For that aim, we conducted anatomical MRI and diffusion tensor imaging (DTI) in elderly hard-of-hearing and age-matched normal-hearing participants. Our results showed significantly lower grey matter volume in the middle frontal cortex in hard-of-hearing compared to normal-hearing participants. Further, higher listening effort was associated with lower grey matter volume and cortical thickness in the orbitofrontal cortex and lower grey matter volume in the inferior frontal cortex. No significant relations between hearing abilities or listening effort were obtained for white matter integrity in tracts connecting auditory and prefrontal as well as visual areas. These findings provide evidence that hearing impairment as well as daily life listening effort seems to be associated with grey matter loss in prefrontal brain regions. We further conclude that alterations in cortical thickness seem to be linked to the increased listening effort rather than the hearing loss itself.Age-related hearing loss is associated with a decrease in hearing abilities for high frequencies and therefore leads to impairments in understanding speech—in particular, under adverse listening conditions. Growing evidence suggests that age-related hearing loss is related to various neural changes, for instance, affecting auditory and frontal brain regions. How the decreased auditory input and the increased listening effort in daily life are associated with structural changes is less clear, since previous evidence is scarce and mostly involved low sample sizes. Hence, the aim of the current study was to investigate the impact of age-related untreated hearing loss and subjectively rated daily life listening effort on grey matter and white matter changes in a large sample of participants ( Age-related hearing loss—also termed presbycusis—is one of the most prevalent chronic disorders affecting older adults. It involves the decrease in hearing abilities for high frequencies and therefore leads to impairments in understanding and processing speech, particularly in difficult listening situations Cardin . Moreoven = 7 and n = 16, respectively). Complementing previous work on hearing abilities, cortical thickness in the prefrontal cortex was positively related to speech perception in older adults . In contrast, Profant et al. (With respect to diffusion tensor imaging (DTI), the role of hearing loss is even less clear since only a few studies have investigated white matter changes in hard-of-hearing participants. Two studies investigated changes in white matter integrity and found significantly lower fractional anisotropy (FA) in hard-of-hearing compared to normal-hearing participants in longitudinal and fronto-occipital tracts  covering normal-hearing participants as well as individuals with mild to moderate age-related hearing loss. For that aim, anatomical MRI and diffusion tensor imaging (DTI) in elderly hard-of-hearing and age-matched normal-hearing participants were analysed to assess grey matter volume, cortical thickness and white matter integrity.To conclude, the evidence regarding neuroanatomical differences between hard-of-hearing and age-matched normal-hearing individuals is scarce and most of the previous studies encompassed relatively low sample sizes. Further, previous work did not focus exclusively on age-related hearing loss and the influence of increased listening effort, as is often experienced by hard-of-hearing participants, on structural brain alterations has not been investigated so far. In contrast to pure-tone thresholds, a measure of listening effort involves the assessment of cognitive resource allocation during speech processing. Thus, listening effort may be a more sensitive marker of neural alterations than hearing abilities determined by pure-tone audiometry  grey matter volume and cortical thickness are lower in the temporal and prefrontal brain regions in hard-of-hearing compared to normal-hearing participants . Subjects with low-frequency hearing loss were excluded, i.e. hearing abilities for all participants were 30 dB HL or better for frequencies up to 1 kHz . High-frequency hearing loss was defined by more than 30 dB HL for at least three frequencies between 2 and 8 kHz. The hard-of-hearing individuals showed a varying bilateral sloping high-frequency hearing loss that was mild to moderate, symmetrical (≤ 15 dB difference between left and right ear for more than three frequencies) and only affecting high frequencies (≥ 2 kHz). Normal-hearing abilities were defined as 30 dB HL or better for each octave frequency between 125 and 3000 Hz and less than 30 dB HL for the mean value over 2000, 4000, 6000 and 8000 Hz . Hearing thresholds for high frequencies (mean 2–8 kHz) were 40.28 (± 8.19) dB in the hard-of-hearing and 19.27 (± 6.03) dB in the normal-hearing group. Individual pure tone audiograms averaged over both ears for the two groups are depicted in Fig. We analysed the structural MRI and DTI data from 38 hard-of-hearing and 33 normal-hearing participants that participated in functional MRI studies within the framework of the Cluster of Excellence Hearing4all. Functional data from these studies have already been published  that was conducted in a soundproof chamber. MRI measurements were conducted in two sessions separated by a short break outside the scanner. In the first run, the functional MRI was measured. After the break, a resting state MRI, anatomical MRI and a diffusion MRI measurement were conducted. Functional MRI results and resting state analyses have been previously published . Further parameters of the diffusion-weighted sequence were (1) TR = 6000 ms, TE = 52 ms, 64 directions, 11 non-diffusion images or (2) TR = 9400 ms, TE = 68 ms, 15 directions, 10 non-diffusion images.A 3T whole-body Siemens Magnetom Prisma MRI machine with a 20-channel head coil was used for all measurements. Data were acquired with slightly different MR sequences due to software updates. The sample measured with the first parameter set included 39 participants  and 32 participants were measured with parameter set two . Structural images were acquired with 3-D T1-weighted MPRAGE sequences with (1) TR = 2300, TE = 4.16, slice thickness 1 mm, 176 sagittal slices or (2) TR = 2000, TE = 2.07, slice thickness 0.75 mm, 224 sagittal slices. Diffusion tensor measurements were acquired with a multi-directional diffusion weighting (MDDW) sequence with a p < 0.05 (FWE cluster size inference with p < 0.001 cluster-forming threshold). Analyses were conducted for the whole brain and the multiple comparison problem is dealt with by using family-wise error correction based on random field theory. Peak coordinates are reported in MNI space.Grey matter metrics were analysed using SPM12  and the CAT12 . Data analysis was performed in SPSS Statistics 25 .DTI data were analysed with ExploreDTI , and no interaction between listening effort sub-category and group (p > 0.1).The mean self-rated listening effort experienced in daily life was rated as rather mild with 3.0 (± 1.4) in hard-of-hearing participants and 2.4 (± 1.2) in normal-hearing participants. The maximum self-rated listening effort in the normal-hearing group was 4.82 and in the hard-of-hearing group 6.81. An additional analysis evaluated the listening effort separately for the three different categories easy, medium and difficult situations. The mean scores were 1.47 (± 1.1) for the easy, 3.29 (± 1.7) for the medium and 4.07 (± 1.9) for the difficult situations in the hard-of-hearing group. In normal-hearing listeners the mean scores were 0.97 (± 0.97) for easy, 2.64 (± 1.3) for medium and 3.38 (1.8) for the difficult situations. The repeated measures ANOVA for the mean values of the listening effort questionnaire as well as the different sub-categories revealed a significant difference between both groups  = 4.045, p < 0.001). Listening effort and hearing loss significantly correlated with each other across all participants .Hearing thresholds for high frequencies  were 40.28 (± 8.19) dB in the hard-of-hearing and 19.27 (± 6.03) dB in the normal-hearing group. The mean high-frequency hearing loss values significantly differed between groups (T(69) = 12.15; p < 0.05 (FWE cluster size inference with p < 0.001 cluster-forming threshold).To investigate the association of high-frequency hearing loss and listening effort with grey matter volume and cortical thickness, we performed between-group comparisons  and regression analyses across all participants . Analyses were conducted on the whole-brain level and effects were determined to be significant when passing a threshold of x = − 50, y = 54, z = 0; cluster size k = 344; z-value = 3.95) in normal-hearing compared to hard-of-hearing participants .Further, self-rated listening effort experienced in daily life was negatively associated with grey matter volume Fig.  and cortT test with default prior option (Cauchy distribution) comparing FA values between both groups. This analysis showed evidence for the null hypothesis in most of the brain regions, except the cingulum . Statistical outcome values can be seen in Table p values, effect sizes and Bayes factors). The Bayesian correlation matrix with FA values and self-rated listening effort showed evidence for the null hypothesis in all brain regions . Note that a post hoc analysis of another common DTI measure, mean diffusivity (data not shown), did not provide any evidence for microstructural changes related to hearing loss or listening effort in the investigated tracts.To investigate white matter integrity, mean FA values from tracts connecting auditory, frontal and visual areas—such as in the superior and inferior fronto-occipital and longitudinal fasciculi as well as in the cingulum and uncinate fasciculus—were computed. Mean values for hard-of-hearing and normal-hearing participants can be seen in Table In a large sample of elderly subjects with age-appropriate hearing and subjects with untreated mild to moderate age-related high-frequency hearing loss, we investigated changes in grey and white matter. Furthermore, the influence of self-rated daily life listening effort on these structural brain alterations was examined. Our results showed decreased grey matter volume in the middle frontal gyrus in hard-of-hearing compared to normal-hearing participants. Additionally, we obtained a negative relation between listening effort and grey matter volume as well as cortical thickness in several prefrontal brain regions. However, our DTI data analysis revealed no significant changes in white matter integrity in age-related hearing loss or any associations with daily life listening effort. The additional Bayesian approach only showed moderate support for differing FA values between both groups in the left middle cingulum.We expected a decrease in grey matter volume and cortical thickness in temporal and prefrontal brain regions in hard-of-hearing compared to normal-hearing participants. Our results showed significantly lower grey matter volume in middle frontal gyrus in hard-of-hearing compared to normal-hearing participants, but no significant differences in cortical thickness.Previous research has shown grey matter decreases in cingulate, superior temporal, superior and medial frontal gyri along with the occipital lobe and hypothalamus in hard-of-hearing compared to normal-hearing participants  and statistical thresholds were more liberal than in our study  with normal hearing as well as mild to moderate age-related hearing loss. The findings in this sample do not indicate a relation between FA values and self-reported daily life listening effort. The Bayesian approach showed a moderate support for differing FA values between hard-of-hearing and normal-hearing participants in the left cingulum, but not in any other brain region. A possible explanation might be that the mild to moderate decrease in hearing abilities and the slight increase in listening effort were not advanced enough to lead to structural changes in white matter metrics such as FA values. Hearing loss in old age is a gradual, slowly progressing process which often remains undiscovered for several years. Accordingly, analyses investigating the impact of the duration of hearing loss on white matter changes are challenging, but would be important to determine whether more severe stages or an extended period of age-related hearing loss is associated with changes in white matter structures. Future longitudinal research may address this question.Contrary to our expectations, we found no evidence for changes in FA values in the superior and inferior fronto-occipital and longitudinal fasciculi as well as in the cingulum and the uncinate fasciculus as a function of hearing loss or listening effort. The additional Bayesian We investigated the neuroanatomical correlates of age-related hearing loss in a large group of elderly normal-hearing and hard-of-hearing participants. Our results showed significantly lower grey matter volume in the middle frontal cortex in hard-of-hearing participants. Furthermore, higher self-rated listening effort was associated with lower grey matter volume in the inferior and orbitofrontal cortex along with lower cortical thickness in the orbitofrontal cortex. In contrast, white matter integrity was not related to age-related hearing loss or daily life listening effort. Hence, we provide evidence that age-related hearing impairment as well as daily life listening effort seems to be associated with grey matter loss in the prefrontal brain regions. Alterations in cortical thickness seem to be linked to the increased listening effort rather than the hearing loss itself. This implies that not only hearing abilities, but also behavioural outcome measures of the decreased auditory input—such as self-rated daily life listening effort—need to be assessed and evaluated with regard to structural alterations in elderly participants. These findings advance our knowledge of underlying structural brain changes in age-related hearing loss. An important question to be addressed in future studies is whether these can be halted or reversed, for instance, by hearing-aid fitting.


Streptomyces griseus HUT6037 (SgChiC). SgChiC is a potential biocontrol agent and a reference enzyme in the study and development of chitinases for various applications. The overexpression of SgChiC was previously achieved by periplasmic localization from where it was extracted by osmotic shock and then purified by hydroxyapatite column chromatography. In the present study, the successful refolding and recovery of recombinant SgChiC (r-SgChiC) from inclusion bodies (IB) by reverse dilution and column chromatography methods is respectively described. Approximately 8 mg of r-SgChiC was obtained from each method with specific activities of 28 and 52 U/mg respectively. These yields are comparable to that obtained from a 1 L culture volume of the same protein isolated from the periplasmic space of E. coli BL21 (DE3) as described in previous studies. The higher yields obtained are attributed to the successful suppression of aggregation by a stepwise reduction of denaturant from high, to intermediate, and finally to low concentrations. These methods are straight forward, requiring the use of fewer refolding agents compared with previously described refolding methods. They can be applied to the refolding of other cysteine rich proteins expressed as inclusion bodies to obtain high yields of actively folded proteins. This is the first report on the recovery of actively folded SgChiC from inclusion bodies.Conventional refolding methods are associated with low yields due to misfolding and high aggregation rates or very dilute proteins. In this study, we describe the optimization of the conventional methods of reverse dilution and affinity chromatography for obtaining high yields of a cysteine rich recombinant glycoside hydrolase family 19 chitinase from  Escherichia coli are a frequently encountered phenomenon with up to 70% of target recombinant proteins reportedly ending up as IB thereby impeding the production and study of such enzymes ) in the ratio of 1:1 and boiled for 15 minutes. The mixture was brought to cooling at room temperature before reading its absorbance at 420 nm. All experiments were carried out in triplicates at least 3–4 times per refolding method. One unit of enzyme activity was defined as the amount of enzyme in mg that released 1 μmol of N-acetyl-D-glucosamine per minute at 37°C.Enzyme assays were carried out by a modified method of Yagishita and Imoto  using coSgChiC with codons optimized for expression in E. coli BL21(DE3) was commercially synthesized and cloned into a pET22-b(+) vector via its NdeI-XhoI restriction sites. SgChiC. The pelB leader peptide was excluded from the expression construct of r-SgChiC (r-SgChiC  to preveE. coli BL21(DE3) cells, upon induction with 0.2 mM IPTG at 30°C for 2 hours, overexpressed the C-terminal His-tagged r-SgChiC exclusively as inclusion bodies. The IB fraction harvested after separating the crude lysate was washed 2 times prior to solubilization with wash buffer  supplemented with 1 M urea  at 340 nm . r-SgChiThe initial refolding protocols attempted for refolding r-SgChiC were the traditional dilution and dialysis methods as described by ,39. ThesThe on-column refolding method carried out by applying the standard AKTA Prime Plus programme and recommended buffer conditions yielded less than 1 mg of purified protein from a 500 mL culture. The yield remained the same even when the culture volume was increased to 1 L. In order to optimize yield, on-column refolding was carried out under varying urea and NaCl combinations in RB and EB respectively . Sodium As observed in all reducing SDS-PAGE gels, samples refolded OnC and by dilution yielded single bands Figs  and 4B oAlthough this was 2 times higher than the RD refolded r-SgChiC, both OnC and RD refolded samples in this study resulted in comparable specific activities as reported previously with SgChiC extracted and purified by osmotic shock .Circular dichroism spectra calculated for r-SgChiC samples refolded by reverse dilution and on-column chromatography revealed similar trends with slight deviations in the detection signal at 190–195 nm and at 210–240 nm .Both refolding methods showed closely related secondary structure components with the presence of positive bands forming helices and beta sheets between 190 and 195 nm and; negative bands at 209 and 222 nm respectively. Helices and beta sheets were estimated to be at 38.4 and 2.2% respectively for RD sample; and 43 and 6% for OnC refolded sample. These results are comparable to the secondary structure component in the crystal structure   with 39–E. coli JM109 host, resulted in extremely low or no yields [E. coli BL21(DE3) with yields up to 10 mg from 1 L culture [E. coil BL21(DE3), SgChiC was retrieved in previous studies by the cold osmotic shock method and further purified by a series of steps including ammonium sulphate precipitation and chromatography on a hydroxyapatite column [SgchiC expression construct was designed with and without the leader peptide for a possible periplasmic and cytoplasmic expression respectively. However, SgChiC was not detected in the periplasmic fraction of E. coli BL21(DE3)::pET-SgChiC construct (with the pelB leader peptide). On the contrary, analysis of cell lysates of the construct without the pelB construct revealed sufficient yields of protein equivalent to the molecular weight of SgChiC in the IB fraction. Therefore, the expression system without the pelB signal peptide, subsequently referred to as pET22-b(+)::SgchiC was retained and used continuously for the expression of r-SgChiC throughout this study. r-SgChiC IB was subsequently subjected to different refolding conditions. Finally, actively folded r-SgChiC was recovered by a reverse dilution and an on-column affinity chromatography method.The expression of r-SgChiC exclusively as inclusion bodies even after optimizing conditions of expression such as temperature, induction time and IPTG concentration necessitated the development of a refolding protocol. Similarly, in previous studies, initial attempts to express the protein in an o yields . Subsequ culture ,40. Thes culture . Protein culture –47. Upone column ,32,40,47E. coli BL21(DE3) cells harbouring pET22-b(+)::SgchiC was terminated after a period of only 2 hours at 30°C. IBs isolated at much later periods did not result in significantly higher yields but posed a greater challenge during solubilization. A similar observation was reported in a previous study whereby the solubility of inclusion bodies harvested within 2 hours of post induction resulted in higher solubility than those isolated beyond 2 hours [E. coli proteins that were soluble or that may have accumulated with r-SgChiC as inclusion bodies. This eliminated the need for the use of detergents like Triton X-100 which would otherwise have required further wash steps especially before on-column refolding can be performed as described elsewhere [While developing a refolding routine either on-column or by dilution it was observed that harvesting cells in their early stationary phase resulted in IB with less impurities. Hence post induction growth of  2 hours . Another 2 hours  partiallr-SgChiC was optimally solubilized with 6 M Gdn-HCl in 50 mM Tris-HCl buffer, pH8 as determined in a preliminary experiment . r-SgChiThe initial refolding trials in this study resulted in either extremely low yields or overly diluted protein suspensions. This was because r-SgChiC has a high propensity for aggregation during refolding even at low concentrations as it readily precipitated under medium to low denaturing buffer conditions during dilution, on-column refolding and dialysis . AggregaIn the RD method applied here, each step was optimized to suppress aggregation as well as to achieve high yields with the lowest possible volume of buffer. The gradual reduction of denaturant (Gdn-HCl) from 6 M to < 1 M therefore took place in four steps of reverse dilution . The bufRefolding by dilution has always been the easiest and most practiced method for the refolding of solubilized IBs. However, the conventional method of dilution requires the addition of proteins into large volumes of buffers (at least 100-fold) leading to extremely dilute samples. This renders further down-stream processing and retrieval of protein in concentrated forms very difficult. Other disadvantages of refolding by conventional dilution has been clearly reported ,9, henceThe principle of refolding on-column is similar to reverse dilution since buffer is added to sample in both cases except that refolding takes place with the protein bound to a matrix in the on-column method. Additionally, on-column refolding has the advantage of simultaneous purification of the sample, hence it can be completed within a relatively shorter period than first purifying under denaturing conditions before refolding by reverse dilution. Initial direct application of the recommended protocol and default parameters in the AKTA PRIME plus system in this study however resulted in extremely low protein yields. The low yields were attributed to one or all of the following: on-column aggregation resulting from incorrect folding or partially folded intermediates; inefficient elution resulting from a firm hydrophobic attachment of refolded protein to the resin; accumulation of proteins at the upper part of the column resulting in precipitation and eventual loss in yield as highlighted earlier . Hence tDialysis was performed as a necessary step for the removal of excess salts and denaturant. This further enhanced the refolding yield Tables  and 2. TE. coli IBs by gel filtration along with an increasing pH gradient from 2 to 7.5. The study suggested that pH and urea gradient were important for correct refolding. A recent study combined the use of high hydrostatic pressure under alkaline pH in the presence of arginine to refold a recombinant dengue 1 virus NSI IB and found out that high yields were obtained at a high pH of 11 and a slightly higher volumetric yield at a pH of 10.5 in the presence of arginine [It appears therefore that an alkaline condition of pH 8 and above was more suitable for the efficiency of L-arginine in suppressing aggregation during dialysis. Burgess et al.,  also notarginine . In a searginine .This observation provides evidence for the stabilizing effect of arginine on enzymes. Although the mechanism for this is not yet clear, it appears that the basic and hydrophilic nature of arginine enhances solubility by acting as a non-denaturing solvent which interacts with hydrophobic patches in proteins ,57,58. TThe success of the refolding methods developed in this study was achieved by suppressing unfavorable interactions at every stage. Aggregation is suppressed in traditional dilution by adding aliquots of protein directly or in pulses into large volumes of buffers with very little or no denaturant ,59 whileS1 Fig(TIF)Click here for additional data file.S2 FigM Protein molecular weight marker. Lane 1, r-SgChiC. Lanes 2 and 3 are supernatants from 2 washing steps.(TIF)Click here for additional data file.S3 FigLow absorbance at 340 nm indicates less aggregation.(TIF)Click here for additional data file.S4 FigSolubilized protein is diluted in the ratio 1:10 and the absorbance at 340 nm. Low absorbance indicates better refolding and less aggregation.(TIF)Click here for additional data file.S5 FigM—molecular weight marker, Lane 1—Onc refolded sample with buffer C4, C6 and C5 respectively, Lane 4—Reverse dilution refolded sample.(TIF)Click here for additional data file.S6 Fig(A) Previous method whereby protein is refolded in buffer containing very low concentration of denaturant. This resulted in loss of protein due to aggregation when applied to the refolding of r-SgChiC in this study (B) Refolding by reduction of denaturant from high–intermediate–low concentrations. Refolding is afterwards completed by dialysis, during which all residual denaturant is excluded from the protein.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Presbycusis, or age‐related hearing loss (ARHL), occurs in most mammals with variations in the age of onset, rate of decline, and magnitude of degeneration in the central nervous system and inner ear. The affected cochlear structures include the stria vascularis and its vasculature, spiral ligament, sensory hair cells and auditory neurons. Dysfunction of the stria vascularis results in a reduced endocochlear potential. Without this potential, the cochlear amplification provided by the electro‐motility of the outer hair cells is insufficient, and a high‐frequency hearing‐loss results. Degeneration of the sensory cells, especially the outer hair cells also leads to hearing loss due to lack of amplification. Neuronal degeneration, another hallmark of ARHL, most likely underlies difficulties with speech discrimination, especially in noisy environments. Noise exposure is a major cause of ARHL. It is well‐known to cause sensory cell degeneration, especially the outer hair cells at the high frequency end of the cochlea. Even loud, but not uncomfortable, sound levels can lead to synaptopathy and ultimately neuronal degeneration. Even in the absence of a noisy environment, aged cells degenerate. This pathology most likely results from damage to mitochondria and contributes to degenerative changes in the stria vascularis, hair cells, and neurons. The genetic underpinnings of ARHL are still unknown and most likely involve various combinations of genes. At present, the only effective strategy for reducing ARHL is prevention of noise exposure. If future strategies can improve mitochondrial activity and reduce oxidative damage in old age, these should also bring relief. Presbycusis, the age‐related increase in hearing thresholds and increased difficulty in discriminating speech in noisy environments (ARHL), is the most common sensory deficit of aging in humans. Social isolation and cognitive decline accompany this disability. Establishing the etiology and biological mechanisms that underlie the pathology will drive any successful attempt to prevent or remediate this condition.1The effects of the passage of time on the structure and function of the inner ear are certainly complex and extremely variable among species and individuals within each species, but in general, most mammals, especially humans, lose hearing sensitivity, more profoundly at high frequencies. Humans also lose the ability to discriminate speech in noisy environments. Variability in the age of onset and the magnitude of degenerative changes are major features of ARHL. Obviously, the interactions among underlying genetics and environmental exposures contribute to the phenotype that is observed at the end of life. This review will provide an overview of the pathology, genetic, metabolic and environmental variables known to play a role in cochlear aging.2Degenerative changes in the inner ear of aged humans and other mammals occur among the sensory hair cells, the primary sensory neurons or spiral ganglion cells, and the cells of the stria vascularis and spiral ligament including the vasculature. The degenerative patterns of these structures were initially described in human temporal bones , it seems reasonable to expect that ARHL is also influenced by genetics. In fact, using the population of Framingham Heart Study participants, it was concluded that about 55% of the variance in auditory thresholds in aged siblings and parent/child relations was attributable to genetic causes , have been assessed using immunohistochemistry as a measure of mitochondrial “fitness.” Decreased immuno‐label, an imprecise measure of protein concentration, was seen in temporal bone sections from aged individuals, though the decrease did not correlate with the amount of hearing loss, nor with the number of remaining ganglion cells in the temporal bone  resulted in a larger loss of hearing in aged mice than the background strain and a greater loss of auditory neurons and strial cells  and guinea pigs, of similar age, exposed to 100 dB octave‐band noise, a level that causes only temporary threshold shifts, experience unexpected amounts of hearing loss at old ages. The hearing loss is accompanied by synaptic damage (synaptopathy) at the inner hair cell‐spiral ganglion cell ribbon synapse that leads to loss of the peripheral processes and neuronal cell bodies in the absence of inner hair cell degeneration (Kujawa & Liberman, 1 peak of an ABR to click stimuli in animals was used to provide a more useful estimate (Prendergast et al., While these experimental data clearly demonstrate this phenomenon, called hidden hearing loss (Schaette & McAlpine, In spite of the controversy, it seems that the experimental data in laboratory mammals, coupled with the human temporal bone data that show loss of radial fibers, and a greater loss of neurons than inner hair cells (Viana et al., 3.4Exposure to ototoxic drugs, especially chemotherapeutic agents and aminoglycoside antibiotics (Jiang, Karasawa, & Steyger, 3.5There has been little attention paid to immune responses in aged cochleas, although in the central nervous system, it is postulated that immune function can have a negative effect on neurons (Patterson, 4Diet and exercise are often discussed in relation to healthy aging and the condition of the inner ear is no exception. Caloric restriction, long known to prolong life, beginning at a young age was effective in reducing hearing loss and cell degeneration in aged rats and mice Seidman, . In coll5ARHL of cochlear origin occurs in most mammalian species, with differences in the time of onset and the magnitude of loss among species. It is associated with degenerative changes including the highly energetic cells of the stria vascularis, the vasculature, auditory neurons, and sensory cells, especially the outer hair cells. The major factor contributing to increased auditory thresholds, in the absence of chronic noise‐induced damage, is the loss of the endocochlear potential (Dubno et al., While genetic causes of cellular degeneration in the cochlea have not been identified, they certainly contribute to the aging phenotype (Gates et al., The magnitude of ARHL could best be reduced by protection from noise exposure that directly leads to outer hair cell degeneration (Liberman & Kiang, The author has no conflicts of interest to declare.Transparent Peer Review Report.Click here for additional data file.


Recent research has demonstrated frontal cortical involvement to co-occur with visual re-organization, suggestive of top-down modulation of cross-modal mechanisms. However, it is unclear whether top-down modulation of visual re-organization takes place in mild hearing loss, or is dependent upon greater degrees of hearing loss severity. Thus, the purpose of this study was to determine if frontal top-down modulation of visual cross-modal re-organization increased across hearing loss severity. We recorded visual evoked potentials (VEPs) in response to apparent motion stimuli in 17 adults with mild-moderate hearing loss using 128-channel high-density electroencephalography (EEG). Current density reconstructions (CDRs) were generated using sLORETA to visualize VEP generators in both groups. VEP latency and amplitude in frontal regions of interest (ROIs) were compared between groups and correlated with auditory behavioral measures. Activation of frontal networks in response to visual stimulation increased across mild to moderate hearing loss, with simultaneous activation of the temporal cortex. In addition, group differences in VEP latency and amplitude correlated with auditory behavioral measures. Overall, these findings support the hypothesis that frontal top-down modulation of visual cross-modal re-organization is dependent upon hearing loss severity. Auditory deprivation, or hearing loss, has been shown to affect adults in several ways, including increasing risk for social isolation, listening effort and fatigue, cognitive decline, and cross-modal cortical re-organization ,2,3,4,5.Later, as cochlear implantation became a successful option for the restoration of auditory input via electrical stimulation of the VIII nerve, implanted adults with acquired profound hearing loss were found to also demonstrate evidence of visual re-organization, with visual input processed in both the tvisual and auditory cortices ,22,23,24While visual cross-modal re-organization clearly occurs in profound hearing loss, several studies have reported this type of plasticity to be intiated in the early stages of auditory deprivation, or mild-moderate hearing loss ,15,28,29In addition to visual recruitment of auditory networks in mild-moderate hearing loss, recent research has identified frontal and pre-frontal cortical involvement in the processing of visual information for this population ,31, raisTaken together, it appears that both bottom-up sensory mechanisms  and top-down modulatory mechanisms  are involved in the processing of visual information in hearing loss. However, the role of the degree of auditory deprivation in this interaction remains unknown. For example, it is unclear whether top-down cognitive mechanisms are active in visual cross-modal re-organization in mild hearing loss, or whether moderate auditory deprivation is required for initation of this compensatory mechanism. Given that age-related hearing loss is typically insidious and long-standing before it becomes a clinical problem, a better understanding of the underlying trajectory of compensatory networks will provide clinically relevant information. Therefore, in the current study, we aimed to observe frontal and pre-frontal top-down resources activated by visual input as a function of mild versus moderate hearing loss and speech perception outcomes. We recorded visual evoked potentials (VEPs) via high-density electroencephalography EEG in response to apparent motion in a group of adults with mild and moderate hearing loss. Source localization analyses were conducted to view activated cortical networks between frontal and temporal regions in the two groups, and VEP responses in frontal regions of interest (ROIs) were created to assess frontal cortical responses. VEP responses were correlated with auditory behavioral measures, including speech perception in noise and pure-tone thresholds. We hypothesized that as hearing loss and speech perception worsened, frontal cortical regions, in addition to auditory cortical areas in the temporal cortex, would be increasingly activated to assist in processing of visual sensory information.Seventeen adults in the age range of 38 to 78 years participated in this study, which was approved by the University of Colorado at Boulder Institutional Review Board. The research was conducted in accordance with the Human Subjects Institutional Review Board (IRB) protocol 0906.16, approved 25 June 2010, at the University of Colorado at Boulder. All participants provided written consent. Each participant received a clinical audiometric evaluation, followed by a speech perception-in-noise assessment and EEG measurements.Of the seventeen participants, ten participants  presented with a clinically mild, sensorineural age-related hearing loss bilaterally. On average, pure tone thresholds for this group were below 25 dB Hearing Level (HL) for frequencies 500–2000 Hz, denoting normal hearing in this range ,36. For U = 70, Z = 3.42, p < 0.001). There was no significant difference in age between the participants in the MILD and MOD groups , and no impairments in visual acuity or neurological function were reported.Average audiograms for the two groups are shown in The QuickSIN™ measure , which iAll participants were presented with a high-contrast sinusoidal concentric grating that continually transitioned into a radially modulated grating (a circle-star pattern) ,40,41,42® 2.0, , stimulus software compatible with Net Station 4 .Participants were seated in a comfortable reclining chair in an electromagnetically shielded sound booth and fit with a high-density 128-channel EEG electrode recording net . The visual stimulus was presented via E-Prime® . In EEGLAB, data were downsampled to 250 Hz, implementing a 125 Hz (Nyquist frequency) anti-aliasing filter. This was followed by a pre-stimulus baseline correction and artifact rejection with a criterion of amplitude greater than +/− 100 μV. The data were also analyzed for eye blinks and saccades to remove ocular artifacts. Bad channels were removed from the recording and replaced with interpolated data from the remaining channels via a spline interpolation algorithm. Remaining epochs were averaged and re-referenced using common average reference.The sampling rate for the EEG recordings was 1000 Hz, with a band-pass filter set at 0.1–200 Hz via Net Station 4 default settings. Individual continuous EEG data were high-pass filtered offline at 1 Hz using a FIR filter set to Net Station 4 default settings. Low-pass filtering at 30 Hz was only performed for VEP figures created in EEGLAB (see below) using a FIR filter via the function pop_eegfiltnew default settings. Continous EEG data were epoched according to the EEG activity surrounding the stimulus presentation. Each epoch contained a 100 ms pre-stimulus and 495 ms post-stimulus interval. Epoched data were then exported from Net Station and imported in to EEGLAB  operatinOnce an individual VEP average was obtained, groupings of seven electrodes corresponding to frontal cortical regions of interest (ROI) were averaged together. Please see ® Scan 7 Neuroimaging Suite (Compumedics Neuroscan™) for source modeling.Independent component analysis (ICA) was performed for individual concatenated EEG data in EEGLAB following artifact rejection and common average referencing prior to ROI averaging ,45,46,47In CURRY, the components were averaged according to each VEP peak and categorized into the MILD and MOD hearing loss groups. For example, each group was comprised of a P1 component average, an N1 component average, and a P2 component average. Separate current density reconstructions (CDRs) were then created for the three VEP component averages in each group, using sLORETA (standardized low-resolution brain electromagnetic tomography). sLORETA is a statistical tool used in estimating CDRs that includes variance of cortical sources in combination with variance from the EEG recording ,52. HeadDue to the unequal sample size between groups, non-parametric statistical analyses were applied to the data. Peak latency and amplitude were compared separately across groups in each ROI using the Mann–Whitney U test . A one-tVEP group averages according to ROI are shown in U = 12, Z = −2.27, p < 0.05) and right  frontal regions. The finding of decreased VEP latency outside of primarily visual processing regions is suggestive of cross-modal recruitment as a result of hearing loss [U = 59, Z = 2.34, p < 0.05). Although increased VEP amplitude is typically associated with cross-modal recruitment and indicative of strengthened neural networks [It was found that the MOD hearing loss group demonstrated an earlier VEP P1 latency than the MILD group in both the left , significant correlations were identified between SNR thresholds and VEP components. As seen in r = −0.530, p = 0.014, Due to the CDR results showing frontal generators for VEP P1 and P2 components , as wellr = 0.633, p = 0.003) and right frontal cortices , as seen in In addition to the link between decreased speech perception and VEP characteristics, we found the degree of hearing loss to be positively correlated with VEP P2 amplitude in the left  with VEP component characteristics to evaluate possible relationships between auditory behavioral performance and visual plasticity.Our results are comprised of three main findings: (a) increased recruitment of frontal cortices combined with cross-modal recruitment of temporal auditory regions for visual processing in moderate hearing loss, (b) significantly decreased VEP latency and amplitude in frontal cortices for moderate hearing loss, and (c) significant correlations between VEP characteristics and auditory behavioral measures. These results are all suggestive of top-down mechanisms which likely modulate cross-modal visual processing, at least to some degree, according to hearing loss severity.Our previous findings have consistently shown that the visual stimuli described in the present study activate cerebello-occipital visual networks in individuals with normal hearing ,28,40,41Previous studies have identified specific frontal networks to be related to listening effort and hearing loss severity during auditory and visual tasks. For example, the left IFG, a region that was active during the VEP P2 component in the MILD group and across all VEP components in the MOD group, appears to act as a supplementary network for speech perception recovery in cochlear-implanted adults  and has While top-down modulation of auditory, visual, and audio-visual networks is indicated to supplement auditory cognitive function ,31,66,67Evoked potential latency has been consistently reported as a biomarker of both visual and auditory cortical plasticity in hearing loss . For exaEvoked potential amplitude changes have also been taken as evidence of cortical plasticity. For example, increased VEP amplitude in auditory cortical regions occurs in visual cross-modal re-organization and is correlated with decreased speech perception ,19,21,22Visual cross-modal reorganization of auditory cortices has been previously illustrated to correlate with poor speech perception performance and cognitive performance ,22,23,28While bottom-up visual cross-modal re-organization and top-down modulation of visual function is indicated to correlate with poor auditory behavioral outcomes, this plasticity may be beneficial to audio-visual integration in hearing loss in real life situations ,15,29,31Our results present evidence of early top-down modulation of visual processing in temporal areas as hearing loss severity progresses from mild to moderate degrees. At the same time, visual cross-modal reorganization of auditory regions is observed to be stable in both mild and moderate hearing loss. This frontal modulatory network, which includes areas of executive function, is negatively related to speech perception in background noise. Taken together, it is apparent that top-down modulation of visual function increases according to the degree of hearing loss, following bottom-up-driven visual recruitment of auditory systems, for compensatory purposes. At the same time, this increasing draw upon cognitive resources may co-occur with re-allocation of auditory function to frontal cortices for low-level sensory processing in hearing loss. It is therefore possible that these compensatory mechanisms may contribute to cognitive decline as finite resources continue to be taxed over time. Future studies should address the relationship of frontal and cross-modal compensation with cognitive performance in persons with hearing loss.


Given an increase in the aging population and its impact on healthcare systems, policy makers for provision of health and social services are aiming to keep older adults in good health for longer, in other words towards ‘healthy aging’. Our study objective is to show that rehabilitation with cochlear implant treatment in the elderly with hearing impairment improves the overall health-related quality of life and general well-being that translate into healthy aging.The multicentre, prospective, repeated measures, single-subject, clinical observational study will accrue 100 elderly, first-time, unilateral CI recipients (≥ 60 years) and analyze changes on specific measurement tools over ca. 20 months from preimplant to postimplant. Evaluations will consist of details collected through case history and interview questionnaires by clinicians, data logging, self-report questionnaires completed by the recipients and a series of commonly used audiometric measures and geriatric assessment tools. The primary indicator of changes in overall quality of life will be the HUI-3.The protocol is designed to make use of measurement tools that have already been applied to the hearing-impaired population in order to compare effects of CI rehabilitation in adults immediately before their implantation, (pre-implant) and after gaining 1–1.5 years of experience (post-implant). The broad approach will lead to a greater understanding of how useful hearing impacts the quality of life in elderly individuals, and thus improves potentials for healthy aging. Outcomes will be described and analyzed in detail.NCT03072862.This research has been registered in ClinicalTrials.gov , 7 March 2017 under the n°  Worldwide demographic shifts indicate that the burden of an aging population on healthcare systems will be affected by the number of babies being born into the more industrialized populations. That can be illustrated by census findings that reveal both Europe and Asia have the largest number of individuals over the age of 65 but the lowest birthrates. The US is also approaching a similar demographic. This means that the contribution to tax and social systems that fund healthcare supplied by working-age individuals will not be able to contribute enough to help finance the healthcare needs of the elderly because those contributing will be too few . The PRBIn terms of healthy aging, defined by WHO  as “The process of developing and maintaining functional ability that enables wellbeing in older age” , simply The presence of a HL and its considerable effects on quality of life and healthy aging has been established in various epidemiological studies , 27, 43,Laureyns M.  estimateThe most common path to remediating hearing loss is through evaluations that may lead to the use of hearing aids (HA). However, in cases where HL cannot be compensated by hearing aids, cochlear implant (CI) may be a viable option ; howeverWith respect to benefits from cochlear implantation, strong supporting evidence suggests that an improvement in functional hearing status via the use of cochlear implants in older adults has far-reaching effects on hearing-related quality of life and other health-related domains. Earlier studies 2002–2009) were most interested in psychosocial factors such as depression and isolation , 57, 72; were mosBenefits associated with quality of life as a primary aim of investigation have been studied . was theHere, we describe the study design of a multicenter prospective study in a large cohort of elderly individuals with equivalent CI experience. As to its unique features, it utilizes a full array of tests that include assessments in the physical, mental, psychosocial domains but with the primary focus on QoL.The primary aim is to identify significant improvement in the overall health-related quality of their life compared to their preimplant condition as measured by the HUI-3. The secondary aim is to investigate the influence of cochlear implant use on a variety of healthy-aging domains and, therefore, the overall well-being of elderly individuals. Specific healthy-aging domains to be investigated are hearing ability and communication, dependency, cognition, risk of falls, loneliness and depression/mood. Further, the study considers the impact of consistent daily use of CI communication through evaluation of data-logging outcomes.Clinicaltrials.gov identifier: NCT03072862.A repeated measure, single-subject design assesses the changes in health-related quality of life and overall well-being as the primary end-point of the study. As an observational study, no additional intervention is applied to our specific population of CI recipients. The study design is multicentre and includes language-appropriate materials relative to the participating CI recipient and has been registered at The evaluation protocol is designed to coincide with routine clinical visits. Each subject will be assessed with the full battery of tests that are repeated at each of the three visits  with the most recent implant technologies that are compatible with processors offering data logging. Subjects are aged ≥60 years with hearing loss that meets all local criteria for CI treatment. Subjects are enrolled into the clinical investigation only after signing the Patient Informed Consent Form prior to the pre-implant assessment . The expected duration for each subject from enrolment is 20 months (+/− 1 month). The study aims to accrue participants distributed across all study sites and languages to avoid site and cultural bias. A maximum of 100 individuals are to be included.Selection Criteria:Unilateral CI candidates with bilateral postlingual deafness with intention to treatContralateral ear: average pure tone thresholds indicate a moderately-severe to profound hearing loss .Willingness to participate in and to comply with all study proceduresFluency in languages used to assess clinical performanceAppropriate expectations from routine CI treatmentAble to decide on study participation personally, and independently sign their consentInclusion:Significantly/severely dependent or fragileUnable to provide consent personallyUnable to complete questionnaires for self-assessment independentlyUnilateral hearing lossRecipients of sequential and simultaneous bilateral CIOssification or other cochlear anomalies preventing full electrode insertion or medical contraindications to surgeryRetro cochlear or central origins of hearing impairmentSignificant comorbidities preventing study participation Clinic Standard fail criteria for CI candidacy in regards to chronic depression, dementia, and cognitive disordersUnrealistic expectations on the part of the subject regarding the possible benefits, risks and limitations inherent to the procedure and prosthetic device.Exclusion:Demographic and hearing impairment characteristics, such as the aetiology of deafness, the duration of profound hearing loss, the hearing aid use, will be collected. The education level will be assessed using the ISCED . In addition to these, general healthcare and patient-profile data and a selection of observational clinical assessment tools were chosen for repeated evaluations of the overall health status of the elderly individual at pre- and post-CI treatment intervals. The measures are commonly used in audiology and/or geriatric practices and are administered in the appropriate language. Translations of questionnaires have been controlled for via a validated translation process thus enabling collation of the data gathered cross culturally.A short description of assessment tools administered at baseline are shown in Table These include standard unaided (dB HL) and aided (dB SPL) threshold measures for frequencies 250–4000 Hz and speech discrimination in quiet and noise.Categories of Auditory Perception II (CAP-II) is an auditory skill rating index consisting of nine hierarchical categories completed by the clinician as an observation of the individuals hearing abilities. The auditory skills increase in complexity ranging from perception of environmental sounds to telephone conversation with an unfamiliar speaker [ speaker .Data Logging: Cochlear sound processors feature an in-built data-logging function. Specifics of the data to be collected, along with anonymous identifying reference, include the average time on-air per day and average times in noise, speech in noise, speech, quiet, music and wind; i.e. product usage data including the use of product features and acoustic environments [ronments .De Jong Loneliness is the six-item validated version [ version  with thrDigit-Symbol Substitution Test (DSST) is pencil and paper test in which the subject is given a key grid of numbers and matching symbols  and a test section with numbers and empty boxes. The task is to fill-in as many empty boxes as possible with a symbol matching each number. The score is the number of correct number-symbol matches achieved in 120 s.Geriatric Depression Scale-15 (GDS-15) is a self-administered questionnaire to record symptoms of depression in older adults. For each of the 15 questions of the scale, the subjects answer Yes or No, where ten questions indicate the presence of depression when answered positively while the other five are indicative of depression when answered negatively [gatively .Hearing Handicap Inventory in the Elderly Screening test (HHIE-S) is a short form, self-assessment scale designed to indicate the effects of hearing impairment on emotional and social adjustment in everyday life of the elderly individual [dividual .Health Utility Mark III (HUI3) is applied to assess health-related quality of life and is completed by the recipient. It considers, in this case, the impact of CI treatment across eight health domains  [nd pain) .Lawton Instrumental Activities of Daily Living Scale (L-IADL) is completed by the clinician mainly to determine a person’s ability to care for him or herself without any help from others. It measures the functional impact of emotional, cognitive, and physical impairments and their need for personal care services [services .Mini Mental State Examination (MMSE)-Short form- was derived from the six memory domains of the full MMSE. It can be used to estimate the severity and progression of cognitive impairment and cognitive change in an individual over time [ver time .Speech Spatial Qualities (SSQ) is a self-assessment scale of hearing ability and communication in daily environments, completed by the patient. It is divided into the three subcategories of speech (comprehension), spatial (hearing in space) and quality (speech and sounds) [ sounds) .The Timed Up and Go test (TUG) measures the time a person takes to stand up from a standard armchair, walk three meters, turn around, walk back to the chair and then sit down again [wn again .Trail Test B (TMT-B) is neuro physiological test assessing executive function. It is a more cognitively demanding task than the Trail A test [The l A test .The study is monitored by an external clinical research organization. A clear data management program has been set into place to validate and verify electronic data capture. Study sites and documentation may be subject to Quality Assurance audits during the course of the observational study. In addition, regulatory bodies, at their discretion, may conduct inspections, during and after study completion.For primary and secondary study objectives, an intra subject endpoint comparison is used: All pairwise comparisons are of interest; i.e. preimplant (Pre)-to-12 months postimplant (Post1), Pre-to-18 months postimplant (Post2), and the change from Post 1 to Post 2.The HUI3 will serve as the primary outcome measure for this study to determine the added health utility gain for the multi- attribute health status between Pre, Post1 and Post2. A one-way ANOVA will be performed on HUI3 multi-attribute scores, with visit as the main factor. Post-hoc tests will be performed to determine significant differences between visits.In  the authOur study objective is to show the potential for positive effects of CI treatment according to local indication criteria on overall health-related quality of life and general well-being that ultimately translate into healthy aging. The study outcomes are intended to provide transparent and comparable, evidence-based information to healthcare policy makers in support of guiding informed decisions on the provision of health services for the treatment of hearing loss.In 2014, Prince et al.  publisheThe comprehensive protocol considers a range of widely accepted, interrelated metrics associated with aging beyond functional hearing. Use of this protocol does not propose to investigate causal effects but rather the pre-post implant status of CI candidates who have 1–1.5 years of experience using their unilateral implants. In line with existing study designs for CI recipients, we have chosen both self-report and clinician-report questionnaires that are essentially non-verbal, thus removing challenges directly associated with utilizing newly acquired functional hearing . All theThe HUI-3, as the primary instrument in assessing CI implant treatment effects on overall health-related quality of life of elderly individuals, has been recommended as the most sensitive and useful tool for CI users , 34, 61.Further, the protocol investigates the major categories of psychosocial, mental, physical and communication behaviors with a range of other assessment tools currently applied to hearing-impaired individuals. The psychosocial domain regarding the issue of depression has been measured by the GDS-15 in CI users , 57. DepData logging is an important addition as it may provide new insights and potential correlations with healthy-aging domains. As has been pointed out by , there hA potential limitation of the study is the lack of a control group. In fact, establishing a randomized-controlled study design is not straightforward as it would imply the need not to give a CI to those control patients who meet criteria for cochlear implantation, which is far from ethical. The single-subject, repeated-measures design allows for subjects to serve as their own controls, and thus increases statistical power. But, the lack of preoperative repeated measures does not allow an estimate of within-subject variations in a non-intervention state. To counter this, the relatively large population size allows for a good estimate of effect size and would make the results broadly applicable. Another limitation is that the investigator or their team may have access to preoperative evaluation information at the time of the follow-up evaluations. However, many of the test results require coding in order to obtain a score and places a layer of protection against investigators making comparisons at the time of evaluation.Ultimately, the study aim is to provide clinical evidence that CI treatment in aging adults currently has the potential to improve the overall health status including, but not exclusive to, hearing function, which in turn can create cost savings from a payer and societal perspective. Age-related HL is often ignored as a stigma associated with aging or just accepted as a natural side-effect of it. Therefore, growing evidence may remove these boundaries and support referring professionals and potential CI candidates in their decision for CI treatment as soon as significant hearing loss is diagnosed in the hopes of reducing the overall impact of hearing disability.


Patient satisfaction with care has become increasingly important since Medicare’s shift to value-based reimbursement models using the Hospital Care Quality Information from the Consumer Perspective survey. Effective communication plays an underappreciated role in satisfaction with care. Hearing loss impacts two-thirds of adults over 70 years and is a barrier to communication. Previous research has found that adults with hearing loss have poorer health outcomes and incur higher medical expenditures. The present study aims to explore the association of hearing loss on satisfaction with care. Nationally representative data from the 2015 Medicare Current Beneficiary Survey (MCBS) collected information on self-reported hearing with a hearing aid  and satisfaction with quality of health care (very satisfied/satisfied and dissatisfied/very dissatisfied) for all adult Medicare beneficiaries. A weighted sample of 48.6 million Medicare Beneficiaries was analyzed using logistic regression, adjusted for sex, race, educational attainment, income, general health, and functional limitations in instrumental activities of daily living. The adjusted model found that compared to adults with no hearing trouble, those with a little trouble hearing and a lot of trouble hearing had 1.47 times  and 1.74 times higher odds  higher odds of reporting dissatisfaction with care, respectively. Hearing loss, possibly mediated by its impact on communication, is associated with satisfaction with care. Given the emphasis placed on patient-reported satisfaction value-based reimbursement programs, hearing loss may represent a potential, low-risk/high-reward area of intervention to improve satisfaction.


Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal egress from the red blood cells by rupturing the two surrounding membranes, the parasitophorous vacuole and the red blood cell membranes. Proteins of the so‐called osmiophilic bodies, (OBs), secretory organelles resident in the cytoplasm, are important players in this process. Once gametes emerge, the female is ready to be fertilized while the male develops into motile flagellar gametes. Here, we describe the function(s) of PBANKA_1115200, which we named Gamete Egress Protein (GEP), a protein specific to malaria parasites. GEP is restricted to gametocytes, expressed in gametocytes of both genders and partly localizes to the OBs. A mutant lacking the protein shows aberrant rupture of the two surrounding membranes, while OBs discharge is delayed but not aborted. Moreover, we identified a second function of GEP during exflagellation since the axonemes of the male flagellar gametes were not motile. Genetic crossing experiments reveal that both genders are unable to establish infections in mosquitoes and thus the lack of GEP leads to a complete block in Plasmodium transmission from mice to mosquitoes. The combination of our results reveals essential and pleiotropic functions of GEP in Plasmodium gametogenesis.Male and female  gep(‐) were not motile.PBANKA_1115200, which we named Gamete Egress Protein (GEP), is a gametocyte protein found only in malaria parasites. GEP partly localizes to osmophilic bodies. A mutant lacking this protein showed aberrant rupture of the parasitophorous vacuole and the red blood cell membranes. Axonemes of the male flagellar gametes  In a few minutes, gametocytes develop into gametes of the two genders that fuse to form a zygote. In 18–20 hr, it develops into an elongated motile ookinete that passes through the midgut epithelium and forms a sporogonic oocyst. During the next two weeks, approximately, oocysts rupture releasing infectious sporozoites that travel to the salivary glands from where, in turn, they are injected into a new host.The sexual stage of 2+ mobilization from internal stores and activation of the calcium‐dependent protein kinase 4 (CDPK4). This master regulator is essential for male gamete maturation, which entails three mitotic divisions, and assembly of axonemes to form eight motile gametes   localizes to vesicles distinct from OBs and is also involved in gamete egress  have been identified as having specific roles in the egress process. In the rodent Flagellar proteins are also required for proper axoneme formation. PF16 functions in maintaining microtubule structure, and in its absence, flagellar movement is abnormal and fertility is reduced , here named Gamete Egress Protein (GEP) due to its essential role(s) in gametogenesis of both male and female gametocytes. GEP is expressed in sexual blood stages and partly localizes to the OBs. When GEP is lacking, gametocytes develop normally but their maturation into gametes is severely affected. In males, axonemes are formed though they do not beat. Egress from the host RBC is also delayed in both genders and transmission to the mosquito is completely abrogated. Overall, our results suggest an important and pleiotropic function of GEP during Plasmodium transmission to the vector.In this work, we characterized a 22.1Anopheles gambiae mosquitoes were reared as described in Facchinelli et al., Plasmodium berghei ANKA 8417HP strain was used throughout the study  parasites.The pBAT plasmid  in the restriction sites SacII‐EcoRI upstream to the mCherry coding region. A PCR fragment of 692 bp , 1:2,000 in Western blot; α‐MDV1 mouse polyclonal, 1:400 in IFA, 1:2,000 in Western blot; α‐SET rabbit polyclonal  and incubating the samples for 15 min at 19°C.Gametocytes were activated after diluting infected blood 1:10 in activation medium (RPMI1640 with 2.5gep(‐) parasites with anti‐SEP1 antibody activated for 15 min, samples were permeabilized with 0.1% saponin for 30 s. The TER‐119 antibody was added diluted 1:200 in PBS with 2% normal goat serum for 1 hr incubation. Hoechst 33342 was used to stain DNA. Samples are washed twice with 1 × PBS before mounting in Vectashield (Vector laboratories).The TER‐119 antibody was used to label the outer leaflet of the RBCM, and anti‐SEP1 antibody was used as a marker for PVM. Gametocytes were activated, and samples were fixed in 4% formaldehyde at different time points after the activation. All steps were carried out at room temperature. For immunolabeling of WT and 2.62 in 0.1 M sodium cacodylate buffer, pH 7.4, overnight at 4°C and processed according to Perry and Gilbert  for 1 hr, in transfer buffer  onto Protran 0.22 µm membrane (Whatman). Incubation with antibodies was done. Primary and horseradish peroxidase‐conjugated secondary antibodies were incubated 1 hr in PBS‐Tween (0.05%), and the membrane was developed using the ECL system  according to the manufacturer's instructions.33.1Plasmodium genus as revealed by multiple sequence alignment comparing five different Plasmodium species cl1 and cl2  clones, both gametocytemia and morphology of male and female gametocytes were also roughly similar to WT as scored in Giemsa stained blood smears from infected mice  lines were inspected under the light microscope 15–20 min after activation. In a total of 13 experiments, we did not detect a single exflagellation event in the mutant parasites  in vitro and their adherence to RBCs forming the so‐called exflagellation centers. Exflagellations from the WT and the two clonal s Figure .gep(‐)cl1 with either a ruptured PVM and/or RBCM  males had vesiculated PVM and in only 5.5% the RBCM ruptured. In females, distinguished by their smaller nucleus, we detected similar results whereby only 4% of the gep(‐) females were fully released, while 38% had a ruptured PVM compared with the WT in which 91% had a ruptured PVM and 69% breached RBCM  clones neither in blood samples of the P. berghei line HPE which does not produce gametocytes  clones  gametocytes indicated no evident morphological alteration of organelles, including male and female OBs  gametocytes, double IFA with SEP1 and MDV1  gametocytes to 30–35 min and evaluated the egress efficiency by Western blot analysis comparing the amount of the OB‐resident proteins MDV1 and G377 released in the exflagellation medium. As shown in Figure gep(‐) parasites but not abrogated. In the activated gametocytes of the WT, only a small amount of GEP was released in the exflagellation medium, while the large majority of the protein was still detected inside the activated gametocytes  gametocytes even though a fraction of male gametes emerged from the host erythrocyte  mutant while moving actively in WT gametes. This could be due to a defect in the structural organization of flagella or timing of assembly of their components. To distinguish between these possibilities, we investigated the formation of the axonemes in gep(‐)cl1 activated male gametocytes by electron microscopy. The ultrastructural analysis confirmed that the majority of observed male gametocytes were still inside the PVM 20 min postactivation gametocytes were also examined in parallel by IFA using anti‐tubulin antibodies that stain male gamete flagella  parasites, male gametocytes were still enclosed in the membranes 20 min postactivation. Distinct nuclei were not visible while axonemes were fully formed consistent with the EM data. Overall, these data suggest that in gep(‐) mutant, axoneme formation and nuclear divisions are not coupled.The same preparations of activated WT and a Figure . In the 3.6gep(‐) transgenic line, both male and female gametocytes were affected in egress and that male gametocytes did not form motile flagella suggesting an additional role of GEP, not limited to the breakdown of cell membranes during gametogenesis. To determine whether the absence of GEP affects ookinete formation, we carried out genetic crosses by mixing infected blood of the gep(‐)cl1 and parasite lines forming either only fertile males (Δ47) or fertile females (Δ48/45)  line were unable to produce ookinetes in three independent experiments, while a cross of Δ47 and Δ48/45 done as a control produced fully mature ookinetes.Our experiments indicated that in gep(‐) parasites, An. gambiae mosquitoes were fed on mice infected with WT or gep(‐) cloned lines. As expected, no oocysts were detected in 2 independent experiments using the two gep(‐) clones, while in both experiments, WT parasites were infective to mosquitoes  protein. Our functional analysis revealed that mutants lacking gep had a pleiotropic phenotype with defects in gametogenesis resulting in the inability of both genders to establish mosquito infections. Both male and female gametocytes had a severe delay in egress from the red blood cell. At 20 min postactivation, the PVM was still intact in about half of the cells. A more dramatic effect was seen in RBCM rupture with only a few mutant parasites being released from the host cell. Furthermore, during male gametogenesis, no motile flagella were formed, although axonemes were assembled. The ultrastructural analysis confirmed this finding and also revealed that nuclear division did not take place. Genetic crosses confirmed that GEP was essential for the fertility of both genders.In this study, we identified the protein GEP (PBANKA_1115200) as a novel factor required for the successful gametogenesis of malaria parasites. GEP is a well‐conserved protein within the Plasmodium‐specific proteins is required for egress of mature gametes from the host RBC. In P. berghei, OB‐resident proteins MDV1  parasites are also severely affected in egress from the host cell, in contrast to MDV and GEST, the protein is mostly retained inside the cytoplasm of the gametocytes. However, we cannot exclude a minor portion of the protein being secreted due to the lack of a suitable marker to investigate this aspect. We observed that OBs also in our mutants moved to the cell membrane, but the release of cargo was delayed though not abolished. This suggests that GEP has a role upstream of OB discharge. Interestingly, a function in membrane fusion and secretion has been shown for the OB‐resident small solute transporter PAT , mutant axonemes were formed but not activated and cytokinesis did not take place. Similar phenotypes have been observed previously. Functional studies on the Anaphase Promoting Complex/Cyclosome (APC/C) revealed a role in cell cycle control during male gametogenesis; a conditional mutant was severely affected in chromosome condensation and cytokinesis ; Validation . Tomasino Pace: Data curation ; Methodology ; Validation . Felicia Grasso: Methodology (supporting). Inga Sidén‐Kiamos: Conceptualization (supporting); Writing‐original draft (supporting). Stefania Mochi: Methodology (supporting). Leonardo Picci: Methodology (supporting). Lucia Bertuccini: Methodology (supporting). Marta Ponzi: Conceptualization ; Data curation ; Funding acquisition ; Writing‐original draft (supporting). Chiara Currà: Conceptualization (lead); Data curation ; Formal analysis ; Funding acquisition (supporting); Investigation ; Project administration ; Supervision ; Writing‐original draft (lead); Writing‐review & editing (lead).All work was carried out in full conformity with Greek regulations and laws on animal experiments. In Greece, these issues are covered by the Presidential Decree (160/91) and law (2015/92) which implement the directive 86/609/EEC from the European Union and the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes and the new legislation Presidential Decree 56/2013. The experiments were carried out in a certified animal facility license (EL91‐BIOexp‐02), and the protocol has been approved by the FORTH Ethics Committee and by the Prefecture of Crete .


Cdh23. The cadherin 23 (Cdh23) gene is a key contributor to early-onset hearing loss in humans. In this study, we tested hearing across a large family of 54 BXD strains generated from B6 to D2 crosses. Five of 54 strains maintain the normal threshold (20 dB SPL) even at 2 years old—an age at which both parental strains are essentially deaf. Further analyses revealed an age-related hearing protection (ahp) locus on chromosome 16 (Chr 16) at 57~76 Mb with a maximum LOD of 5.7. A small number of BXD strains at 2 years with good hearing correspond roughly to the percentage of humans who have good hearing at 90 years old. Further studies to define candidate genes in the ahp locus and related molecular mechanisms involved in age-related resilience or resistance to AHL are warranted.Inbred mouse models are widely used to study age-related hearing loss (AHL). Many genes associated with AHL have been mapped in a variety of strains. However, little is known about gene variants that have the converse function—protective genes that confer strong resistance to hearing loss. Previously, we reported that C57BL/6J (B6) and DBA/2J (D2) strains share a common hearing loss allele in  Age-related hearing loss (AHL), or presbycusis, is a major sensory impairment  generallahl , 4519, 4Cdh23 , 18, 46.alh8) shows enhanced signals at all 8, 16, and 32 kHz. We and others have reported that the locus on Chr11 (ahl8) contributing to progressive hearing loss in D2 mice is a missense variant of the Fscn2 gene [c.753ACdh23 mutation is shared by both D2 and B6 [Arl6, Crybg3, Epha3, Epha6, Filip1l, Gabrr3, Gbe1, Gpr15, Hspa13, and Samsn1. Htr1f is identified as a frameshift variant.QTL analysis was used in our current research. We screened 54 aged BXD strains and performed a QTL analysis for hearing phenotypes at 8, 16, and 32 kHz. This analysis unveiled one novel QTL on chromosome 16 (Chr 16) and one known QTL on chromosome 11 (Chr 11) for all 3 measurements . Neverthcn2 gene , 35. The2 and B6 , and it ahp at the gene and molecular levels.MSM derived Ahl3 in B6-Chr17 (MSM) consomic mice showed a prominent hearing loss resistance and was mapped on chromosome 17 in 2004 . Apparenc.753ACdh23 allele [Cdh23 gene are involved in a spectrum of hearing impairments, including hearing loss with vision loss, Usher syndrome 1D, early-onset progressive hearing loss, and AHL in humans or mouse models. Two recent studies have shown that Cdh23 mutations significantly contribute to AHL in humans [Cdh23 is an important gene linked to hearing loss. Thus, the protective locus may prevent hearing loss in humans.Many of our commonly used hearing loss inbred mouse models carry the A allele , 47, whin humans . As a reIn summary, using QTL mapping, we have identified a novel locus on Chr 16 that is a significant contributor to the protection of hearing. It lessens the decline of the auditory function and pathology related to AHL, a disease affects the quality of lives in more half of the elderly over 75 years old. The discovery of this locus will help to better understand the molecular mechanism of AHL and provide clues for identifying new candidate genes responsible for human senile deafness and other hearing impairment.


Among the previous studies about the ADL recovery and its predictors, the researches and resources used to study and protect the baseline-independent older patients from being permanently ADL-dependent was few. We aimed to describe the level of activities of daily living (ADL) at discharge and ADL change within 6 months after discharge in older patients who were ADL-independent before admission but became dependent because of acute illness, and to identify the predictors of early rehabilitation,so as to provide the basis to early intervention.Stratified cluster sampling was used to recruit 520 hospitalised older patients who were ADL-independent from departments of internal medicine at two tertiary hospitals from August 2017 to May 2018. Demographics, clinical data, and ADL status at 1, 3, and 6 months after discharge were collected. Data were analysed using descriptive statistics, Student’s t-test, Pearson’s chi-square test,Spearman’s correlation analysis, binary logistic regression analysis, and receiver operating characteristic (ROC) curve analysis.p < 0.001). ADL score at discharge , age , post-discharge residence , and cognition status at discharge  were predictors of ADL recovery. The area under the curve of the four predictors combined was 0.763 (p < 0.001).There were 403 out of 520 patients completing the 6-month follow-up, and 229 (56.8%) regained independence at 6 months after discharge. There was an overall increasing trend in ADL with time. The recovery rate was the highest within the first month after discharge, gradually declined after 1 month, and changed less obviously from 3 to 6 months after discharge (Studying ADL recovery rate and its predicting indicators of the baseline independent inpatients at different time points provide a theoretical reference for the formulation of nursing plans and allocation of care resources. Disability is a common health problem in the elderly that brings a heavy burden to individuals, families, and society. Acute admission and subsequent hospitalisation are important factors of disability among elderly persons. Many studies have shown that those patients with dependence in activities of daily living (ADL) persisting longer than 6 months rarely experience functional recovery –3. ThereAlthough the above-mentioned studies examined the post-discharge function of older inpatients for a period of 1 month to more than 1 year, even 18 months, samples included older patients who were independent and dependent at the baseline. However, in order to prevent long-term dependence, it is more meaningful to examine ADL changes in older persons who are independent at baseline rather than including those already dependent. However, at present, few studies like this have been conducted. Hansen  investigIn consequence, we targeted older patients who were ADL-independent before admission and aimed to describe their ADL changes from admission to 6 months after discharge and identify the early stage of disease predictors of ADL recovery. This knowledge can provide a theoretical basis for the prevention of long-term dependence and the rational allocation of care resources in the future.Stratified cluster sampling was used to recruit 520 hospitalised older patients from August 2017 to May 2018 from departments of neurology, cardiology, respiratory, geriatrics, and emergency at two Grade A tertiary hospitals in the central and southern regions of Zhejiang Province, China. Participants were patients aged 60 years or over who were independent at baseline (Barthel Index score = 100 points) and became dependent (i.e. Barthel Index score < 100 points) because of an acute illness or an acute attack of a chronic disease, and provided informed consent. Patients who had a length of stay under 48 h, were difficult to follow up, had an injury or surgical operation, or were critically ill were excluded. During the investigation period, a total of 13,380 older patients were discharged from the relevant departments of the two hospitals. There were 2417 (18.06%) ADL-dependent patients at the time of discharge, of which 1548 were dependent before admission, and 869 were independent before admission but dependent at the time of discharge . Among the 869 cases, 549 questionnaires were sent out at the time of discharge, 520 were effectively responded, but only 403 cases completed the 6-month follow-up finally. Amongst them, 229 (56.8%) were independent in ADL after 6 months of discharge, and 174 (43.2%) were still dependent. The sampling procedure is detailed in Fig. The Demographic Information Questionnaire was designed by the researchers and included sociodemographic data  and clinical information such as discharge diagnosis, therapeutic drugs during hospitalization, chronic diseases, laboratory indicators, and other disease data as well as body mass index (BMI) and other nutritional indicators.The Barthel Index  includesThe Abbreviated Mental Test includes 10 items; if answered correctly, each item is scored as 1 point; a total score of 8 to 10 indicates normal cognitive ability, less than 8 indicates cognitively abnormal .The 5-Item Geriatric Depression Scale includes five yes/no questions, scored as 0 or 1 point; a score of 2 points or greater indicates a depression tendency; the sensitivity and specificity of the scale were 97 and 85%, respectively .The Instrumental Activities of Daily Living (IADL) Scale is a self-reported instrument including eight items .Level of IADL independence was measured by the number of IADLs requiring assistance.The full score is 8 , and the minimum 0  .Investigators were uniformly trained research group members . The investigators were led into the hospital by the head of each local hospital and entered the departments with the consent of the principal in charge. At the time of discharge or 1–2 days before discharge, after obtaining written informed consent from the patient or his/her family, data were collected.The baseline assessments (i.e. 2 weeks before admission) on ADL (Barthel Index) and IADL as well as the cognition status and depression at discharge were completed with the patients face to face.The ADL assessment included patients’ ADL level 2 weeks before admission to determine whether they were ADL-independent at baseline (inclusion criteria), and the ADL level at discharge. Laboratory indexes and ADL scores at admission were obtained from the electronic medical records. Patients’ families or primary caregivers were interviewed when patients were unable or too ill to communicate .ADL were assessed again by home visit or telephone follow-up at 1, 3, and 6 months after discharge. The duration of each assessment was 5–10 min. Since all patients were independent at baseline (i.e. 2 weeks before admission), we defined ADL recovery as a Barthel Index score of 100 points during follow-up. The study was approved by Ethics Committee of Wenzhou Medical University .p < 0.05) were then subjected to stepwise logistic regression analysis to evaluate the predictors of ADL independence. Spearman’s correlation analysis, binary logistic regression analysis, and ROC curve analysis were performed to identify the predictors and predictive value. For missing data, the mean substitution method was used for data interpolation was used. Categorical data were described as frequency (%), and Pearson’s chi-square test was applied to assess the differences between groups. Significant variables from the univariate analysis  years. The number of males (209) was slightly higher than that of females (194). Other sociodemographic information was detailed in Table  ± 7.69 y2)), the range was 13.33–32.87 kg/ m2, and the mean was (22.71 ± 3.64). According to Chinese evaluation criteria [There were 403 patients with neurological, respiratory, and cardiovascular conditions and patients with neurological diseases accounted for the majority of admissions. Other diseases were digestive and urinary (2 cases (0.5%), respectively). As for BMI (BMI = weight (kg)/height . The cognitive scores ranged from 0 to 10 points, with a mean of (7.58 ± 2.35). There were 161(39.9%) patients with abnormal cognition, and 242 (60.1%) patients with normal cognition. The depression scores ranged from 0 to 5 points, with a median of 0 . A total of 35 (8.7%) patients had a depressive tendency, and 368 (91.3%) did not.F = 284.111, p<0.001). From the perspective of the change trend, the ADL of older patients showed an upward trend from admission to 6 months after discharge, with the recovery rate being highest within 1 month after discharge, and gradually slowing down after 1 month after discharge, and showing no obvious change from 3 to 6 months after discharge. The functional change diagram was shown in Fig. This study compared the ADL level at admission, discharge, and 1, 3, and 6 months after discharge. The ADL status of participants was presented in Table Taking ADL independence at 6 months after discharge (binary variable) as the dependent variable, univariate analyses were carried out with sociodemographic data, clinical data, pre-admission IADL score, ADL scores at admission, the depression score, cognition score and ADL scores at discharge as the independent variables. The results showed that age, post-discharge residence, number of medicine, glycerin trilaurate, blood glucose, length of stay, the cognition status, depression at discharge and ADL scores at admission and discharge had an significant effect on ADL independence at 6 months after discharge. The results are presented in Table Taking ADL independence at 6 months after discharge as the dependent variable and the significant variables in univariate analyses as independent variables, binary logistic regression was carried out. The results showed that age < 65 years, returning home after discharge, the high cognitive scores and high ADL scores at discharge were significantly associated with ADL independence at 6 months after discharge. The results are presented in Table p < 0.001). These results are presented in Fig. In order to further examine the predictive value of the above factors for the recovery of ADL in older patients at 6 months after discharge, the prediction model was examined by ROC curve analysis. Taking ADL scores at discharge, age, post-discharge residence, and cognition scores at discharge as independent variables and ADL independence at 6 months after discharge as the dependent variable, the logistic regression analysis model was established, and the predictive efficiency of the ROC curve was fitted by the probability value in the model. The results showed that the area under the curve (AUC) for the four predictors combined was 0.763 , which was higher than the results of Boyd et al. , 22, 23.Similar to the results of previous studies , 7, 24, As for the factors related to functional recovery after discharge, previous studies had shown that age , 26, 27,ADL score at discharge and post-discharge residence were predictors of ADL recovery in elderly patients at 6 months after discharge, which had not been reported in previous studies. The higher the ADL score of older patients at discharge, the greater the probability of ADL independence recovery at 6 months after discharge. Although previous studies had confirmed the correlation between physical function at discharge and prognosis , 30, ADLThis study also found that the ADL score at 6 months after discharge of older patients who returned home (87.64 ± 21.51) was significantly higher than that of those going to nursing homes or other care institutions (62.22 ± 31.31). It might be that patients going home could experience care, companionship, and support from their families, which might play a role in the recovery of their functions; or might be the ADL and diseases of the two above-mentioned groups were distinct at discharge. Patients who went to institutions after discharge had a score of (24.35 ± 19.35), which was significantly lower than that of those who went home after discharge (55.93 ± 19.44), which could be the reason why patients going home had a higher ADL score at 6 months after discharge.Depression, albumin, and IADL 2 weeks before admission had no significant effect on the recovery of ADL at 6 months after discharge. In terms of depression, patients with a depressive tendency accounted for a small proportion of the sample (8.7%), which was lower than in previous surveys (17.3%) . This maAs for IADL 2 weeks before admission, some scholars held that patients who had much more IADL dependent were less likely to regain ADL independence. However, there was no association between the two in this study, which may need to be further examined in future research.In this study, the ROC curve analysis of age, cognition, post-discharge residence, and ADL score at discharge showed that the AUC for the combined predictors was 0.763, which is higher than in previous studies (0.640–0.784) . This inDue to practical limitations, this study could not examine other factors that may have an impact on ADL in early stages of disease, such as the comorbidity index. In addition, this study did not consider factors that may affect ADL after discharge in older patients, which need to be further studied in the future. It have been reported that patients with neurological diseases accounted for the majority of the hospitalized in this study, so the population of this study cannot represents all of acute hospitalized elderly patients.The ADL of hospitalised ADL-dependent older patients who were independent before admission showed a trend of gradual recovery within 6 months after discharge, but nearly half of the patients were still unable to regain ADL independence at 6 months after discharge. ADL score at discharge, age, post-discharge residence, and cognition were significantly associated with the recovery of ADL at 6 months after discharge in hospitalised ADL-dependent older patients, which could be used as a theoretical reference for the formulation of nursing plans and the allocation of care resources in the future.Additional file 1:. Table S1 Description of Missing Data. For missing data, the mean substitution method was used for data interpolation. The missing data were detailed in Table S1.