-N-acetylglucosaminyltransferase (beta4-GlcNAcT) is an enzyme with structural similarity to mammalian UDP-Gal:GlcNAcbeta-R beta1-->4-galactosyltransferase (beta4-GalT). Here, we report that also the exon organization of the genes encoding these enzymes is very similar. The beta4-GlcNAcT gene (12.5 kilobase pairs, spanning 10 exons) contains four exons, encompassing sequences that are absent in the beta4-GalT gene. Two of these exons (exons 7 and 8) show a high sequence similarity to part of the preceding exon (exon 6), suggesting that they have originated by exon duplication. The exon in the beta4-GalT gene, corresponding to beta4-GlcNAcT exon 6, encodes a region that has been proposed to be involved in the binding of UDP-Gal. The question therefore arose, whether the repeating sequences encoded by exon 7 and 8 of the beta4-GlcNAcT gene would determine the specificity of the enzyme for UDP-GlcNAc, or for the less preferred UDP-GalNAc. It was found that deletion of only the sequence encoded by exon 8 resulted in a completely inactive enzyme. By contrast, deletion of the amino acid residues encoded by exons 7 and 8 resulted in an enzyme with an elevated kinetic efficiency for both UDP-sugar donors, as well as for its acceptor substrates. These results suggest that at least part of the donor and acceptor binding domains of the beta4-GlcNAcT are structurally linked and that the region encompassing the insertion contributes to acceptor recognition as well as to UDP-sugar binding and specificity.

A Lymnaea stagnalis gene, with sequence similarity to that of mammalian beta 1-->4-galactosyltransferases, encodes a novel UDP-GlcNAc:GlcNAc beta-R beta 1-->4-N-acetylglucosaminyltransferase.

A cDNA encoding a novel glycosyltransferase, that may be involved in a variant pathway for the synthesis of complex type oligosaccharide chains, was cloned from the pond snail Lymnaea stagnalis. By heterologous hybridization, using bovine beta 1-->4-galactosyltransferase cDNA as probe, a genomic clone from a snail library was isolated. This genomic clone was subsequently used to clone the corresponding cDNA from a prostate gland library. The isolated cDNA encodes a polypeptide of 490 amino acids with a type II membrane protein topology typical for glycosyltransferases. The carboxyl-terminal part, encoding the putative catalytic domain, reveals considerable sequence similarity with the corresponding region of mammalian beta 1-->4-galactosyltransferases, suggesting an evolutionary relationship. Expression of this cDNA in COS cells and insect cells revealed that the encoded enzyme transfers GlcNAc, rather than Gal or GalNAc, from the corresponding nucleotide sugars to several beta-N-acetylglucosaminides. Structural characterization by 1H NMR spectroscopy of products formed in vitro demonstrated that the enzyme can be identified as a UDP-GlcNAc:GlcNAc beta-R beta 1-->4-N-acetylglucosaminyl-transferase. A new family of glycosyltransferases has hereby been discovered, consisting of enzymes that act on acceptor substrates with a terminal beta-linked GlcNAc residue and establish a beta 1-->4-linkage, but have a different nucleotide sugar requirement.

Isolation and characterization of three cDNAs coding for Rab proteins from the albumen gland of the mollusc Lymnaea stagnalis.

Three cDNA clones encoding small GTP-binding proteins, LS-Rab1, LS-Rab2 and LS-Rab18a were isolated from a cDNA library from the albumen gland of the pulmonate snail Lymnaea stagnalis. Comparison of the deduced amino acid sequences with available sequences from the EMBL/Data Bank revealed that LS-Rab1 and LS-Rab2 show a sequence identity of 89-90% to the mammalian Rab1 and Rab2 proteins, and can therefore be regarded as the L. stagnalis homologs. LS-Rab18a may be considered a new member of the Rab subfamily, closely related to mouse Rab18 (74% amino acid identity). Interestingly, LS-Rab1 and LS-Rab2 share a very high sequence conservation with their mammalian homologs (95-97%) over the first 178-191 N-terminal amino acids, whereas the C-terminal part is almost completely divergent, except for their extreme ends (2-4 amino acids). The implications of these observations for the understanding of Rab-targeting signals are discussed. The LS-rab cDNAs were expressed in COS-7M6 cells. The resulting 22-kDa products were shown to bind GTP. In the albumen gland mRNA, levels of LS-rab1 appeared to be much higher than those of LS-rab2 and LS-rab18a, suggesting an important role for the LS-Rab1 protein in the albumen gland.

Use of the enterobacterial outer membrane protein PhoE in the development of new vaccines and DNA probes.

PhoE protein is a major outer membrane protein of Escherichia coli. The polypeptide spans the membrane 16 times, thereby exposing 8 regions at the cell surface. Insertions in these regions did not affect the biogenesis of the protein. Therefore, we considered the possibility of using PhoE as a vector for the exposure of foreign antigenic determinants at the cell surface, with the ultimate goal of constructing new (live oral) vaccines. Via recombinant DNA techniques, B-cell epitopes of VP1 protein of foot-and-mouth-disease virus were inserted in the exposed regions of PhoE. The inserted epitopes were antigenic and immunogenic in the PhoE-associated conformation. Guinea pigs, immunized with such a hybrid protein were protected against viral challenge. Similarly, a T-cell epitope of the 65 kDa heat-shock protein of Mycobacterium tuberculosis remained antigenic and immunogenic in the PhoE-associated conformation, although recognition by the cells of the immune system was dependent on the amino acids, flanking the epitope. When the amino acid sequences of the PhoE proteins of different members of the family of Enterobacteriaceae are compared, the cell surface-exposed regions are hypervariable. Therefore, we considered the possibility that the DNA segments encoding these regions are species-specific. By using synthetic oligonucleotides corresponding to such DNA segments, primer couples for the specific detection and identification of different enterobacterial species, including Salmonella, by polymerase chain reactions have been developed.

Outer membrane protein PhoE as a carrier for the exposure of foreign antigenic determinants at the bacterial cell surface.

PhoE protein is an abundant outer membrane protein of the Escherichia coli K-12 outer membrane. This protein can be used as an exposure system to produce foregin antigenic determinants and for their transport to the bacterial cell surface. The system is very flexible, since insertions varying in length and nature could be made in different cell surface-exposed regions of PhoE, without interfering with the assembly process of the mutant proteins into the outer membrane. Two antigenic determinants of the structural VP1 protein of foot-and-mouth disease virus were inserted in different combinations in four cell surface-exposed regions of PhoE. The epitopes were exposed at the bacterial cell surface and they keep their antigenic and immunogenic properties in this PhoE-associated conformation. Immunization of guinea pigs with one hybrid protein, containing a combination of the two epitopes inserted in the fourth exposed region, resulted in complete protection against challenge with the virus. A T-cell epitope of the 65 kDa heat shock protein of Mycobacterium tuberculosis was inserted in the fourth exposed region of PhoE and in vitro proliferation of two T-cell specific clones was demonstrated. Thus, the PhoE exposure system has been shown to be suitable for presentation of both B-cell and T-cell determinants to the immune system. Furthermore, good expression of the hybrid protein in attenuated Salmonella strains, which can be used as live oral vaccines, was shown.

Efficient recognition by rat T cell clones of an epitope of mycobacterial hsp 65 inserted in Escherichia coli outer membrane protein PhoE.

PhoE is a pore-forming protein, abundantly expressed in the Escherichia coli outer membrane. Previous investigations have shown the possibility of inserting antigenic determinants in cell surface-exposed regions of PhoE by recombinant DNA techniques without disturbing the biogenesis and the functioning of the protein. This method proved to be successful for foot-and-mouth disease virus B cell determinants. We have now shown for the first time that PhoE can also be used as a carrier molecule for T cell epitopes. A well-characterized T cell epitope (180-188) of the 65-kDa heat-shock protein (hsp 65) of Mycobacterium tuberculosis was expressed in PhoE and tested for recognition by specific T cell clones. Specific and efficient T cell proliferation was found after stimulation with this protein construct in vitro. Interestingly, paraformaldehyde fixation of antigen-presenting cells did not abrogate T cell recognition. Thus, in contrast to hsp 65 itself, recognition of epitope 180-188 in the context of PhoE appeared to be independent of antigen-processing events. At the level of polyclonal T cell responses the epitope in the context of PhoE is recognized more efficiently than 180-188 as synthetic peptide or in the context of the hsp 65 molecule itself. These findings indicate that PhoE may serve as attractive vaccine carrier not only for B, but also for T cell epitopes. Furthermore, the possibility for expression of PhoE constructs in attenuated Salmonella typhimurium strains offers the exciting prospect of new types of live oral vaccines expressing selected combinations of B and T cell epitopes.

Protection of guinea-pigs against foot-and-mouth disease virus by immunization with a PhoE-FMDV hybrid protein.

A hybrid protein was constructed containing two antigenic determinants of the structural protein VP1 of foot-and-mouth disease virus, inserted in a cell surface-exposed region of Escherichia coli outer membrane protein PhoE. Immunization of guinea-pigs with partially purified protein resulted in high levels of neutralizing antibodies and complete protection against challenge with the virus.

Outer-membrane PhoE protein of Escherichia coli K-12 as an exposure vector: possibilities and limitations.

The phosphate-limitation-inducible outer-membrane protein (PhoE) of Escherichia coli K-12 can be used in an expression system as a carrier for foreign antigenic determinants, facilitating their transport to the bacterial cell surface. The system is very flexible, since insertions varying in length and nature can be made in different cell-surface-exposed regions of PhoE protein, without interfering with the assembly process into the outer membrane. Multiple insertions of an antigenic determinant can be made in the second and eighth exposed regions, resulting in a total insert length of up to 30 and 50 amino acid (aa) residues. Insertions can be made in two exposed regions, simultaneously. However, some limitations were encountered, e.g., insertion of eight or more hydrophobic aa residues affected both the translocation process across the inner membrane and the assembly process into the outer membrane. Also, the insertion of sequences containing many charged residues resulted in accumulation of precursor protein in the cytoplasm.

Outer membrane PhoE protein of Escherichia coli as a carrier for foreign antigenic determinants: immunogenicity of epitopes of foot-and-mouth disease virus.

Outer membrane protein PhoE of Escherichia coli was used for the expression of antigenic determinants of foot-and-mouth disease virus. Five hybrid PhoE proteins were constructed containing different combinations of two antigenic determinants of VP1 protein of the virus. The hybrid proteins were expressed in two E. coli strains and the proteins were correctly assembled into the outer membrane. The inserted epitopes were exposed at the surface of the cell and were antigenic in this PhoE-associated conformation. Immunization experiments, performed with partially purified protein, resulted in all cases in a significant anti-peptide antibody titre. In one case in which the hybrid protein with the largest insert was used, a neutralizing antibody response was detected.

Role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein.

To investigate the role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein, deletions were generated in two presumed cell surface-exposed regions of the protein. Intact cells expressing these mutant proteins were recognized by PhoE-specific monoclonal antibodies, which recognize conformational epitopes on the cell surface-exposed parts of the protein and/or were sensitive to a PhoE-specific phage. This shows that the polypeptides were normally incorporated into the outer membrane. When the deletions extended four amino acid residues into the seventh presumed membrane-spanning segment, the polypeptides accumulated in the periplasm. In conclusion, exposed regions of PhoE protein apparently do not play an essential role in outer membrane localization, which is consistent with the observation that these regions are hypervariable when PhoE is compared to the related proteins OmpF and OmpC. In contrast, the membrane-spanning segments are essential for the assembly process.

Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12.

Amino acid residue arginine-158 of the outer membrane protein PhoE of Escherichia coli K-12 has been shown to be cell-surface-exposed [Korteland et al. (1985) Eur. J. Biochem. 152, 691-697]. To study the effects of small insertions in this region of the protein on its biogenesis and characteristics, a unique restriction site was created by site-directed mutagenesis in a plasmid carrying the phoE gene and oligonucleotides of 12-74 bp were inserted. The insertions did not interfere with incorporation into the outer membrane since (a) several monoclonal antibodies, directed against the cell-surface-exposed part of PhoE protein, bound to whole cells producing the altered proteins and (b) the proteins formed functional pores for the uptake of beta-lactam antibiotics. The binding of one monoclonal antibody and of the PhoE-specific phages TC45 and TC45hrN3 was disturbed by the insertions, showing that this region of the protein is immunogenic and is involved in the binding of both of these phages. The functioning of the mutant pores was characterized both in vivo by studying the uptake of beta-lactam antibiotics and in vitro after the reconstitution of the proteins in black lipid films. The pore characteristics changed depending on the nature of the inserted amino acids. Addition of a negatively charged amino acid resulted in decreased anion-selectivity, whereas insertion of a positive charge and deletion of a negative charge had only a small influence.

Analysis of structure-function relationships in Escherichia coli K12 outer membrane porins with the aid of ompC-phoE and phoE-ompC hybrid genes.

To study structure-function relationships in the outer membrane pore proteins OmpC and PhoE of Escherichia coli K12, we have constructed a series of phoE-ompC hybrid genes in which DNA encoding part of one protein is replaced by the homologous part of the other gene. The hybrid gene products were incorporated normally into the outer membrane, allowing their functional characterization. Combined with previous studies, the present results permit the identification of regions involved in determining functions and properties in which the native PhoE and OmpC proteins differ, such as pore characteristics, receptor activity for phages and binding of monoclonal antibodies. Most of these properties were found to be determined by multiple regions clearly separated in the primary structure. The combined phage and antibody binding data have demonstrated that at least five distinct regions in PhoE and OmpC are exposed at the cell surface. The locations of these regions are in agreement with a previously proposed model for porin topology.

Use of outer membrane protein PhoE as a carrier for the transport of a foreign antigenic determinant to the cell surface of Escherichia coli K-12.

PhoE protein is an abundant transmembrane protein from the Escherichia coli K-12 outer membrane. A synthetic oligodeoxynucleotide corresponding to an antigenic determinant of the C-terminal part of the VP1 protein of foot-and-mouth disease virus was inserted into the phoE gene in an area corresponding to a cell surface-exposed region of the PhoE protein. The level of expression of the hybrid protein was normal and the protein was incorporated into the outer membrane. The VP1-epitope was exposed at the cell surface since intact cells were recognized by a monoclonal antibody which was raised against the virus.

Localization of functional domains in E. coli K-12 outer membrane porins.

The genes ompC and phoE of Escherichia coli K-12 encode outer membrane pore proteins that are very homologous. To study the structure-function relationship of these proteins, we have constructed a series of ompC-phoE hybrid genes in which the DNA encoding part of one protein is replaced by the corresponding part of the other gene. These hybrid genes were easily obtained by using in vivo recombination. The fusion sites in the hybrid genes were localized by restriction enzyme mapping. The hybrid gene products were normally expressed and they were characterized with respect to functions and properties in which the native OmpC and PhoE proteins differ, such as pore characteristics, the receptor activity for phages and the binding of specific antibodies. Three regions within the N-terminal 130 amino acids were localized which determine pore characteristics and a segment between residues 75 and 110 contains amino acids which determine specificity for PhoE phages. A major cell surface-exposed region is located between residues 142 and 267. This region contains residues which are required for the binding of monoclonal antibodies directed against the cell surface-exposed part of PhoE and residues which determine specificity for OmpC phages.