Escherichia coli SMC protein, MukB, which is essential for its in vivo function, requires its interaction with the dimeric kleisin, MukF that in turn interacts with the KITE protein, MukE. Here we demonstrate that, in addition, MukB interacts specifically with Acyl Carrier Protein (AcpP) that has essential functions in fatty acid synthesis. We characterize the AcpP interaction at the joint of the MukB coiled-coil and show that the interaction is necessary for MukB ATPase and for MukBEF function in vivo.Structural Maintenance of Chromosomes (SMC) complexes act ubiquitously to compact DNA linearly, thereby facilitating chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE proteins that interact with kleisins. The ATPase activity of the  E. coli MukBEF is an SMC complex that plays key roles in chromosome organization-segregation. Here the authors show that the interaction between MukBEF and the Acyl Carrier Protein (AcpP) is essential for MukBEF activity in vitro (ATPase) and in vivo. In Escherichia coli, the SMC complex, MukBEF, is composed of three proteins, the SMC protein MukB, the kleisin, MukF and the KITE protein, MukE6. Although divergent in primary sequence from other SMC proteins, MukB shares common ancestral and architectural features including an ABC-like ATPase head domain, a ~50 nm long antiparallel coiled-coil and a dimerization hinge domain . This enables the joining of two dimeric MukBEF complexes into dimer of dimer (DoD) complexes that are essential for in vivo MukBEF function16. MukE dimers interact with MukF; thus the complete MukBEF complex has a 4:4:2 B:E:F stoichiometry16. MukB ATP hydrolysis results from the engagement of two head domains that create two shared ATP binding sites. MukB alone has minimal ATPase activity, but is activated in the presence of MukF and further modulated by the interactions with MukE and DNA14. ATPase activity is essential for in vivo function, as mutant MukB proteins that are deficient in ATP hydrolysis  or ATP binding  display ΔmukB phenotypes18. Cells lacking functional MukBEF, grown in a minimal medium under permissive conditions, have disorganized chromosomes with misplaced genetic loci, and fail to properly segregate sister chromosomes, leading to anucleate cell production20. Furthermore, ΔmukB cells are inviable during rapid growth that supports overlapping replication cycles, apparently because of failures in chromosome segregation20. Because the lack of functional MukB can result in reduced proliferation in organisms that cause disease phenotypes, such as Vibrio vulnificus21, MukB is a potential target for new antimicrobial therapeutics22.SMC complexes act by linearly compacting chromosomal DNA, to organize chromosomes and facilitate their segregation prior to cell division, through individualizing newly replicated sister chromosomesain Fig. . In addiead Fig. 5,14,15. 25. Since AcpP is a highly abundant E. coli protein 26, it was not clear from early reports whether this reflected a specific interaction or a fortuitous association. AcpP is an essential hub protein that through a covalent interaction with its phosphopantetheine (PPant) arm, shuttles intermediates along the fatty acid biosynthesis pathway by a series of acyl transfer reactions . In addition, AcpP has been shown to interact with other unrelated protein partners including SpoT, IscS, and SecA30. Searches for binding partners of AcpP have also indicated an interaction with MukB, although any functional significance to this interaction was not explored31.Acyl Carrier Protein (AcpP) has been repeatedly reported to co-purify with MukBons Fig.  (revieweter), consistent with the impaired ATPase function. We propose that AcpP is an essential partner for MukBEF action in chromosome organization segregation.Here, we identify the AcpP binding site on MukB and analyze the functional consequences of this interaction in vitro and in vivo. We show that the interaction of AcpP with a conserved region of the coiled-coils, in the MukB joint, is essential for MukB ATPase activity. MukBEF complexes of wild-type stoichiometry assemble in the absence of AcpP. The binding of AcpP to MukB inhibits higher-order intermolecular coiled-coil interactions between MukB molecules in vitro, consistent with a hypothesis in which AcpP binding to the MukBEF joint facilitates essential conformational changes in the complexes during cycles of ATP binding and hydrolysis. Mutations within the MukB–AcpP binding site reduce AcpP association and thus impair MukB ATPase activity. Importantly, these mutations result in an altered pattern of MukBEF complex localization within cells, including an increased association with the replication termination region  MukB and three truncated variants were purified and tested for the presence of associated AcpP using SDS-PAGE , consisting of the ATPase head and the first ~30% of head-proximal coiled-coils, but not with MukBH (MukB Head), consisting of just the ATPase head domain. Even with the addition of recombinant AcpP, no MukBH-AcpP binary complexes were detected  consisting of just the head-proximal coiled-coils  revealed AcpP interacts with MukB with a 1:1 monomer-monomer stoichiometry , we focused our analysis on residues surrounding K1125. K1125 is located within the C-terminal helix in the coiled-coil proximal to the ATPase head domain . Treatment of MukBain Fig. . Crystal 13 Fig. . Sequenc 13 Fig. .36  and MukBKRK . We observed a reduction in the levels of co-purified AcpP in MukBK1114E, MukBW1117E, and MukBC1118E samples, as judged by SDS-PAGE, confirming the importance of these residues to the MukB–AcpP interface , to a level comparable to MukBF co-purified with AcpP (27.2 ± 1.2 ATP molecules/MukB2/min) and similar to that reported previously (where the samples will have contained co-purified AcpP)14. Consistent with this, the addition of MukE to AcpP containing MukBF samples modestly inhibited MukF activation . The relative contributions of these forms are explored later.To characterize the functional significance of the MukB–AcpP interaction, AcpP was depleted from WT MukB during heparin purification using an extended salt gradient, where AcpP-depleted MukB eluted as a second peak with a higher retention time  to MukB, given that both AcpP and MukF are required for MukB ATPase. Since MukB residues K1114–K1125 are in proximity to residues L1219 and L1226, which have been implicated in MukF N-terminal domain binding14 Fig. , we usednMS Fig. . These MnMS Fig. , arise wnMS Fig. . Complemion Fig. . Furtherion Fig. , therebyK1114E and MukBKRK mutant proteins, which are deficient in AcpP binding, were also able to form complexes with MukEF  , demonstrate that the AcpP interaction with MukB is not a prerequisite for MukF binding.MukBkEF Fig. . AdditioHN complexes that form with MukEF in the presence and absence of AcpP and which are more amenable to stoichiometry analysis on native gels. This approach was informed by our initial demonstration that AcpP binding perturbs the formation of a BS3-induced intermolecular cross-link between two K1125 MukBHN residues in the joint . Neither dimeric MukBHN2E4F2 complexes, nor the equivalent of DoD complexes, which form by head engagement in the presence of AMPPNP (MukBHN4E4F2)14 required AcpP  and MukBHN8E8F4 (+AMPPNP); these disappeared in the presence of AcpP  and MukBHN4E8F4 (−AMPPNP) complexes arise from the dimerization of MukBHN4E4F2 and MukBHN2E4F2 complexes, respectively, through coiled-coil interactions in the AcpP binding region of the joint where the K1125 residues were cross-linked by BS3  failed to produce these presumptive higher-order complexes, irrespective of the presence of AcpP, but still formed AMPPNP-dependent MukBK1114E, MukBW1117E, MukBC1118E, MukBKK and MukBKRK)  the activity of MukBKRK was only 7.6 ± 1.4 ATP molecules/MukB2/min (~32% of the WT MukBEF activity in the presence of 50 μM AcpP)  Fig. , d. All RK) Fig. . MukBW11pP) Fig. . These dE. coli results in a mixture of both apo-AcpP and holo-AcpP species . Nevertheless, the formation of this disulfide bond could contribute to the stabilization of the AcpP–MukB interaction.AcpP overexpression in cpP Fig. e, f. FurΔmukB background strain. ΔmukB cells exhibited temperature-sensitive growth in the rich medium at 37 °C, which was restored by basal expression from the multi-copy number plasmid pET21a encoding a WT mukB gene , MukBRK  and MukBKC  cells were Muk+, as assessed by growth at 37 °C, indicating that the temperature sensitivity of MukBKRK is likely due to a lack of AcpP interaction, although we cannot eliminate the possibility that the phenotype is influenced by protein conformational changes induced by the combined mutations. Consistent with our observations, multiple substitutions in other AcpP-target protein interfaces are required to abolish AcpP binding with other AcpP binding proteins in addition to MukB41.Next, we assessed the viability of MukB mutants impaired in AcpP binding by transforming plasmid-borne genes of the mutants into a ion Fig. . MukBKR ΔmukB cells containing a functional mYpet fusion to the endogenous MukE protein and fluorescent markers located near oriC (ori1) and close to the middle of ter (ter3)17. In cells expressing WT MukB, fluorescent MukBEF foci were associated with the ori1 locus, as reported previously by ourselves and others, for MukBEF expressed from the endogenous chromosomal locus , had its MukBEF distributed over the whole nucleoid, with few, if any, defined fluorescent foci  and those that had a reduced ori1 association and increased ter3 association. These latter variants all contained the MukBK1114E mutation either alone, or in combination with one or two further mutations in the AcpP binding region, MukBKK and MukBKRK, respectively. MukBK1114E, showed a small reduction in association with ori1 (47.7 ± 2.0%) and a complementary increase in association with ter3 (14 ± 1.3%) . Despite these similarities, only MukBKRK cells exhibited temperature-sensitive growth, while the double mutants, like the single ones, grew at 37 °C. The behavior of the mutants in relation to ori1/ter3 localization was independent of whether there was a single ori1 locus present (in cells soon after birth that had not replicated or segregated the ori1 locus), or whether there were two sister ori1 loci, after replication and segregation  Fig. . MukBKK ion Fig. . Neverthion Fig. , suggestori1 to ter3 colocalization in mutants carrying the MukBK1114E mutation was further evident when the normalized distribution of ori1, ter3 and MukBEF foci along the longitudinal cell axis was plotted , or abnormally positioned foci (mukBEQ). MukBKRK expressing cells contained 2.4 ± 2.0% anucleate cells, likely higher than WT cells, but less than the Muk- cells, whereas MukBK1114E and MukBKK expressing cells had close to WT levels of anucleate cells . Overall, these observations are consistent with an increasing impairment in MukBEF function as AcpP binding is decreased in a stepwise fashion by a progressive mutation in the AcpP binding region of MukB. Despite MukBKRK cells still being able to form chromosome-associated MukBEF complexes, we propose that at least a substantial fraction of these are impaired in MukBEF function, consistent with a defect in ATP hydrolysis, temperature-sensitive growth, increased anucleate cell formation, and consequent preferential location within ter.We next analyzed the percentage of anucleate cells within each population, assessed by lack of DAPI staining and absence of G1116E and MukBV1124E also exhibited temperature sensitivity, although the defect was not as complete as for ΔmukB cells. <10% of MukBV1124E plated cells yielded colonies at 37 °C, with the surviving colonies being relatively small. A higher proportion of MukBG1116E expressing cells grew at 37 °C, but the colonies were again smaller  complexes of full-length MukB complexed with MukEF, the functional unit in vivoent Fig. , demonstent Fig. , generatmer Fig. . An alte46. AcpP is highly abundant in E. coli cells26  and is involved in a wide range of essential steps in fatty acid biosynthesis, along with other specific interactions. Since it exists in a wide range of acylated and unacylated forms, it is challenging to imagine how any modulated MukBEF activity on chromosomes results from cellular changes in AcpP as a consequence of changes in fatty acid metabolism. Parenthetically, MukBEF function only becomes essential for cell viability under condition of rapid growth during which overlapping rounds of replication occur18. Indeed, the MukBEF clade of SMC complexes is largely confined to bacteria that support overlapping rounds of replication as part of their lifestyle. Nevertheless, MukBEF is clearly active and important for normal chromosome organization-segregation under conditions of slow growth, when each round of replication is initiated and terminated in the same cell cycle18. Although our work has not identified any specific form of AcpP that preferentially interacts with MukB or influences its activity, any connection between cellular metabolism and the activity of MukBEF complexes on the chromosome, is likely to involve a specific form (or forms) of AcpP whose abundance and activity is under metabolic control. In this scenario, levels of fatty acid biosynthesis could be coordinated in some way with chromosome organization segregation mediated by MukBEF. Our assays have found no evidence for this; apo-AcpP and holo-AcpP had comparable activities in stimulating MukB ATPase in vitro, while a disulfide between the PPant free thiol and MukBC1118 is not essential for either ATPase or in vivo function. An alternative scenario to one in which the AcpP–MukB interaction modulates MukBEF action with fatty acid and lipid synthesis is one in which this is an “accidental” recruitment of a protein during evolution, just like the recruitment of the “metabolic enzymes”, ArgR, ArcA and PepA, as essential accessory factors in site-specific recombination essential for multi-copy plasmid stability48.Given that other SMC complexes can act in the absence of AcpP binding to the joint, it is difficult to rationalize why this requirement has evolved in the MukBEF clade; there is no obvious connection between AcpP and the other MukBEF co-evolved players that include MatP, SeqA, Dam, and topoisomerase IV32. Similarly, the interaction between AcpP and the SecA component of the protein-membrane translocase machinery could couple fatty acid–lipid metabolism with protein transport through the inner membrane. Although it has been proposed that binding of AcpP to MukB might mediate interactions with the SecA component of the protein-membrane translocase machinery, to allow for correct oriC positioning within cells50, in our opinion this appears unlikely. A Turing patterning mechanism positions the largest cluster of MukBEF complexes on the chromosome at either midcell or ¼ positions and the ori association with these clusters results directly from the depletion of MukBEF complexes from ter as a consequence of their dissociation directed by their interaction with MatP-matS18. We are unaware of any compelling evidence that replication origins are associated either with SecA complexes or the inner membrane.Elsewhere, it has been proposed that the interaction of AcpP with proteins uninvolved in acyl transfer may contribute to the coordination of cellular metabolism. For example, the SpoT–AcpP interaction may help coordinate the cells protein synthesis stringent response to fatty acid starvationori1 positioning in AcpP binding defective MukBKRK expressing cells is similar to that observed in other situations where MukBEF function is impaired sufficiently to give a temperature-sensitive growth phenotype, regardless of whether it is a defect in ATP binding (MukBDA), hydrolysis (MukBEQ), or where there is a complete lack of MukB. The ability of MukBKRK expressing cells to form fluorescent clusters of MukBEF complexes demonstrates that under conditions of impaired AcpP binding, these complexes can still associate with the chromosome, despite a substantial fraction of these being impaired in MukBEF function. These observations are consistent with a defect in ATP hydrolysis arising from impaired AcpP binding, with the consequent preferential location of chromosomal-bound complexes to within ter, similar to ATP hydrolysis-defective MukBEQEF complexes that cannot be displaced from MatP-bound matS sites within ter, because of impaired ATP hydrolysis18. Since a proportion of cellular MukBKRK is likely to be bound by AcpP, given the latter’s abundance, we believe this explains why some MukBKRK complexes are ori-associated and at least partly functional, albeit with cells having a Muk− phenotype as assessed by temperature sensitivity. In a situation where MukB could not bind AcpP at all, we do not know whether the disposition of the heads would allow sufficient ATP binding to associate with ter as in mukBEQ cells, or whether ATP binding would be so transient that few if any chromosome-associated complexes would be present, as in mukBDA cells. The demonstration of a progressive defect in the ability of MukB to respond to increasing AcpP in ATPase assays when the MukBK1114E substitution is combined with one (MukBKK), or two further substitutions (MukBKRK) in the AcpP binding region of MukB, correlates well with the increasing in vivo defects that indicate impaired ATPase hydrolysis. Also, the MukBKK mutant, which had 60% of WT ATPase activity in vitro at 50 μM AcpP has a Muk+ phenotype in vivo, while MukBKRK, with 32% ATPase activity in vitro at 50 μM AcpP was Muk-. By comparison, a study of the relationship between the in vitro ATPase activity of B. subtills mutant SMC complexes and their rate of DNA translocation in vivo, which can be assessed directly in this organism, showed mutants that had 6-20% residual ATPase activity in vitro retained 60–80% of DNA translocation activity in vivo and had an almost normal Smc+ phenotype assessed by growth51.The perturbed The work reported here, provides the platform for future studies of the MukBEF mechanism and how it is influenced by AcpP. This will require an integrated combination of structural, biochemical, biophysical, and genetic studies and may elucidate more mechanistic and functional insights into the MukBEF clade of proteins, which has evolved an apparently unique architecture, along with a distinctive family of co-evolved partners.mukB 3xFLAG C3013I (NEB)14. Proteins were purified as previously described14. Briefly, after purification using TALON superflow resin, protein samples were further purified using either a HiTrap DEAE FF column  for MukE and MukF, or HiTrap Heparin HP column  for MukB and derivatives. Appropriate fractions (selected by 4–20% gradient SDS-PAGE) were pooled and concentrated by centrifugal filtration  for loading onto a Superdex 200 Increase 10/300 GL  column equilibrated in storage buffer  glycerol). Peak fractions were assessed for purity (>90%) by SDS-PAGE/Coomassie staining, snap-frozen as aliquots, and stored at −80 °C.MukB-His, MukE-His and His-MukF were overexpressed from pET vectors in C3013I cells (NEB), MukB-His variants were expressed in strain FL01, which is acpP with a thrombin-cleavable N-terminal 6xHis tag in C3031I cells (NEB). 2 L cultures of LB supplemented with kanamycin (25 μg/mL) were grown at 37 °C to an OD600 of 0.5–0.6 and induced with β-d-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM. After overnight incubation at 18 °C, cells were harvested by centrifugation, resuspended in lysis buffer  supplemented with a protease inhibitor tablet and homogenized. Cell debris was removed by centrifugation and cell lysate mixed with ~5 mL of TALON Superflow resin and incubated for 30 mins at 4 °C. The slurry was poured into a column and washed with 10× volume lysis buffer, 4× volume wash buffer A , and 1× volume wash buffer B . Bound proteins were eluted using elution buffer  and dialyzed overnight in lysis buffer with the addition of thrombin protease (10 U per 1 mg of AcpP). Uncleaved protein was removed by incubation with TALON Superflow resin before concentrating for loading onto a Superdex 75 Increase 10/300 GL  column equilibrated in lysis buffer. Peak fractions were assessed for purity (>90%) by SDS-PAGE/Coomassie staining, snap-frozen as aliquots, and stored at −80 °C. P. aeruginosa AcpH and B. subtilis Sfp were purified as described37. Briefly, 6×His-tagged proteins were first purified using TALON superflow resin. After elution with imidazole, AcpH was immediately desalted using a PD-10 column (Sephadex G-25) into storage buffer . Sfp was instead dialyzed overnight into storage buffer . Protein samples were then assessed for purity before aliquoting and storing at −80 °C.AcpP was expressed from a pET28a plasmid encoding 2 (12.5 mM), MnCl2 (5 mM) and TCEP (5 mM). Reactions were incubated at 37 °C for 24 h and monitored by 20% Urea-PAGE37. The addition of the PPant group was achieved in a similar manner, except the final reaction contained 50 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 0.5 mM TCEP, 1 mM CoA, and 0.1 mg/mL BsSfp. Protein samples were then purified by size exclusion chromatography, snap-frozen as aliquots, and stored at −80 °C.The removal of the AcpP-PPant group was achieved by mixing purified AcpP (1 mg/mL) with AcpH (0.1 mg/mL) in the presence of MgCl14. 150 µL samples containing standard reaction buffer supplemented with ATP to a final concentration of 1.3 mM were assayed in a BMG Labtech PherAstar FS plate reader at 25 °C. The data were analyzed using MARS data analysis software. Quantitation of phosphate release was determined using the extinction coefficient of 11,200 M–1 cm−1 for the phosphate-dependent reaction at A360 nm at pH 7.0. Final reactions contained 65 mM NaCl. Data were then plotted and analyzed in Prism version 8.3.0.ATP hydrolysis was analyzed in steady-state reactions using an ENZCheck Phosphate Assay Kit (Life Technologies)m/z range, S-lens RF 100%, collisional activation in the HCD cell 50–200 V, trapping gas pressure setting kept at 7.5, temperature 100-200 °C, resolution of the instrument 12500. The noise level was set at 3 rather than the default value of 4.64. No in-source dissociation was applied. Data were analyzed using Xcalibur 4.2 (Thermo Scientific) and UniDec52. Data collection for all spectra was repeated at least three times. Errors on observed masses were calculated by following a previously described method53.Prior to MS analysis, protein samples were buffer exchanged into 200 mM ammonium acetate pH 8.0, using a Biospin-6 (BioRad) column and introduced directly into the mass spectrometer using gold-coated capillary needles . Data were collected on a Q-Exactive UHMR mass spectrometer (ThermoFisher). The instrument parameters were as follows: capillary voltage 1.1 kV, quadrupole selection from 1000 to 20,000 HN (0–4.5 μM) was incubated with MukF (1.5 μM), MukE (3 μM) and AcpP (at the indicated concentrations) in 4× Native PAGE sample buffer  with DTT (1 mM) and MgCl2 (1 mM) for 30 min at 22 ± 1 °C. Samples were then analyzed using 3–12% native Bis-Tris gels with dark blue cathode buffer. Gels were destained in 40% (v/v) ethanol, 10% (v/v) acetic acid for 30 min before destaining with 8% (v/v) acetic acid overnight.MukB or MukBMukB samples were heated to 95 °C in LDS Sample Buffer (4X) (ThermoFisher NP0007) with or without the presence of a reducing agent. Samples were then analyzed using NuPAGE™ 7%, Tris-Acetate SDS-PAGE (ThermoFisher EA03585BOX) followed by western blots using anti-AcpP  as primary and goat anti-rabbit HPR  as a secondary antibody.HN, BS3 (50–250× molar excess over MukBHN) was added to a sample of MukBHN, co-purified with or without AcpP, or with the addition of recombinant AcpP (at various molar ratios). Reactions were incubated at RT for 30 min then quenched with Tris buffer (50 mM) before diluting with SDS-loading buffer and analyzed using SDS-PAGE. Gel bands corresponding to cross-linked species were excised, reduced with TCEP (10 mM), and alkylated with 2-Chloroacetamide (50 mM) before overnight digestion with Trypsin. Peptides samples were speed-vac dried and resuspended in 5% formic acid/5% DMSO before LC–MS/MS analysis. For cross-links involving WT MukB, BS3 (1 mM) was added to samples of AcpP-depleted MukBEF  in the presence and absence of recombinant AcpP and AMPPNP (1 mM). Samples were allowed to react for 2 h at RT before quenching with ammonium bicarbonate (100 mM). Samples were then denatured with urea (4 M) in ammonium bicarbonate buffer (100 mM) before the addition of TCEP (10 mM) followed by 2-chloroacetamide (50 mM). Samples were then pre-digested with LysC (1 μg/100 μg of the sample) for 2 h before overnight digestion with trypsin (1 μg/40 μg of sample). Tryptic digestion was stopped with the addition of formic acid (5%). Digested peptides were centrifuged at top speed for 30 min at 4 °C to remove undigested material. The supernatant was loaded onto a handmade C18 stage tip, pre-activated with 100% acetonitrile. Peptides were washed twice in TFA 0.1%, eluted in 50% acetonitrile/0.1% TFA and speed-vacuum dried. Peptides were resuspended into 2% acetonitrile/0.1% formic acid before LC–MS/MS analysis.For cross-links involving MukBm/z, resolution 70,000; AGC target, 3e6, maximum injection time, 50 ms). The 10 most intense peaks were selected for higher-energy collision dissociation (HCD) fragmentation at 30% of normalized collision energy. HCD spectra were acquired in the Orbitrap at resolution 17,500, AGC target 5e4, maximum injection time 120 ms with fixed mass at 180 m/z. Charge exclusion was selected for unassigned and 1+ ions. The dynamic exclusion was set to 40 s. Tandem mass spectra were searched using pLink software version 2.3.9.54 against an E. coli protein sequence database. Peptide mass tolerance was set at 20ppm on the precursor and fragment ions. Data were filtered at FDR below 5% at the PSM level. Tandem mass spectra of cross-linked peptides were extracted and annotated using pLabel 2.455.Peptides were separated by nano-liquid chromatography (Thermo Scientific Easy-nLC 1000) coupled in line a Q-Exactive mass spectrometer equipped with an Easy-Spray source (ThermoFisher Scientific). Peptides were trapped onto a C18 PepMac100 precolumn  using Solvent A . The peptides were further separated onto an Easy-Spray RSLC C18 column  using a 120 min linear-gradient (15–35% solvent B (0.1% formic acid in acetonitrile)) at a flow rate of 200 nL/min. The raw data were acquired on the mass spectrometer in a data-dependent acquisition mode (DDA). Full-scan MS spectra were acquired in the Orbitrap  glycerol, 2 μg/mL thiamine, and required amino acids  at 30 °C. An overnight culture was diluted ~1000-fold and grown to A600 0.05–0.2 and deposited on a medium containing agarose pad after staining with 1 μg/mL DAPI. The ΔmukB cells used had a functional mYpet fusion to the endogenous mukE gene, fluorescently labeled ori1 (mCherry), and ter3 (mCerulean)  (hyg), tetO240@ter3 (1644) (gen), ΔleuB::Plac-lacI-mCherry-frt, ΔgalK::Plac-tetR-mCerulean-frt, ∆araBAD (AraC+), mukE-mYPet-frt-T1-T2-Para-ΔmukB-kan)18, expressing basal levels of pBAD24 plasmid-borne WT MukB, the indicated MukB mutants, or empty pBAD24 plasmid control (ΔmukB). Epifluorescence images were acquired on a Nikon Ti-E inverted microscope equipped with a perfect focus system, a ×100 NA 1.4 oil immersion objective (Nikon), an sCMOS camera (Hamamatsu Flash 4), a motorized stage (Nikon), an LED excitation source (Lumencor SpectraX) and a temperature chamber (Okolabs). Fluorescence images were collected with 100 ms exposure time using excitation from a LED source. Phase-contrast images were collected for cell segmentation. Images were acquired using NIS-Elements software (Nikon). Cell segmentation and spot detection from the fluorescence channel were performed using SuperSegger56. Low-quality spots were filtered out with a fixed threshold for all data sets (4.5). The threshold was selected to minimize the number of falsely identified MukBEF foci within background signal yet maximize the number of foci analyzed; the threshold ensured ~90% of cells expressing WT MukB contained at least one MukBEF focus, whilst ~90% of ΔmukB cells had none. The percentages of cells containing one or more spots, distances to the closest ori1/ter3 marker, and localization along the long cell axis were calculated using MATLAB (MathWorks) as described18. Percentages of anucleate cell formation were calculated using a custom script. Custom scripts used in this study are available in Supplementary Software The ability of MukB variants to complement the temperature-sensitive growth defect of a Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Software 1Reporting Summary


Sensorineural hearing loss (SNHL) affects approximately 466 million people worldwide, which is projected to reach 900 million by 2050. Its histological characteristics are lesions in cochlear hair cells, supporting cells, and auditory nerve endings. Neurological disorders cover a wide range of diseases affecting the nervous system, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), autism spectrum disorder (ASD), etc. Many studies have revealed that neurological disorders manifest with hearing loss, in addition to typical nervous symptoms. The prevalence, manifestations, and neuropathological mechanisms underlying vary among different diseases. In this review, we discuss the relevant literature, from clinical trials to research mice models, to provide an overview of auditory dysfunctions in the most common neurological disorders, particularly those associated with hearing loss, and to explain their underlying pathological and molecular mechanisms. Histological characteristics of age-related hearing loss include degenerative pathology in cochlear hair cells, supporting cells, and auditory nerve endings, resulting in irreversible damage to the sensory epithelium of the cochlea , moderate (35–50 dB), moderately severe (50–65 dB), severe (65–80 dB), profound (80–95 dB), and total (≥95 dB) hearing loss. Lesions in the cochlea, auditory nerve, and central auditory pathway induce sensorineural hearing loss (SNHL); nearly a third of the population over the age of 65 is suffering from it cochlea . Sound i cochlea . OHCs fu cochlea , and env cochlea . As dama cochlea . These e cochlea . The pul cochlea . The asc cochlea .Neurological disorders include a broad range of diseases that affect the nervous system, of which neurodegenerative diseases and neurodevelopmental disorders have been widely discussed. In the elderly, neurodegenerative diseases are common causes of morbidity and cognitive impairment . The proAlzheimer’s disease is a progressive neurodegenerative disorder and the most common form of dementia. One in 10 people aged over 65 years is affected by AD, and the incidence increases with age . Late-onAPP, PSEN1 (Presenilin 1), and PSEN2 (Presenilin 2) are responsible for the occurrence of FAD, and it was reported that mutations in these genes alter APP processing, induce Aβ formation, and then initiate tau pathology. In contrast, more than 90% of AD patients appear sporadically, which usually presents with late-onset AD  and NFTs are extensively distributed throughout relay stations in the ascending auditory pathway . Many ADClinical literature suggests that midlife hearing loss is independently correlated with accelerated progression of sporadic AD and incident dementia. The degree of hearing loss was positively associated with an increased risk of dementia. AD-related neuropathology was found in the central auditory pathway but was not clinically identified in the peripheral auditory pathway. Although multiple clinical studies have been designed to determine the relationship between hearing loss and AD, there are still some flaws in the experimental design. Most of the important information has been neglected, including the extent of hearing loss, measurements of auditory processing, differences between sexes, and auditory condition in different AD classifications, resulting in restricted access to information to determine the relationship between auditory dysfunction and AD. Mouse model studies further illustrated that hearing loss is associated with AD development. In the APP/PS1 mouse model, the shifts of ABR and distortion product otoacoustic emission (DPOAE) preceded the neuropathy observed in the brain. Loss of hair cells and SGNs was observed in AD mouse models, which was likely induced by the spread of AD-related neuropathology (Aβ deposition and tau pathology) in the cochlea. However, the three AD-related mouse models were all designed with mutations in FAD genes, which could not completely mimic the pathogenesis of sporadic AD. The central auditory pathway has not yet been studied in these mouse models. High-frequency hearing loss has been observed in patients with AD and incident dementia. Moreover, both the central and peripheral auditory pathways are affected by AD-related neuropathology, but the concrete cochlear pathology is still debated. The results vary among studies due to different mouse models, sampling times, and hearing measurements. Hence, for a comprehensive understanding of AD-related hearing loss, standard observation criteria should be established in further studies.Huntington’s disease is an autosomal-dominantly inherited disorder with a mean prevalence of 2.71 per 100,000 individuals worldwide . HD manihuntingtin lead to polyglutamine (polyQ) extension at the N-terminal of Htt protein, and mutant Htt accumulates to cause neuronal loss 150 mice, thresholds measured by click and tone bursts ABR analysis at 15 months of age revealed that approximately 20 dB thresholds were increased for click and tone bursts at frequencies of 4 and 8 kHz. No differences were observed at frequencies of 16 and 32 kHz because wild type mice developed presbycusis at 15 months of age 150 mice. Moreover, pathological studies of the central auditory pathway, including cochlear nuclei, superior olivary complex, and auditory cortex, should be performed to determine auditory-related lesions and guide auditory-related tests carried out for HD patients and high-risk groups.Auditory dysfunction appears to be authentic for HD. Clinical studies have shown that hearing impairments and auditory processing dysfunction are present in HD patients. Delayed ERPs are suggested to be a potential predictor of HD. While there is no consensus that auditory sense, processing, or discrimination is uniparted or jointly present in HD, more research objectives, detailed acoustic measurements, and specified auditory items should be included. Hearing impairment is solid in HD mouse models, and hearing loss precedes the occurrence of motor defects and worsens with the progression of HD in R6/2-HD mice. The loss of hair cells and SGNs was also observed. Hdh(CAG)Parkinson’s disease is a neurodegenerative disorder characterized by static tremor, bradykinesia, rigidity, and postural instability, affecting 1–2 per 1,000 individuals . Before The association between hearing dysfunction and PD suggests a common neuropathological background. Lewy pathology and dopaminergic neuronal degeneration are two primary neuropathological features of PD that spread as PD progresses . Other pThere have been no reports on auditory dysfunction and auditory anatomy in PD mouse model. Although manipulation of specific genes reported in familial PD, including transgenic overexpression for α-synuclein and leucine-rich repeat kinase 2 and knockout models for Parkin, DJ-1, phosphatase, and tensin homolog-induced novel kinase 1, made it possible to establish many mouse models, none of them recapitulate key clinical and neuropathological features of PD entirely, especially in the absence of neurodegeneration of dopaminergic neurons . While tAutism spectrum disorder is a neurodevelopmental disorder characterized by social isolation, stereotypical behaviors, and interests, with a prevalence of approximately 1% worldwide and has a strong male predominance . Sense dCurrently, most previous studies identified increased rates of audiological dysfunctions in ASD, including hearing impairments, hyperacusis, difficulty in sound discrimination with background noise and speech sounds encoding . A higheELMOD3 and FGF2. ELMOD3 is involved in autosomal recessive non-syndromic deafness disability  and concluded that hearing loss was present in these four disorders. However, the related auditory lesions and underlying mechanisms vary among them.150 mice, mHtt aggregation was observed. However, for a comprehensive understanding of auditory dysfunction in AD and HD, more clinical trials involving more subjects and including a variety of detailed auditory measurements should be carried out, and complete studies on auditory circuitry (from the cochlea to the auditory cortex) of mouse models should be conducted in the future.In AD, high-frequency hearing loss was observed in both the patient and mouse models. Moreover, Aβ deposition appeared to be the initial neurological etiology. Auditory studies on AD mouse models raise the possibility that the auditory pathway is more sensitive to AD-related neuropathology and auditory dysfunction, especially hearing loss, presents before the onset of cognitive impairments. Thus, auditory measurements can provide a reference for preliminary diagnosis and early interventions for patients with AD. Hearing impairments and auditory processing dysfunction have been observed in HD patients. In R6/2-HD mice, hearing loss precedes the characterized presentations of HD, and in the SGNs of Hdh(CAG)A broad range of auditory dysfunctions, including hearing loss, abnormal speech discrimination, and sound lateralization, have been reported in PD, and asymptomatic hearing impairments appear to be a new non-motor symptom of PD patients, and hearing measurements may act as a non-invasive potential biomarker and indicator of disease severity. There are no transgenic mouse models that can completely mimic the important neuropathological features of PD, especially the neurodegeneration of dopaminergic neurons. Hence, better PD mouse models should be established, and to gain insight into the underlying molecular mechanisms, auditory studies in existing mouse models still worth exploring.Many auditory dysfunctions, including hearing impairments, hyperacusis, difficulty in sound discrimination, and speech sound encoding, have been detected in patients with ASD. Concomitant hearing loss makes the diagnosis of ASD more challenging. As both disorders affect communication abilities in children and early hearing interventions have been reported to improve social communication and reduce aggression in ASD, comprehensive audiological assessments should be carried out in confirmed and suspected ASD in children. Moreover, approximately 10% of ASD cases have an identifiable genetic background. Clinical and transgenic mouse model studies revealed the involvement of hearing impairments, raising the possibility that associated genes may act as potential deafness genes. Hence, more potential ASD-related candidate genes should be identified, and specific transgenic mouse models should be applied to explore the function of autism-related genes in the auditory system.Sensorineural hearing loss affects a large population of people worldwide, and the impact of hearing loss is broad and profound, including delayed language development in children, social isolation, and psychological illness. Hearing loss is not only present in neurological disorders mentioned above but can also affect the prognosis of these diseases to some extent. Hence, exploring hearing loss in neurological disorders is beneficial for understanding the pathogenesis and improving the prognosis of these diseases.SL and CC wrote the manuscript. LL, XM, XZ, and AL collated the resource. JC, XQ, and XG wrote and reviewed the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hearing loss is a significant health problem that can result from a variety of exogenous insults that generate oxidative stress and inflammation. This can produce cellular damage and impairment of hearing. Radiation damage, ageing, damage produced by cochlear implantation, acoustic trauma and ototoxic drug exposure can all generate reactive oxygen species in the inner ear with loss of sensory cells and hearing loss. Cisplatin ototoxicity is one of the major causes of hearing loss in children and adults. This review will address cisplatin ototoxicity. It includes discussion of the mechanisms associated with cisplatin-induced hearing loss including uptake pathways for cisplatin entry, oxidative stress due to overpowering antioxidant defense mechanisms, and the recently described toxic pathways that are activated by cisplatin, including necroptosis and ferroptosis. The cochlea contains G-protein coupled receptors that can be activated to provide protection. These include adenosine A1 receptors, cannabinoid 2 receptors (CB2) and the Sphingosine 1-Phosphate Receptor 2 (S1PR2). A variety of heat shock proteins (HSPs) can be up-regulated in the cochlea. The use of exosomes offers a novel method of delivery of HSPs to provide protection. A reversible MET channel blocker that can be administered orally may block cisplatin uptake into the cochlear cells. Several protective agents in preclinical studies have been shown to not interfere with cisplatin efficacy. Statins have shown efficacy in reducing cisplatin ototoxicity without compromising patient response to treatment. Additional clinical trials could provide exciting findings in the prevention of cisplatin ototoxicity. The cochlea can be damaged by a variety of insults that can result in acquired sensorineural hearing loss. Hearing loss can result from ototoxic drugs, noise trauma, and injury from cochlear implant insertion, ageing and radiation. A common theme demonstrated by these harmful entities is the production of oxidative stress by free radical generation leading to inflammation and loss of sensory cells and reduction in hearing acuity. This review will address cisplatin ototoxicity. It includes an up to date presentation of the mechanisms associated with cisplatin-induced hearing loss, current treatments to ameliorate cisplatin ototoxicity and novel and exciting future treatments that are being explored.Cisplatin is widely used as a chemotherapeutic agent to treat solid malignant tumors in adults and children . UnfortuAnimal models for cisplatin ototoxicity reveal that outer hair cells (OHCs) of the first row of basal turn of the cochlea are the targets for initial damage ,5,6. InjThe molecular mechanisms by which cisplatin causes cochlear damage and hearing loss are still being studied. Cisplatin needs to enter cells in the cochlea in order to cause cell death. Cisplatin may enter cells by passive diffusion and facilitated transport ,9. CisplMechanoelectrical transduction (MET) channels may provide a route of entry for cisplatin into hair cells. Thus, partial or reversible blockade of MET channels may reduce cisplatin ototoxicity. Cisplatin has been found to block MET channels in chick cochlear hair cells in a dose and voltage dependent manner . IontophThe cochlea has a high rate of metabolism. It requires an active antioxidant defense system in order to manage reactive oxygen species (ROS) generation from normal metabolism to function properly to maintain healthy hearing. This defense mechanism can be overwhelmed when the cochlea is subjected to extreme oxidative stress from noise or ototoxic drugs. Cisplatin ototoxicity is strongly associated with increased oxidative stress in the cochlea ,21. A ke2- produced by cisplatin upregulation of NOX3 to generate the highly reactive peroxynitrite free radical (ONOO-). Cisplatin induced nitrative stress in the cochlea leads to the nitration of LIM domain only 4 (LMO4), a transcriptional regulator, and decreases its cochlear expression levels. LMO4 is a transcriptional regulator that controls the choice between cell survival and cell death. Nitration of LMO4 appears to be related to cisplatin-induced ototoxicity  inhibitor) have been reported to show efficacy against cisplatin ototoxicity in rodent models  in the cochlea . A very There are several potential treatments to explore in the future for prevention of cisplatin ototoxicity. Animal studies which have shown drugs that protect against cisplatin ototoxicity without interfering with cisplatin efficacy should be considered for clinical trials. These include β-lapachone, honokiol, pifithrin alpha, EGCG, capsaicin, and a Src-protein kinase inhibitor.Pediatric oncologists recommend trials of systemic rather than intratympanic route of treatment with protective agents against cisplatin . A stronORC-13661 is a novel high-affinity permeant blocker of the mechanoelectrical transducer (MET) channel in OHCs. In mouse cochlear cultures it provided significant protection of hair cells against cisplatin. It appears to be well tolerated in rats following oral administration . Future Statins do not appear to interfere with cisplatin’s anti-tumor effects in patients . AdditioExosomes are membrane-bound nanovesicles that can be released from various cells in the body including supporting cells in the cochlea. They can provide HSPs to protect against cisplatin ototoxicity . They coCisplatin chemotherapy is associated with a very high incidence of irreversible sensorineural hearing loss. The drug gains access to the cochlea by several transport mechanisms, including the MET channels, which could serve as targets for treating ototoxicity as shown in


Sudden Sensorineural Hearing Loss (SSNHL) is a medical emergency requiring immediate attention as delayed treatment can lead to permanent and devastating consequences. Primary care physicians are likely the first to be presented with SSNHL and therefore have the crucial role of recognizing it and initiating timely and appropriate management. The aim of this study was to gain insight into the current knowledge and practice trends pertaining to the diagnosis and management of SSNHL among family physicians in Canada.An 18-question survey targeting Canadian family physicians was marketed through two, physician-only discussion groups on the social media platform Facebook. Responses were collected between August 1st and December 22nd 2019 then aggregated and quantified.n = 49) reported that in their practice, unilateral SSNHL warrants urgent referral to otolaryngology and 84.6% (n = 44) reported that unilateral sudden-onset hearing loss warrants urgent referral for audiological testing. 73.1% of participants (n = 38) reported that they would attempt to differentiate between conductive and sensorineural hearing loss if presented with unilateral, acute or sudden-onset hearing loss. 61.5% (n = 32) would rely on tuning fork tests to inform management decisions, as compared to 94.2% (n = 49) relying on case history and 88.5% (n = 46) on otoscopy. 76.9% (n = 40) would prescribe corticosteroids if presented with confirmed, unilateral SSNHL.52 family physicians submitted responses. 94.2% (The majority of family physicians in the study would make appropriate referral and treatment decisions in the management of SSNHL, understanding it is a medical emergency. Tuning fork tests are under-utilized for informing management decisions compared to other means of differentiating conductive and sensorineural hearing loss. Further research is needed to understand why some family physicians do not prescribe corticosteroids for treatment of SSNHL, which may then identify any gaps in knowledge or inform improvements in clinical protocol.The online version contains supplementary material available at 10.1186/s40463-021-00498-x. Sudden Sensorineural Hearing Loss (SSNHL) is a medical emergency that can lead to permanent and devastating consequences if left untreated. In the literature, SSNHL is often defined as the rapid onset of hearing loss ≥30 dB HL across three contiguous frequencies, in 72 h or less . The heaSSNHL is often idiopathic in nature but several etiologies have been proposed, including viral infection, vascular impairment, autoimmune disorder, otologic disease and retrocochlear pathology. The viral theory suggests SSNHL occurs when the cochlea or cochlear nerve is compromised by either an invading virus or reactivation of a latent neurotropic virus in the inner ear. Alternatively, a systemic viral infection can trigger an immune-mediated response or activation of the cellular stress pathway in the cochlea . SSNHL cA number of factors that correlate with hearing recovery outcomes in SSNHL have been identified. Degree and configuration of the hearing loss at time of onset are commonly reported to have prognostic value. Those initially presenting with a greater degree of hearing loss are likely to have poorer recovery , 36–38. The current standard for initial treatment of SSNHL is a tapered course of oral corticosteroids , 44. PrePrimary care physicians (PCPs) are likely to be the first point of contact for patients seeking medical attention for SSNHL and therefore have the crucial role of recognizing potential cases and initiating appropriate management quickly. At present, there is a lack of research that explores the management practices of PCPs in Canada when presented with sudden hearing loss. The specific aim of this study was to gain insight into the current knowledge and practice trends pertaining to the diagnosis and management of SSNHL among Canadian family physicians.An 18-question forced answer choice survey targeting family physicians in Canada was marketed through two private, physicians-only discussion groups on the social media platform, Facebook. The two groups were, “First 5 Years in Family Practice – Canada” and “The Canadian Doctors Lounge,” which contained 4765 and 2217 members, respectively, at the study’s commencement. A link to the electronic survey and a brief description of the study was posted in each of the group discussion forums to solicit participation. To ensure responses were only obtained from the target population, the Study Information Letter presented at the beginning of the survey explicitly stated that only participation from family physicians in Canada was being sought. The electronic survey link was available from August 1st through December 22nd 2019. The survey was hosted using Google Forms, the free online secure-link survey platform. Approval for this study was granted by the Sunnybrook Health Sciences Centre Research Ethics Board.see Survey Copy]. The last six questions were used to obtain demographic information about the participants. No information that could be used to identify participants was collected.The first twelve survey questions were designed to assess knowledge and practice trends in the diagnosis and management of SSNHL [Responses were automatically aggregated and quantified by Google Forms. Additionally, all data was tabulated in Microsoft Excel so that responses from each individual participant could be reviewed for any inconsistencies.n = 35) with only two participants having practiced more than 20 years (3.8%). Most participants reported practicing primarily in Ontario  with the remaining responses representing family physicians in Manitoba, Alberta, British Columbia, Newfoundland & Labrador, Nova Scotia and Quebec. Half the participants primarily provided care in urban areas (50%), 12 in suburban (23.1%) and 14 in rural (26.9%). Half the participants reported primarily practicing in Non-Academic Group or Team settings (50%) but nine in Academic Group or Team settings (17.3%) and nine in Urgent Care or Emergency Departments (17.3%) . The remaining six participants reported encountering only 5–10 such patients in the past six months (11.5%). More than half of the family physicians who participated in the survey reported that 1–4 weeks was the typical wait-time for a patient, referred for unilateral, sudden-onset hearing loss, to be seen by an otolaryngologist . 16 reported the typical wait-time to be one week or less (30.8%) . Eight defined SSNHL as hearing loss that develops within 48 h (15.4%) and eight defined it as developing over a period of seven days (15.4%) (Table Only 15 of the 52 participants correctly defined SSNHL as hearing loss that can develop within a period of 72 h (28.8%). Most understood it as developing in less than 24 h  reported that they would attempt to differentiate between conductive and sensorineural hearing loss whereas 26.9% would not (n = 14). Regarding the use of tuning forks, 63.5% of participants (n = 33) reported using tuning fork tests to differentiate between conductive and sensorineural hearing loss but fewer reported feeling confident administering and interpreting the results of these tests    and 88.5% (n = 46), respectively, to be used for informing management decisions. 61.5% would use tuning fork tests (n = 32) and 69.2% would use the results of an audiological evaluation (n = 36)  would refer for an otolaryngology consultation. Almost all participants  reported that in their practice, unilateral SSNHL warrants urgent referral to otolaryngology. 84.6% of participants (n = 44) reported that in their practice, unilateral sudden-onset hearing loss warrants urgent referral for audiological testing  would refer for audiological evaluation and 80.8%  would prescribe corticosteroids for suspected, unilateral SSNHL, even prior to confirmation with audiological testing. 11.5% (n = 6) would prescribe corticosteroids and antivirals. 25% (n = 13) would not prescribe any pharmacologic agents when presented with suspected SSNHL (Table n = 40) would prescribe corticosteroids and 17.3% (n = 9) would prescribe corticosteroids with antivirals. 23.1% of participants (n = 12) would not prescribe any pharmacologic agents for confirmed SSNHL, of which six (11.5%) responded that family physicians should not prescribe any treatment. None of the participants reported prescribing thrombolytics, vasodilators or other pharmacologic agents for either suspected or confirmed SSNHL  reported including possible causes of the hearing loss. 50% (n = 26) include available treatment options and the associated risks/benefits, 25% (n = 13) include the impact of the hearing loss on quality of life and 11.5% (n = 6) include rehabilitation options, such as the use of amplification. Almost half of participants  responded that they do not provide counselling to patients presenting with SSNHL as they are rarely certain of the diagnosis initially. However, of these 22 participants, four also selected additional response options indicating which topics they would include if they were to provide counselling  for sudden hearing loss established by the American Academy of Otolaryngology – Head and Neck Surgery Foundation (AAO-HNSF), both are unnecessary in the initial evaluation of SSNHL. Results of routine blood work are likely to be normal and even if abnormal, would not change initial management. There is also the risk that false-positives may lead to additional unnecessary testing. CT scans lack the sensitivity to detect retrocochlear pathology and expose patients to radiation. MRI scans of the brain, brainstem and internal auditory canal with gadolinium should be considered instead , 51.The responses to two of the study questions indicate that most of the family physicians who participated understand SSNHL is a time-sensitive emergency requiring prompt medical attention. First, a large majority of participants indicated unilateral, sudden-onset hearing loss would warrant urgent audiological evaluation. This is consistent with AAO-HNSF guidelines, which recommends confirming the diagnosis of SSNHL through audiometry as soon as possible after the onset of symptoms . Early dUrgent referral to otolaryngology is contingent on whether the referring physician can recognize SSNHL at the time of initial presentation, which includes ruling out conductive hearing loss (CHL). According to the AAO-HNSF, a crucial first step for clinicians presented with sudden hearing loss is to differentiate between conductive and sensorineural hearing loss (SNHL), as each requires significantly different management . Nearly Another means of differentiating CHL from SNHL recommended in the initial evaluation of unilateral, sudden hearing loss is the use of tuning fork tests (TFTs), specifically the Weber and Rinne , 57, 58.Confirming the sensorineural nature of a sudden hearing loss can also provide the rationale for a family physician to initiate immediate use of corticosteroids, prior to consultation with otolaryngology, thereby increasing the chance of hearing recovery. 76.9% of participants indicated they prescribe corticosteroids when presented with confirmed, unilateral SSNHL. This figure suggests most family physicians who participated correctly understand corticosteroids can be effective for the initial treatment of SSNHL. This is reassuring considering more than half of participants indicated patients referred to otolaryngology for sudden-onset, unilateral hearing loss waited one to four weeks before being seen, a delay in treatment that could reduce or eliminate the likelihood of recovery. However, the initiation of corticosteroid treatment, just like urgent referral to otolaryngology, depends on whether the referring physician recognizes the sensorineural nature of the presented loss. TFTs, along with otoscopy and a thorough case history, can and should be used by family physicians to make this determination. The relatively limited use of TFTs among our participants may be correlated with their low confidence levels in administering and interpreting such tests. One possible explanation offered by our data is that the family physicians who participated do not encounter sudden hearing loss often enough to use these tests regularly, making it difficult to maintain proficiency. If so, family physicians can use the Hum test instead, a simple and valid alternative to the Weber that does not require a tuning fork .Although the majority of participants indicated they prescribe corticosteroids for the treatment of SSNHL, the remaining utilize an alternate pharmacological approach or none at all. Nine participants indicated they prescribe antivirals in addition to corticosteroids even though previous review and analysis of the literature did not find any statistically significant benefit with their use , 63. AntOur study has several limitations. Despite the several thousand members in the two Facebook groups solicited for our survey, our response yield was low with only 52 participants. Given the small sample size, lack of participants from some provinces and territories, and with the majority of participants having less than five years of practice experience, the responses obtained may not necessarily be representative of the knowledge and management practices of all family physicians in Canada. We are also unable to verify participants’ responses to objective questions  which may be inaccurate due to memory and implicit or explicit personal biases. There is also likely the influence of selection bias by utilizing exclusively an electronic survey marketed only to specific discussion groups on a single social media platform. Finally, there is a possibility certain survey questions were susceptible to response bias due to either prior knowledge of the study’s premise , or wording used . When answering these questions, participants may be drawing on their knowledge of how to manage confirmed SSNHL, rather than their actual experience in clinic when a diagnosis is not yet known. If so, responses may overestimate how often family physicians make appropriate management decisions in those situations.Despite these limitations, the study’s findings reveal opportunities to enhance the knowledge and management practices of PCPs with regards to SSNHL. Continuing Medical Education with a focus on how to administer and interpret TFTs, which diagnostic tests to order or avoid, and appropriate use of therapeutic options could benefit family physicians and help them provide better patient care. Otolaryngologists can and should assume a primary role in providing this education. Formal learning experiences, such as seminars or conference workshops, and dissemination through publications could both be effective means of doing so. To ensure patients receive treatment in a timely manner, PCPs and otolaryngologists should establish conventions for communicating SSNHL during the referral process so that urgent priority can be given.In conclusion, the majority of family physicians who participated in our study would make appropriate referral and treatment decisions in the management of SSNHL, recognizing it is a medical emergency requiring urgent care. The majority of participants would prescribe corticosteroids for SSNHL, which is the established standard for initial treatment, but not all do. Further research is needed to understand the rationale for this alternative approach and identify possible gaps in knowledge or improve clinical protocols. Regardless, expeditious referral to otolaryngology or the initiation of corticosteroid therapy is contingent upon the family physician being able to first determine whether the sudden loss of hearing is conductive or sensorineural in nature. TFTs are a quick and reliable means of doing so yet under-utilized by the participants in our study. The majority reported a lack of confidence administering and interpreting the results of TFTs, which may be correlated with their limited use. Our study reveals opportunities for otolaryngologists to provide PCPs with education focusing on the evaluation and treatment of SSNHL that can potentially improve patient outcomes.Additional file 1. Survey. A copy of the study survey.


Self-report outcome measures at each assessment captured fatigue, listening effort, hearing handicap, auditory lifestyle, social participation restrictions, and work, social and physical activity levels. Hearing-aid fitting led to a significant reduction in listening-related fatigue, but not general fatigue, in the intervention group compared to the control group. Additionally, social activity level increased and social participation restriction decreased significantly after hearing aid fitting in the intervention group compared to the control group. No significant interaction was found between working status and change in listening-related fatigue score. This study is the first to make a longitudinal measurement of fatigue before and after first-ever hearing aid fitting and to identify an increase in social activity level after hearing aid fitting. These findings have important implications for future research and the clinical practice of hearing aid fitting.People with hearing loss experience fatigue, and it is unknown whether this is alleviated by treatment with hearing aids. The objective of this study was to address this issue and to investigate the possible concomitant effect of hearing-aid fitting on activity levels. An intervention group ( Hearing loss can affect people's lives beyond their difficulty hearing . One effThe basic and most intuitive theory of listening-related fatigue states that the cognitive effort required in challenging listening situations constitutes a drain on finite cognitive resources, resulting in fatigue . HoweverLogically, an increase in audibility through the fitting of a hearing device should reduce the listening effort required in any given situation, and in turn reduce fatigue. Evidence regarding the impact of hearing aid fitting (as opposed to cochlear implant fitting) on fatigue has focused to date mainly on the cross-sectional study of people wearing and not wearing hearing devices . Such reListening-related fatigue affects people in different ways, and may also be managed differently from person to person . As list(Q1) Does first-ever hearing aid fitting have an impact on fatigue?We hypothesized that fatigue would reduce post-hearing aid fitting.(Q2) What variables may influence the impact of hearing aid fitting on fatigue?Demographic, hearing, and lifestyle factors could be associated with the impact of hearing aid fitting on fatigue. It was hypothesized that age, gender, hearing loss, hearing handicap, social activity level, work activity, and need for cognition (inclination towards effortful cognitive activity) would be associated with fatigue scores. It was hypothesized that listening effort, being functionally linked to fatigue, would correlate temporally with fatigue.(Q3) Do levels of social or physical activity change after hearing aid fitting, and is this related to fatigue?We hypothesized that social and physical activity would rise after hearing aid fitting. Associated variables of interest for social activity were age, gender, hearing handicap, auditory lifestyle demand, and need for cognition. Associated variables of interest for physical activity were age, gender, and hearing handicap. We hypothesized that social and physical activity would rise for participants who experienced a reduction in fatigue.Longitudinal assessment of fatigue before and after hearing aid fitting has not previously been reported. Therefore, we conducted a longitudinal study to address the following research questions:This research received ethical approval from the West of Scotland Research Ethics Committee (18/WS/0030) and the NHS R&D (GN18EN073).Fatigue was measured before, and at three timepoints after first-ever hearing aid fitting in an intervention group. A control group of people with hearing loss, some with and some without hearing aids, also completed the same measurement regime . AdditioParticipants were between 18 and 75 years old. For inclusion in the intervention group participants had to have self-reported hearing difficulties, be receiving their first-ever hearing aid(s), and not be attending the audiology clinic with a primary complaint of tinnitus or vestibular issues. For inclusion in the control group participants had to have self-reported hearing difficulties and have experienced no change in hearing aid status for at least 1 year . Though not individually matched, the control group was kept as close as possible to the intervention group for gender, mean age, and mean four-frequency average hearing loss see .Participants for the intervention group were recruited from the waiting list for audiological evaluation at the Glasgow Royal Infirmary audiology department, except for two participants recruited through word of mouth. The first (baseline) assessment was conducted before each participant's audiological screening, and for this reason, not all potential intervention group participants ended up receiving hearing aids. Those who were not fitted with a hearing aid became part of the control group. The remaining participants in the control group were selected from the Hearing Sciences—Scottish Section participant database. The stability of hearing aid status  was checked at the start of each assessment to ensure participants remained eligible for their designated group.A minimum sample size of 90 participants (45 per group) was estimated based on a between-group analysis of covariance (ANCOVA), with a power of 80% (β = 0.2), capable of detecting an effect size of 0.3 and a type one error rate alpha of 0.05. The power calculation was conducted using G* power calculator version 3.1. The sample size calculation was based on the Fatigue Assessment Scale (FAS) as this was the questionnaire used in the most relevant previous research .In total 129 participants completed at least one assessment, and 106  completed all four assessments. Altogether 89 participants were recruited for potential inclusion in the intervention group. Seventeen of those participants did not complete all four assessments, dropping out for reasons such as personal circumstances, failure to respond to communications, and failure to meet the inclusion criteria that were not initially detected. Seven participants were offered a hearing aid at their clinical appointment, but chose not to be fitted, and 12 were told that a hearing aid would not be appropriate. Data collection ran from March 2018 until December 2019. Each participant made four visits to the department. For the intervention group there was one assessment before hearing aid fitting, and then 2 weeks, 3 months, and 6 months post fitting (equating to 3 months between each assessment). These time points were chosen as the first few weeks after a hearing aid fitting involve potentially troublesome sensory and psychosocial adjustments , whereasDuring assessment 1, which took ∼1 h, participants provided written informed consent and were then asked about their hearing history (including hearing aid use and tinnitus), underwent ear examination and audiometric assessment, and completed research questionnaires. At subsequent assessments, participants completed the same questionnaires apart from the “Need for Cognition Scale,” and there was no repeat of the pure tone audiometry or history taking. Continued hearing aid use (or non-use for certain members of the control group) was ascertained at each assessment by asking how many hearing aids they own and how often they wear them . While some participants rarely used their new hearing aids, no participants gave up on their hearing aids entirely. At the final assessment, participants were debriefed and offered the opportunity to ask additional questions about the study. Appointments were arranged by either phone or email, depending on the participant's preference, and appointment reminders were also provided in the same way. Participants received £50 compensation in total; £10 at each of the first three visits, and £20 at the final visit.Supplemental Digital Content 1.The outcome measures used in the study were 13 self-report questionnaires which addressed fatigue, listening effort, hearing handicap, activity , social participation restrictions, auditory lifestyle, and need for cognition. For the purposes of this study, participants were asked to respond with respect to their current level of hearing aid usage . Full questionnaire details are provided in Three self-report questionnaires in total were utilized to measure fatigue. Two of the questionnaires assessed long-term fatigue. The FAS  is a widListening effort was assessed using the Listening Effort Assessment Questionnaire (EAS)  which meSocial activity level was assessed using two self-report questionnaires. The social activity log (SAL)  measuresThe experiential component of social activity was assessed using the Social Participation Restrictions Questionnaire (SPaRQ)  which coStatistical analyses were carried out using R version 3.6.1 . For anat-tests, or Mann-Whitney U-tests. Group mean differences at each subsequent time point, controlling for baseline score, were analyzed using ANCOVA or non-parametric equivalent. The sm package was used to analyze non-parametric ANCOVA (rτ) were used to assess the relationships between the magnitude of change in relevant variables.Associations between-group (intervention vs. control) and outcome measure scores at baseline were assessed using independent-sample c ANCOVA . SeparatSupplemental Digital Content 2.In all following text, the pre-fitting assessment is termed “baseline” with the post-fitting assessments denoted as 2, 3, and 4. A Spearman rank correlation matrix of all baseline questionnaires is provided in H = 2.82, p = .012), but this was not the case at assessments 3 or 4   . Effect F = 4.56, p = .039; F = 6.19, p = .015). VFS-A-40 scores (baseline corrected) for cognitive fatigue, emotional fatigue, and social fatigue were lower in the intervention group than the control group at assessment 2, , assessment 3, , and assessment 4, . There was no group difference for physical fatigue. Based on these results, there does appear to be a beneficial impact of first-ever hearing aid fitting on fatigue (Q1).Analysis of the multidimensional scales of the MFSI, after controlling for individual participants’ baseline scores on the outcome measure in question, found no significant differences between groups in any assessment for physical fatigue, mental fatigue, or emotional fatigue. The vigor scale of the MFSI showed that after controlling for individual participants’ baseline scores on the outcome measure in question, intervention group scores were significantly higher (better) than the control group at the final two assessments, , x2(2) = 3.92, p = .14, suggesting that participants in the intervention group tended to follow the same trend across time. A logarithmic function of time was found to be a better fit than linear, quadratic, and cubic, SD = 0.4 , x2(2) = 4.66, p = .03.To further investigate the impact of hearing aid fitting on fatigue by breaking down the interaction term (group × assessment) and re-assessing the relationship between fatigue and associated variables, a new multilevel growth model was created for the intervention group only . The sloSupplemental Digital Content 2).Although the growth model analysis suggested a logarithmic trend of VFS-A-40 over time, visualization of the individual VFS-A-40 trajectories highlights that individuals had a wide variety of score trajectories from baseline to assessment 4 . Effect sizes based on adjusted means at each assessment were d = 0.5, d = 0.82, and d = 0.66. In the intervention group, the change in VFS-A-40 score from baseline was significantly correlated with the change in EAS score at all follow-up assessments .After controlling for individual participants’ baseline scores on the outcome measure in question, listening effort significantly decreased in the intervention group post-fitting compared to the control group , but not at the final two post-intervention assessments, . The lack of a consistent pattern suggests that post-intervention VFS-A-40 scores changed at a similar rate in both working and non-working participants.Mean VFS-A-40 scores for intervention group participants who work were higher at each assessment than for those who were not in work. However, this difference was not statistically significant at baseline, In summary, hearing handicap at baseline is the only pre-fitting variable which was significantly associated with a change in fatigue, and which therefore may influence the impact of hearing aid fitting on fatigue (Q2).To answer this compound question, we analyzed the activity levels across time between the intervention and control groups, followed by a correlation analysis of the temporal changes in activity and fatigue.d = 0.36, d = 0.58, and d = 0.52.ANCOVA analysis found that SAL scores, controlling for baseline score, were significantly higher in the intervention group than the control group at assessments 2, 3, and 4 . HoweverGroup, assessment number, baseline ALDQ, and NFC were significantly and positively associated with SAL score . BaselinAs group was significantly associated with SAL score, further analysis was conducted on the intervention group alone. For this group, assessment and baseline ALDQ score were significantly and positively associated with SAL score . BaselinF = 8.2, p = .005; F = 21.5, p = <.0001; F = 17.26, p = < .0001). The social perception subscale also reduced (improved) significantly in the intervention group at each post-intervention assessment, .Given that social activity increased significantly after hearing aid fitting, the change in social participation restriction was also investigated. It was found that the social behavior subscale of the SPARQ reduced (improved) significantly more in the intervention group than the control group when controlling for baseline scores at each assessment after hearing aid fitting, . However, when the intervention group alone was analyzed, the correlation became marginal . Therefore, social activity levels increased with hearing aid fitting, but there is no evidence that decreasing fatigue is associated with increasing social activity (Q3).For participants in both groups, the changes in VFS-A-40 and SAL scores from baseline to assessments 2 and 3 were not significantly correlated, but at assessment 4 the changes from baseline were significantly correlated  = 0.45, p = 0.5; F = 0.58, p = .45; F = 1.05, p = .31. Therefore, there is no evidence of physical activity being affected by hearing aid fitting.There was no mean difference in IPAQ scores between groups at baseline, This study was designed to examine the impact of hearing aid fitting on fatigue, and variables related to changes in fatigue (research question Q1), whether activity changed after hearing aid fitting (Q2), and whether this had any link to fatigue (Q3). The two general fatigue questionnaires (FAS and MFSI) showed no difference between groups post-fitting. This finding supports the results of Due to the very recent development of the VFS-A-40 scale, our finding of a significant impact of hearing aid fitting on VFS-A-40 scores at both the group and individual levels stands alone, to date. The VFS-A-40 measures listening-related fatigue, and as such seems more sensitive to the fatigue experienced by people with a hearing loss than general fatigue questionnaires, despite scores on all three fatigue questionnaires being significantly positively correlated at assessment 1. The VFS-A-40 does not measure transient fatigue explicitly, but it does pose hypothetical situational questions. As a result, this study can offer no further insights into the distinct impacts of hearing aid fitting on transient and long-term fatigue. With the exception of the MFSI vigor subscale and the VFS-A-40 physical subscale, the scores on all of the other constituent subscales of the MFSI and the VFS-A-40 followed the same pattern as the total scales. MFSI subscale scores were not significantly different between groups across time points, whereas VFS-A-40 subscale score changes were significantly different between groups at each time point post fitting. These results could mean either that hearing aid fitting impacts all dimensions of listening-related fatigue similarly, or that people tend to report subjective fatigue as unidimensional, as has been previously suggested . The lack of correlation between baseline hearing handicap and MFSI vigor does not support the previous finding of a correlation between HHIE and MFSI vigor . However, the significant improvement at the final two assessments in the intervention group suggests that vigor may play a role in listening-related fatigue.Although a reduction in listening-related fatigue is clearly to be welcomed, it is unclear whether the change is of clinical significance. Median VFS-A-40 scores were negative for both groups at baseline , which suggests that listening-related fatigue was not high for most participants, and therefore may not have been problematic. It is currently not possible for us to report VFS-A-40 data to establish whether the scores were normal for a population with audiometrically determined hearing loss, or how they compare to normal hearing norms. However, previous research has established that the threshold of minimally important difference for changes in health-related quality of life is approximately half a population standard deviation . At baseIn line with Alhanbali et al. (2018), hearing handicap correlated with fatigue. The HHIE/A was the only questionnaire to correlate significantly with all three fatigue questionnaires at baseline, and significantly associated with VFS-A-40 scores in the multilevel growth models. The absence of any relationship between fatigue and audiometric hearing loss reported by Alhanbali was also reflected in the current study. However, while Previous cross-sectional studies suggest that hearing device fitting has a positive effect on social activity . This waPrevious research has concluded that the control and enjoyment one has during social activity could affect the fatigue experienced . The sigThe study recruited participants to the intervention group from people who were due to visit an audiology clinic as potential recipients of a hearing aid. As is normal, some of these ended up not being fitted with hearing aids, so they became part of the control group instead. There was no significant difference in VFS-A-40 score at any assessment between this subgroup and the control group participants recruited from the participant database. However, there was also a possibility that participants who did not receive a hearing aid could respond differently at the second assessment, either with improved scores due to relief (having been told that they did not need hearing aids), or worse scores (having been told they could not be helped at this point). While scores consistent with this behavior were displayed in some individuals, additional analysis showed that this had no impact on subgroup VFS-A-40 scores. Additionally, due to the variable nature of audiology appointments, the time from the first assessment to the second assessment was longer than three months for some individuals. While this was not a major issue, it could have distorted some analyses.The difference between groups regarding age range occurred due to the extended period of recruitment and data collection, with some participants finishing as others began. Thus, any dropouts were problematic to match in the opposing group. Most participants had mild to moderate hearing losses, as the study required participants who were only just receiving their first hearing aid. It is possible that hearing aid fitting would result in more dramatic listening-related fatigue reductions for people with severe hearing losses, which this study could not fully investigate. On the other hand, this study represents a realistic sample of people who are likely to receive a first-ever hearing aid and therefore is more clinically relevant than one involving participants with more severe hearing losses.The study measured hearing aid use to ensure that participants remained eligible for their assigned groups. It would be interesting to know if the level of hearing aid use  influences fatigue or even the change in fatigue post-fitting. However, the present study was not designed to investigate this question and therefore future studies would be needed to gain a better insight.The longitudinal design of the study allowed the investigation of changes at the individual level across time. By doing so, many of the potential confounding variables in previous research were reduced or eradicated. As the FAS has been used in relevant studies of people with hearing loss before, it was the basis for sample size and power analysis. However, the FAS has, to the authors’ knowledge, never been used in longitudinal assessment before. Despite this, the FAS has been shown to have excellent internal consistency, convergent validity, and test-retest reliability for various populations . By usinThe present study is the first to use longitudinal methodology to measure fatigue before and after first-ever hearing aid fitting. It is also the first study to demonstrate a longitudinal increase in social activity level after hearing aid fitting. Hearing aid fitting showed no effect on long-term general fatigue, but there was an improvement from before fitting to six months post-fitting for listening-related fatigue. This provides a more detailed result compared to the mixed outcomes from previous research. Like social activity level, social participation restrictions improved post fitting. While the design of this study does not allow an assessment of a direct impact of social activity on fatigue, the increase in social activity and reduction in listening-related fatigue after hearing aid fitting are important findings for clinical practice as more focus should be given to extra-auditory needs and outcomes during hearing healthcare. Future research should assess whether current fatigue scales measure what we want to know in relation to hearing loss and hearing aid fitting. Research should also further investigate the impact of hearing aid use on social activity level, as well as on experiential aspects of social activity such as restriction.Click here for additional data file.Supplemental material, sj-docx-1-tia-10.1177_23312165211052786 for Hearing Aids Reduce Daily-Life Fatigue and Increase Social Activity: A Longitudinal Study by Jack A. Holman, Avril Drummond and Graham Naylor in Trends in HearingClick here for additional data file.Supplemental material, sj-docx-2-tia-10.1177_23312165211052786 for Hearing Aids Reduce Daily-Life Fatigue and Increase Social Activity: A Longitudinal Study by Jack A. Holman, Avril Drummond and Graham Naylor in Trends in Hearing