Mixed hearing loss associated with a sensorineural component and an impaired conductive mechanism for sound from the external ear canal to the cochlea represents a challenge for rehabilitation using either surgery or traditional hearing amplification. Direct stimulations of the ossicular chain and the round window (RW) membrane have allowed an improved hearing in this population. The authors review the developments in basic and clinical research that have allowed the exploration of new routes for inner ear stimulation. Similar changes occur in the electrophysiological measures in response to auditory stimulation through the traditional route and direct mechanical stimulation of the RW. The latter has proven to be very effective as a means of hearing rehabilitation in a group of patients with significant difficulties with hearing and communication. Hearing loss is a common malady that affects people of all ages. Throughout the last century, many different interventions have successfully improved communication skills for deaf and hard-of-hearing individuals. Methods employed have varied, depending on the etiology, degree of hearing loss, and the status of the middle ear. Mild-to-moderate sensorineural hearing loss is easily treated with traditional hearing aids, while cochlear implantation may be required in cases of severe-to-profound loss. A purely conductive hearing loss is often managed with middle ear operative procedures, amplification, or a combination of the two. However, the mixed conductive and sensorineural hearing loss remain a difficult condition to ameliorate. Traditional amplification is often stretched to its limits, having to overcome both a conductive and a sensorineural loss with outcomes often distorted and unappreciated by the patient. Hearing physician-scientists have turned to alternative pathways of stimulation, most commonly osseointegrated bone stimulators . In the The contribution of the RW to hearing was first suggested in the late mid-17th century with early clinicians proposing that it was a possible route for transmission of vibratory energy to the inner ear , 3. The In early patient observations , it was via the RW membrane.With the dissatisfaction of the standard hearing aid and the low acceptance rate in many deafened individuals, interests turned to devise potential alternative methods to stimulate the inner ear. During the latter decades of the 20th century, multiple investigators began to stimulate the ossicular chain directly with mechanically translating devices to produce ossicular chain movement. The devices used different technologies to stimulate the inner ear, such as piezoelectric crystals , 10, eleRecorded cortical-evoked potentials were similar in response to a broadband click in the external auditory canal and a vibrational stimulation of the RW membrane. The latter group noted that sound stimulation of the ear was more effective than direct mechanical RW stimulation in producing cortical responses.After multiple attempts to re-establish the traditional ossicular route of stimulation of the inner ear due to disease, further attempts are often doomed to failure. The work of the above investigators –8 led GaIn such a group of patients who have previously undergone multiple surgeries, Colletti et al.  removed The vibroplasty has met skepticism by others , complaiSkarzinski et al.  in a lonReviewing the early investigative works  and the Rosowski et al.  outlinedCochlear microphonic represents primarily the outer hair cell response of the cochlea, allowing an estimate of the stimulus energy received. CM waveforms were recorded in response to the two different stimulation modes in this live model, and representative waveforms are shown in Ossicular chain disruption, a common scenario, improved CM for frequencies near 3 kHz, however, the differences were relatively minor and were not statistically different. Electro-vibrational transfer functions, defined as stapes velocity normalized to the input signal to the transducer as a function of frequency, were similarly calculated during RW stimulation. With an intact ossicular chain, the peak velocity occurred at 0.68 mm/s/V at 2.8–3 kHz and fell off quickly above and below this peak. With chain disruption, the peak velocity at 2.8–3 kHz was reduced by −6.3 dB to 0.33 mm/s/V, but velocities were nearly doubled across the rest of the range of frequencies tested after disarticulation of the chain, being significant in all animals. Stenfelt et al.  had prevVibroplasty is subject to potential compromise based on surgical techniques applied . The potClinically, fixation of the stapes footplate or RW closure occurs with a high frequency in chronic ear disease. The effect of fixation of the oval and RW on the cochlear response was measured in the adult fat sand rat . RW fixaCompound action potential (CAP) measurements, which represent synchronous firing of auditory nerve fibers, are similar in morphology, as shown in A more in-depth look at the ABR in a live Guinea pig model comparing responses to piezoelectric transducer RW stimulation with that of an acoustic stimulus in the external auditory canal showed significant differences in amplitudes and latencies of the ABR to the two stimulations . At compCoupling of the prostheses has been speculated as a cause of the failure by many. Mancheno et al.  stressedIn more extensive studies of the multiple variables of RW exposure and transducer tip diameter in stimulating human cadaveric temporal bones , stapes The two diameter tips resulted in the same stapes velocity outputs in the low-to-medium frequencies. However, the 0.5-mm tip was 5–6 dB better than the 1 mm at high frequencies both before and after drilling the RW niche. The lack of improvement at the low-to-mid frequencies for the 1 mm ball tip was speculated as potentially resulting from interference of the bony dimensions of the RW, measured as 0.92 mm on average compared to 1.0 mm stimulating ball electrode. With the smaller tip, the entire circumference of the RW membrane could be observed.Backside loading has been stressed as influencing the performance of an FMT in enhancing its vibratory performance and retention of long-term coupling . Using aPlacing FMT perpendicular to the footplate, i.e., the direction of vibration toward the interposed tissue gave the best results in stapes movement . DisartiTwo different pathways for forward and reverse stimulations have been proposed , due larNakajima et al.  determinThis review has summarized the utility of the vibroplasty operation and its scientific validation. While many points are still controversial in parameters of surgical techniques and actual changes that may be occurring in forward vs. retrograde stimulation, clinical outcomes have been excellent in general in a population with very little else available to improve inner ear stimulation. The size of the FMT is a problem in many situations, approximately equaling the RW membrane in size . The intMultiple devices developed for ossicular chain stimulation have been modified for direct stimulation of the RW with successful outcomes. However, while being able to drive the inner ear retrogradely with the improvement of sound perception is very helpful to patients, it is not always commercially feasible to produce such devices. Even though results from essentially all the devices over the years have been significant improvement , third-pMany refinements need to be made to all components of these devices, including implantable microphones and the problems of designing a translating device to be used long term in a biological system. Though successful results by patients with the APHABs  would seFailure of obtaining third-party reimbursement throughout the world will continue to prevent future production and advancements. Even though these needed improvements could be made with present day advances in technology, the decision to withdraw or not the market was made by the company, based on marketing feasibility and needed expenditure to perfect the stimulating devices. Today, only the FMT is being manufactured and marketed worldwide, while all others have been removed.HJ primary author in developing concept research and writing. NG and DT advised on concept and aided writing 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.


Grooming is an essential health maintenance activity that is fundamental to the welfare of many companion animals. Despite the potentially serious consequences of inadequate grooming for pets and their caregivers, few studies have examined the role of access to pet grooming services and supplies in promoting and maintaining companion animal health and welfare. The goal of this paper was 2-fold: (1) To provide preliminary findings demonstrating the scope of grooming and matting concerns among animals served by a large, non-profit animal welfare organization and (2) to provide a call for research to guide effective prevention of and responses to grooming-related omissions of care. We retrospectively extracted data from five American Society for the Prevention of Cruelty to Animals (ASPCA) programs serving the New York City area: ASPCA Animal Hospital (AAH), Community Medicine (CM), One ASPCA Fund, ASPCA-NYPD (New York City Police Department) Partnership, and the Community Engagement (CE) Program. The prevalence of grooming–related concerns was relatively consistent across all three veterinary service programs . Thirteen percent of the ASPCA-NYPD Partnership's cruelty cases involved general hair matting concerns and/or strangulating hair mat wounds (93% were long-haired dog breed types). Five percent of CE cases received grooming-related supplies to support pet caregivers' in-home grooming capabilities. Our findings underscore the need to understand the scope of grooming-related concerns among animals served by veterinarians and other community programs to improve animals' access to health-related services. Pet grooming is a health maintenance activity that is fundamental to the welfare of companion animals. Most companion animals require some degree of grooming, which can include basic hygiene care such as brushing, clipping, and trimming hair, bathing, cleaning the ears, and trimming claws. Inadequate grooming can lead to pain and discomfort for the animal and other threats to animal health and wellbeing. For example, when animals' claws are not adequately trimmed, they may alter the gait of the animal and make walking uncomfortable or challenging. In extreme cases, the claws may grow in a circular pattern and penetrate the paw pads on the underside of the feet causing painful wounds. Overgrown claws can alter the normal anatomic position and function of the feet . AdditioThere are diverse reasons why a pet owner may not maintain their pet's grooming needs, some of which may be unintentional  and/or due to circumstances beyond their control . StiNationally representative studies suggest that nearly 60% of U.S. households report having at least one pet, with dogs and cats being most prevalent . Lack ofThere is limited empirical data on pet grooming in the U.S. However, a recent report from the American Pet Products Association  indicateInformation regarding grooming practices of U.S. cat owners is limited despite evidence that brushing cats' hair is an important practice that serves to remove dead hair, aerate the skin, and disentangle knots that can cause pain and discomfort such as skin tightness and pruritus (an itching sensation), particularly in long-haired breeds . The APPIt is important that veterinary and animal welfare professionals and researchers consider access to grooming services and supplies when discussing animals' access to health-related services. This is of particular importance at the current time given that a recent nationally representative study suggests that one in five households acquired a new pet during the COVID-19 pandemic (13%). Moreover, “designer breeds” , some of which require more intensive at-home and professional grooming for coat maintenance, are increasing in popularity –20. To aThis study was retrospective in design. All data reported on in the current paper were collected between 2018 and mid-2021 and reflect animals served in the New York City area. For the current study, we extracted data stored in electronic databases maintained by the ASPCA. Specifically, we examined data from five ASPCA program areas, which were stored in two databases (CiviCore and ImproMed). Each of the programs is described below.AAH accepts cat and dog patients who require urgent or emergency care and belong to pet owners who are New York City residents and are experiencing financial hardships or constraints. Pet owners can schedule an appointment for their pets for veterinary care and/or be referred by other ASPCA programs in New York City and private veterinarians in the area. Services are either low or no-cost.CM provides high quality, high volume spay/neuter and primary veterinary care via the ASPCA's Community Veterinary Centers (CVCs) and mobile clinics in communities that experience barriers to veterinary care. Focus areas include the Bronx and Brooklyn. In addition to this work, CM, Community Engagement (described below), and the ASPCA Adoption Center collaborated to conduct a soft launch of grooming services at the CVCs and at weekly vaccine events in May 2021. Services available included basic grooming and nail trims, educational demonstrations for owners, and supplies . These appointments were exclusively for clients who had brought their pet(s) in for vaccines or preventive care. All services provided by CM are partially or fully subsidized. For grooming services rendered as part of the soft launch, we examined data from May 26, 2021 through July 28, 2021.The One ASPCA Fund is a subsidy program for veterinary care facilitated by the ASPCA's Client and Member Support team to support and improve welfare for as many animals as possible. This program provides services for medical conditions that have a good prognosis and require short-term care. Clients qualify if they are referred by the ASPCA's Community Engagement or CM teams, the NYPD, or social service agencies .The ASPCA partners with the NYPD to prevent and respond to animal cruelty in New York City. The NYPD responds to animal cruelty complaints, and the ASPCA directly cares for the animal victims by providing forensic evaluations, medical treatment, housing and placement, behavior assessments and treatment.The CE team works with New York City residents who lack access to vital care, services, and supplies for their pets. Often, these pet owners are referred to the team by the NYPD as cases that would benefit from receiving services rather than criminal justice action. CE provides families with resources to help them create and sustain a safe and healthy environment for their pet(s). The team also accepts referrals from social service and other allied agencies and conducts outreach throughout the community to increase awareness and access to the ASPCA's veterinary care and spay/neuter services, among other pet-related resources.To identify grooming-related appointments among our veterinary medicine programs, we examined all appointments with medically necessary grooming or nail trim noted in the animal's record. When a grooming service was not explicitly captured in the appointment data as a distinct field, we identified grooming-related appointments by reviewing the appointment reason as reported by the client, reviewing the DVM's appointment notes, and/or searching for phrases and words including medical grooming, sedated grooming, matting, matted, strangulation, unkempt, overgrown, ingrown, embedded, curled, curling, and other variations of these words and phrases. Cases and appointments involving ear cleaning and infections were not included because it was not possible to determine whether these appointments were related to grooming-related omissions of care. For data from the ASPCA-NYPD Partnership, cruelty cases that included any version of the words “matting”  or “strangulation”  in the case description were included. A detailed overview of the procedures for identifying strangulating hair mats is provided in Watson and Niestat .Data were exported to Microsoft Excel, which was used to produce descriptive statistics on grooming and matting-related cases and appointments for each program.One ASPCA Fund included grooming-related observations by the DVM and/or provision of grooming services.The number of cases and appointments per ASPCA program and the corresponding percentage involving grooming and/or hair matting concerns are provided in Since 2018, more than 1 in 10 (13%) of the ASPCA-NYPD Partnership's cruelty cases have involved general hair matting concerns and/or concomitant strangulating wounds. Five percent of CE cases received grooming-related supplies to support in-home grooming capabilities . During the soft launch of the grooming services pilot program, 204 grooming appointments were provided at CVCs and weekly vaccine events; moreover, all available appointments were filled. At least 47% of these appointments served long-haired dog breeds or mixes ; 20% of appointments did not capture the breed.To our knowledge, this is the first paper to provide preliminary findings demonstrating the scope of grooming-related concerns among animals served by a large, non-profit animal welfare organization. We found that the prevalence of grooming–related concerns was relatively consistent across all three veterinary service programs (between 4 and 6%). Our results suggest that for many pet owners served by our animal welfare organization, especially by programs that aim to improve access to veterinary care through fully or partially subsidized services, earlier access to grooming care can help promote animal health and welfare. The level of need for medical grooming identified across these programs suggests that preventable issues, such as hair matting, likely diverts limited resources that could be better positioned for other veterinary care. For example, grooming is not on the menu of services provided by the ASPCA's veterinary programs. Thus, pets are typically brought in for another medical concern, and yet the animals' grooming need cannot be ignored and must be addressed by the veterinary staff at the same time. This could divert doctors' and staffs' time and resources away from treating other patients, which is of particular concern given the current veterinary staff shortage , 22.Consistent with Watson and Niestat's  researchOur findings suggest that the provision of basic grooming services, facilitating clients' access to grooming services and supplies, and increasing clients' knowledge of their pet's grooming needs should be considered an important aspect of veterinary and animal welfare professionals' ability to provide a spectrum of care. Increasing clients' access to basic grooming may help to prevent more expensive and advanced care in the long-term and is particularly important for pet owners with financial limitations and those from underserved communities , 24. AccThis study also identified that the prevalence of grooming-related cases among animals served by the CE team was consistent with those served via the ASPCA's veterinary care programs (5%). This comparable rate is interesting to consider given that individuals who receive CE services and supports are often referred by social service and other allied agencies and by the ASPCA-NYPD Partnership in situations where non-criminal interventions can improve the health and safety of the animal. In contrast to the other ASPCA programs examined in this study, the rate of grooming-related concerns among animals served by the ASPCA-NYPD Partnership was notably higher and nearly double the rate found among the other programs at 13%. In addition, 97% of the ASPCA-NYPD Partnership's related cases involved dogs. The low rate of cases involving cats could be explained by prior evidence that cases of cruelty are more likely to receive prosecutorial attention if they involve dogs, despite other evidence that cats are the species most often involved in these cases –27. The Findings from our examination of CE and ASPCA-NYPD Partnership cases suggest that programs aimed to improve access to health-related care among pet owners and those that provide direct human services would benefit from developing collaborative relationships with animal welfare organizations that can connect pet-owning clients with supplies and resources that help foster their ability to groom their pets. In addition, animal welfare organizations can be proactive in efforts to prevent grooming-related omissions of care. Examples of proactive efforts include providing trainings for allied professionals , educational materials for staff and clients , and no- and low-cost services for their clients, as these populations likely face increased barriers to accessing health-related information and services for their pet. With more proactive support to meet pets' grooming needs, pet owners may intervene before hair matting or other identified medical problems significantly impact the animal's quality of life.To prevent grooming-related omissions of care, it is important that the veterinary science and animal welfare fields consider how the social determinants of health that impact human health and wellbeing  have a direct and indirect effect on pet owners' ability to groom their pets, particularly among marginalized communities. Pet owners may not be able to provide basic grooming care due to the conditions of the environment in which they live . For exaIt is common for private, for-profit veterinary practices to include non-medical services  within their business model as both an additional means of profit and as a convenience for pet owners. Our findings suggest that grooming services warrant consideration in the non-profit model as well, particularly in communities that lack access to pet care and/or have been historically excluded from vital pet care services. Given that many animal welfare organizations and shelters offer emergency sheltering and/or pet food pantries, it is conceivable that no- and low-cost pet grooming services could be added to expand the continuum of care and services provided for underserved animals and their caregivers. In addition to providing grooming services and supplies, programs that provide grooming demonstrations for pet owners and teach them to groom pets could be an effective way to prevent grooming-related omissions of care. Furthermore, it is possible that animal welfare organizations could partner with existing non-profit organizations that provide hygiene kits, access to mobile showers, and self-care resources for individuals who are housing insecure and expand the scope of services to provide animal-inclusive services that help to preserve the bonds between marginalized and underserved people and their pets.Collectively, our results suggest that improving access to grooming services and supplies and improving caregivers' knowledge of their pets' grooming needs may improve the welfare of a significant number of companion animals served by programs that aim to improve underserved pet owners' access to veterinary care. Previous work has explored the concept of social determinants of animal health, a model that emphasizes health determinants that are important or unique to animals. For example, Card et al. discussed the intersection of human and animal social determinants of health and the importance of access to veterinary services in pet health equity . As sociThere are several limitations of our study that warrant consideration. First, our study was retrospective in design. Due to differences and changes in services, client eligibility parameters , and data entry and storage programs across ASPCA programs, our data points  vary slightly across programs. Second, we did not evaluate all aspects of grooming-related care, such as ear cleaning, or related medical issues such as myiasis. This was not possible due to inconsistencies in how programs collect these data. Third, it is important to note that there may be overlap between clients served through our veterinary services and those served by CE and the ASPCA-NYPD Partnership. Although we were able to identify the number of appointments, cases, and organizational resources serving animals who present with grooming-related concerns, we cannot identify the exact number of animals served as it is possible that some animals were double-counted due to how many times they were seen, both within and across programs. Fourth, our data reflect clients of a large non-profit organization in a large, urban city. Thus, our data may not generalize to other regions of the U.S. or animals served by organizations with fewer resources. Moreover, there is little marketing for the ASPCA's programs, and the services are limited, which likely makes the rates reported in this paper an underestimate of the scope of need. Finally, our datapoints span the time periods prior to, during, and after the height of the COVID-19 pandemic, which had major impacts on the operations of animal welfare organizations, veterinary practices, and law enforcement agencies.We conclude this paper with a call for research that aims to prevent and adequately respond to grooming-related omissions of care. First, to prevent grooming-related omissions of care and increase animals' access to health-related services, it is important to understand the scope of these concerns in general veterinary practice and in community and shelter medicine settings. We encourage other animal welfare professionals and researchers to examine and report on the scope of grooming-related concerns within animal care services. Such data will be critical to informing programs and policies that enhance grooming-related care. Furthermore, future work should aim to identify whether there are shared characteristics  among pet owners whose animals are at risk for grooming-related omissions of care. Relatedly, understanding pet caregivers' beliefs and knowledge about grooming is essential to developing resources and programs that can successfully prevent and adequately respond to grooming-related omissions of care. Such information could help identify who may be most likely to benefit from knowledge and resources on grooming-related care and access to no- or low-cost services and/or how services and resources can be adapted to be culturally appropriate and responsive. Understanding discrepancies between veterinarian-identified grooming concerns and pet owners' awareness and concerns about these issues is also an important direction for future research.As previously discussed, several individual and contextual factors likely serve as obstacles to adequate pet grooming, such as the owners' financial resources, proximity to grooming services, and access to pet-friendly transportation. Research is needed to understand these barriers and how they can be prevented, eliminated, and/or mitigated by non-profit animal welfare organizations, private veterinary clinics, and allied professionals. There is also a need to understand how pets' behavior impacts caregivers' ability to groom pets. For example, transporting pets to grooming appointments and/or attempting to groom them at home may be particularly difficult when animals have behavioral problems and behavioral conditions, such as aggression, anxiety, reactivity, and/or fear of being handled . MoreoveFinally, the physical, mental, and cognitive health of pet owners and characteristics of their social relationships also have implications for the quality of care that animals receive and there is substantial need to understand how the psychological and physical health of pet owners may contribute to grooming-related animal welfare issues. For example, it may be unsafe for older pet owners and/or those with physical limitations or disabilities to groom pets at home. These owners may require assistance with traveling to grooming appointments and/or purchasing grooming supplies and performing grooming activities, such as bathing and nail trimming . RegardiCompanion animals' grooming needs are an important aspect of their health-related care. Our findings provide preliminary evidence that improving access to grooming services and supplies and improving caregivers' knowledge of their pets' grooming needs is likely to improve the welfare of a significant number of companion animals. As few studies have examined pet owners' knowledge of their pet's grooming needs and/or barriers and facilitators of access to grooming services and supplies, we strongly advocate for continued research in this area. In addition, there is a great need for research aimed at establishing best practices for implementing programs that provide no- and low-cost grooming-related services and supplies for animals and their owners, particularly among underserved and low-resourced populations and communities. Consistent with prior work, our findings suggest that improved inter-agency and cross-services collaboration can help to ensure the health and welfare of multispecies families , 47.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SM and JS: conceptualization and methodology. JS: analysis. SM, JS, LN, and CD: writing, review, and editing. All authors contributed to the article and approved the submitted version.This study was funded by the ASPCA.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.


Sensorineural hearing loss in beta-thalassemia is common and it is generally associated with iron chelation therapy. However, data are scarce, especially on adult populations, and a possible involvement of the central auditory areas has not been investigated yet. We performed a multicenter cross-sectional audiological and single-center 3Tesla brain perfusion MRI study enrolling 77 transfusion-dependent/non transfusion-dependent adult patients and 56 healthy controls. Pure tone audiometry, demographics, clinical/laboratory and cognitive functioning data were recorded.p < 0.04). Relative auditory cortex perfusion values showed a significant U-shaped correlation with PTA values among hearing loss patients, and a linear correlation with the full scale intelligence quotient  with its domain related to communication skills  in controls but not in beta-thalassemia patients. Audiometric test results did not correlate to cognitive test scores in any subgroup.Half of patients (52%) presented with high-frequency hearing deficit, with overt hypoacusia (Pure Tone Average (PTA) > 25 dB) in 35%, irrespective of iron chelation or clinical phenotype. Bilateral voxel clusters of significant relative hypoperfusion were found in the auditory cortex of beta-thalassemia patients, regardless of clinical phenotype. In controls and transfusion-dependent (but not in non-transfusion-dependent) patients, the relative auditory cortex perfusion values increased linearly with age (In conclusion, primary auditory cortex perfusion changes are a metabolic hallmark of adult beta-thalassemia, thus suggesting complex remodeling of the hearing function, that occurs regardless of chelation therapy and before clinically manifest hearing loss. The cognitive impact of perfusion changes is intriguing but requires further investigations.The online version contains supplementary material available at 10.1186/s13023-021-01969-0. Beta-thalassemia is an inherited blood disorder caused by defective production in the beta chain of hemoglobin. The most severe forms require regular blood transfusions to survive  exacerbating multisystem iron overload. Overall survival has strikingly changed after the introduction of iron chelators that enabled the prevention and management of iron related systemic complications . HoweverIn this scenario, there is a growing need to better define the hearing loss features, including type, prevalence, pathogenesis, evolution, and impact on cognitive functioning. This effort would be important for disentangling the possible pathogenic links and compensatory mechanisms, to identify new targets for prevention and management of some of the disease related aspects that might considerably impact on patients’ quality of life. We performed a multicenter single-scanner cross-sectional MRI study analyzing non-invasively in beta-thalassemia patients and healthy controls the auditory cortex perfusion changes occurring in relation with clinical phenotype, age, audiological findings and cognitive performances.Study subjects were patients with transfusion-dependent and non-transfusion-dependent Thalassemia  aged more than 16 years and healthy controls who had participated in previous multimodal brain MRI and neurocognitive studies , 14. TheMost patients were undergoing annually pure tone audiometry as per clinical practice ; the evaluations closest to the MRI assessments were collected; for those patients who did not have an audiological test available (i.e. NTDT patients naive for ICT), a pure-tone audiometry was purposefully performed.Pure Tone Average (PTA) was calculated as the mean in dB of the four central frequencies tested in the tonal audiometric examination . Normal hearing was defined if PTA was between 0 and 25 decibels (dB nHL); the degree of hearing loss was classified according to World Health Organization—Grades of Hearing Impairment in different levels of severity: mild hearing loss (26–40 dB nHL), moderate hearing loss (41–60 dB nHL), severe hearing loss (61–80 dB nHL) and profound hearing loss (> 81 dB nHL).Additionally, three main groups were identified based on audiometric profile: patients with normal audition , patients with hearing loss in at least one frequency but normal PTA (fHL), and patients with overt hearing loss .All study participants underwent brain MRI on the same 3.0 T scanner  with a 20-channel head and neck coil.3, bolus-duration 700 ms, inversion-time (TI) = 1990 ms, 2 repetitions, 50 slices, . This series was acquired with the subject at rest with eyes open. For anatomical reference a 3D-T1-weighted magnetization prepared rapid gradient echo (MPRAGE) sequence was also acquired with TR = 2400 ms, TE = 2.25 ms, resolution = 1 × 1 × 1 mm3, matrix-size = 256 × 256, generalized autocalibrating partially parallel acquisitions (GRAPPAs) factor of 2 in phase-encoding direction.Cerebral Perfusion weighted maps (PWI) were obtained from brain images acquired using a 3D Pulsed Arterial Spin Labeling (PASL) sequence with labeling scheme FAIR Q2TIPS, TR = 5000 ms, TE = 16.38 ms, matrix-size 64 × 64 voxels, voxel-resolution = 3 × 3 × 3 mmThe perfusion analysis method is reported in the online material.p < 0.001 voxel-level and only clusters at p < 0.05 family-wise error corrected at the cluster level were considered significant.Since the interest was focused regionally on the auditory cortex, to reduce the intrinsic inter-subject variability related to both physiological and technical aspects of ASL acquisition, relative cerebral blood flow (rCBF) values were obtained after normalization to the mean regional rCBF value of a non-auditory sensory brain region. For this purpose the mask of bilateral occipital cortex was obtained using pickatlas toolbox in SPM  and the Study subjects underwent a pool of psychometric tests included in the Wechsler Adult Intelligence Scale-Fourth Edition (WAIS-IV) . For thip < 0.05. Finally, an analysis of covariance (ANCOVA) was used to assess the linear dependence of the mean rCBF values in the AC and the PTA separately for the groups with hearing impairment (HL and fHL). Only descriptively, the same dependence was also investigated using a polynomial fitting (bsquare fit).Comparisons between groups were performed using the T test, the Mann–Whitney U test and the Chi-square test (or the Fisher Exact test when required) for respectively normally distributed, ordinal and qualitative non ordinal variables. The linear correlation between two variables was tested using Pearson's correlation. Statistical significance was set at p < 0.01).Seventy-seven patients (54 TDT and 23 NTDT) and 56 healthy controls were enrolled; demographics and clinical characteristics are shown in Table Seventy-one patients  underwent auditory assessment; air conduction charts are presented in Fig. p > 0.05). Mean-age of patients with hearing deficit was 37.8 ± 9.5 versus normal hearing patients 30.5 ± 10.5 (p = 0.003). Three out of seven NTDT patients with abnormal audiometry never took iron chelation therapy. Overt hypoacusia (PTA > 25 dB) was found in 25/71 patients  and 12/71  were classified as fHL . Among patients with PTA > 25 dB, hypoacusia was mild in 18/25 , moderate in 5/25 and severe in 2/25 .Any degree of hearing deficit was noted in 37/71 patients : it was bilateral and symmetric in 50% of the subjects. Conductive deficits were found in 3/37 ; three a mixed deficit, one had SNHL at one side and conductive at the other, two had SNHL one side and mixed the other.Whole brain perfusion is expected to be globally increased due to anemia  and thisAccordingly, the auditory cortex in beta-thalassemia patients showed a significant relative hypoperfusion in the Heschl’s gyrus and the neighboring cortex bilaterally , that was replicated in TDT patients ; in NTDT patients no significant changes appeared in the auditory cortex perfusion values with increasing age . , relative hypoperfusion was confirmed compared to healthy controls (left cluster size = 722 voxels and right cluster size = 264 voxels).Among patients, after regression on age, there was no significant difference in regional perfusion values among patients with normal audition, fHL and HL , taken separately, correlated with rCBF values. At first, a linear regression was performed accounting for the groups , that revealed a significant interaction effect (HL-group × PTA) on the rCBF values. Observing the different behavior of the two groups the correlation was further described with a second-order polynomial fitting using the curve fitting toolbox of MATLAB Fig. .Fig. 4Pop = 0.01, left side p = 0.02) and with the cognitive domains primarily involved in communication skills .No correlation was found in the patients' group between cognitive functioning scores and audiometric profile  most likely hearing loss worsens and becomes more frequent with aging, as hypocusic patients’ mean-age was 8 years higher than what observed in normal hearing patients; (2) differently from what has been reported for the pediatric age, in adulthood most patients present with sensorineural hearing loss, and (3) even patients naive for iron chelation therapy may present with sensorineural hearing impairment.The main novel result of the present study is the robust evidence of decreased relative perfusion of auditory cortex in beta-thalassemia. This finding takes into account (and is therefore not affected by) the fact that the global (whole brain) perfusion was expectedly increased in beta-thalassemia as a (known) compensatory mechanism to lower mean hemoglobin levels.To overcome the confound of global hyperperfusion, a ratio between the rCBF in each brain location and the rCBF in the occipital cortex was first obtained, assuming that the optic pathway and, more generally, the visual function are not affected in this condition . Interestingly, the relative hypoperfusion of the auditory cortex was observed in both clinical phenotypes (TDT and NTDT), thus highlighting that this phenomenon does not depend on disease severity and affects all forms of beta-thalassemia regardless of transfusion dependency. In addition, the ratio between auditory and visual cortex perfusion values increases with age in healthy controls in line with what observed by Hugenschmidt et al.  In theirAccordingly, TDT patients showed a similar trend of increasing relative auditory cortex perfusion, although this trend remained at any age below what was observed in healthy controls.In contrast, NTDT patients showed perfusion values close to healthy controls at a young age. However, relative perfusion values remain substantially constant with increasing age, so that during adulthood (i.e. starting at age 40) they become even lower than those of TDT patients.If confirmed on larger age-ranges samples, our findings reveal a different time course of perfusion among patients treated conservatively without blood transfusions, that strikingly differs from what observed in normal controls.In this case, the pathogenic link between age related perfusion changes and NTDT related features  would then need to be further explored.Hypoperfusion in the auditory cortex did not correlate with the audiological phenotype as patient subgroups with normal hearing, fHL and HL did not differ significantly in terms of mean relative perfusion values. However, auditory cortex perfusion values were intriguingly linked with the hearing loss severity in the patient subgroups with auditory deficits.These apparently contradictory findings  are partly explained by the decreasing perfusion with increasing severity in the fHL subgroup and the concomitant increasing perfusion with increasing severity in the HL subgroup. Considering hearing deficit in beta-thalassemia patients as a single phenomenon that worsens by involving both more, and more severely, the tested frequencies, the correlation between hearing deficit and perfusion is well depicted by a curve that associates the lowest perfusion levels to an intermediate hearing loss severity (i.e. a PTA close to 25 dB). These findings seem to indicate that the perfusion initially correlates with the hearing deficit, but that other compensatory mechanisms likely act whenever the deficit overcomes a PTA threshold of 25 dB. Interestingly, a compensatory increase in perfusion occurs at this threshold, at which individuals experience difficulty in understanding everyday speech .Our findings among normal hearing beta-thalassemia patients prompt the counterintuitive hypothesis that auditory cortex hypoperfusion could be not secondary to hearing loss, rather it might pre-date the onset of clinically detectable hearing deficits. This hypothesis could also explain the common detection of sensorineural hearing deficit among NTDT patients naive for iron chelators.Normal hearing is crucial in cognitive functioning as it allows effective responses to external stimuli, contributing to comprehension, vocabulary and memory; it is therefore conceivable that the metabolic activity of the auditory cortex in healthy subjects correlates linearly with the cognitive scores such as the FSIQ. In contrast, the relative auditory cortex perfusion was found in both beta-thalassemia phenotypes persistently low, independently of cognitive functioning. The lack of increasing perfusion with increasing cognitive functioning in beta-thalassemia made the difference in perfusion vs healthy controls even larger among those with higher cognitive performances, as if the auditory cortex metabolism were not further recruitable in response to increased cognitive functional needs. Actually, the inability to tune the auditory cortex metabolic level to the cognitive functioning could be a possible, so far unexplored, explanation of the generally reduced cognitive performances observed in the beta-thalassemia population, especially in the TDT subgroup and regarding primarily the verbal-comprehension tasks.. CompareTwo other points deserve to be further addressed. The fact that, notwithstanding the above mentioned metabolic impairment, some beta-thalassemia patients reach high level cognitive performances might suggest the existence and recruitment of alternative neural networks for compensating the low response of the auditory cortex. Indeed, the lack of correlation between hearing loss severity and cognitive functioning among beta-thalassemia patients supports the hypothesis of alternative recruitments of less efficient extra-auditory neural networks. Secondly, the more severe metabolic involvement of the left auditory cortex  seems to further underline the link between the observed metabolic derangement and the cognitive language-related scores, as the left is known to be the dominant hemisphere for most language tasks.Notably, auditory cortex hypoperfusion has been recently shown to distinguish patients with age-related hearing loss due to hair cell loss, that typically involves the high frequencies with a pure tone audiogram rather similar to what observed in beta-thalassemia patients . HoweverThe phenomenon of auditory cortex hypoperfusion independent from hearing deficits is rather difficult to explain, even considering differential development paths of the regional brain cortex or regional differences in brain vasculature. It is well known, for example, that the auditory cortex is one of the few regions that maturate early during the fetal life. A regionally different timing of brain cortex maturation could result in a selective vulnerability of the early developing cortices to early ischemic and metabolic events. However, an effect of thalassemia on the auditory cortex appears unlikely during gestation or early postnatal months as fetal hemoglobin production is preserved. In addition, the visual cortex, that shares a similar maturation course during fetal brain development, does not seem to be concomitantly affected.Analogously, the anatomic features of the intracranial vascular system do not seem to expose the auditory cortex to specific perfusion injuries. The auditory cortex does not belong to watershed territories; on the contrary, this region is well supplied by branches of the middle cerebral artery, the main cerebral artery of the developing and mature brain.Therefore, the hypothesis of a disease-related selective regional vulnerability is intriguing but not likely. To make this issue even more puzzling, data on the pediatric brain perfusion are still lacking in beta-thalassemia and it is unknown whether the perfusion deficit of the auditory cortex appears at birth or even earlier. The present study focused on adults because of reasonable ethical concerns in investigating small children requiring sedation and because normative perfusion MRI data on the developing brain are still lacking and difficult to be achieved. Auditory cortex hypoperfusion predisposing to subsequent hearing loss is a hypothesis that could partly explain the high rate of hearing deficit in beta-thalassemia. So far, hearing loss in beta-thalassemia has been considered a peripheral sensorineural high-frequency auditory problem mostly due to chelation treatment. However, data on the link between chelation therapy at current dosages and hearing deficits are rather conflicting , 8, 23. Even though this is the first study that provides a detailed picture of the metabolic status of the auditory cortex in beta-thalassemia in relation to the audiogram profile and the cognitive scores, there are limits that should be highlighted to define the possible future development in this field of research. First of all, we used a non-invasive ALS-MRI technique that does not provide absolute perfusion values. For this reason, we considered a ratio with a likely not involved brain cortex  introducing possible confounders. Secondly, the present study included only a couple of dozens of NTDT patients; as they likely represent a distinct beta-thalassemia patient population  studies focusing on a larger sample of NTDT are advisable. We used the WAIS assessment for cognitive functions, that is preferred in the clinical practice, though other cognitive tests used in the research context could be more appropriate in characterizing the cognitive profile of patients. Finally, our study did not include pediatric patients leaving unsolved the timing regarding the development of the observed perfusion change in beta-thalassemia. This knowledge will help us to better define, if present, possible actions to prevent brain perfusion changes in this complex hereditary hemoglobinopathy.Bilateral auditory cortex hypometabolism seems to represent an early hallmark of beta-thalassemia regardless of the clinical phenotype and the presence of hearing loss. The complex cognitive, audiological and metabolic scenario resulting from our study appears novel, extremely intriguing and needing to be further investigated with longitudinal studies. The latters will help to disentangle the pathogenic link between auditory cortex perfusion and cognitive and audiological impairment that characterize brain metabolism and function in beta-thalassemia.Additional file 1. Audiological, Cognitive functioning, MRI data evaluation. S. Fig.1. Relative auditory cortex perfusion values among patients with auditory evaluation. e-Table. Pearson’s correlations between cognitive scores derived by the WAIS and right and left auditory/visual cerebral area perfusion ratio in healthy controls and patients.


The present study investigated how visuospatial working memory (VSWM) is involved in the construction of a spatial situation model for spatial passages presented auditorily. A simple spatial tapping condition, a complex tapping condition as a target-tracking task, and a control condition, were used to analyze the role of VSWM. To understand how individuals who differ in verbal working memory (VWM) capacity (determined with a listening span test) process spatial text during dual-task performance, individual differences in VWM capacity were analyzed. In two experiments, the participants listened to a spatial text at the same time as performing a spatial concurrent task or no concurrent task. The results of the free recall test in Experiment 1 showed that there were no differences between the tapping conditions in the high VWM capacity group; the low VWM capacity group had a lower performance in both spatial tapping tasks compared to the control condition. The results of the map drawing test in Experiment 2 showed that complex spatial tapping impaired performance in comparison to simple spatial tapping and the control condition in the high VWM capacity group; in the low VWM capacity group, both spatial tapping tasks impaired recall performance. In addition, the participants with high VWM capacity demonstrated better performance. Overall, the results suggest that individuals with high VWM capacity have more resources to process verbal and spatial information than those with low VWM capacity, indicating that VWM capacity is related to the degree of the involvement of VSWM. In the processing of text comprehension, three levels of representation are constructed  developed by Daneman and Carpenter , is consConversely, VSWM capacity, as commonly measured by forward and backward spatial span tasks, also known as the Corsi block-tapping task Corsi , reporteTherefore, it is conceivable that the construction of the spatial situation model in listening comprehension, which is considered to consist of two temporally related processes, i.e., construction of the propositional text-base through language processing and construction of the situation model through the constructed propositional text-base  and a map drawing test for imagery memory (Experiment 2), and the spatial tapping tasks were predicted to have different interference effects in each test.In Experiment 1, we investigated whether the degree of involvement of VSWM depends on VWM capacity when the verbal memory of spatial descriptions is required. The materials used in this study consisted of a protagonist's movements and relevant landmarks, which leads the semantic representations of each sentence to be connected spatially to the next sentence temporally along the time axis. Therefore, to recall a complete verbal memory, the production of bound verbal and imagery representations in the episodic buffer is expected. The reconstruction of linguistic information requires the retrieval of the processed sentences in their input order, which needs additional processing resources to retain the verbal representations. On the basis of the typical interference effect of the tapping tasks observed in the processing of spatial texts found in the general population . There were four males and 16 females in both groups. All of the participants were paid 500 yen as a reward.A total of 40 undergraduates and graduates  from Hiroshima University participated in Experiment 1. Their mean age was 25.2 years (standard deviation [According to studies showing strong VSWM involvement in route perspective descriptions (Brunyé and Taylor SD = 6.07), and the mean length of the listening materials was 30 s (SD = 6.07).Each text contained five landmarks, three descriptions about the protagonist’s movements such as “turn left” and “walk straight” and three descriptions about relative location among the landmarks such as “on the left” and “on the right” (see excerpts in Table SD = 8.27), and the lack of a significant difference with one-way ANOVA confirmed a comparable completion level of the spatial situation model  = 0.02, p = 0.981). After the free recall task, a survey on the difficulty of the texts with a five-point evaluation method was also completed by the participants, with one point indicating “extremely easy” and five points indicating “extremely difficult.” The mean score of the three texts was 3.56 (SD = 0.51), and the lack of a significant difference with one-way ANOVA also confirmed a comparable level of difficulty  = 1.43, p = 0.869).A pilot study, with six Chinese L1 speakers who did not take part in Experiment 1, was conducted to assess the equivalency of difficulty and confirm the construction of a connected and integrated spatial mental model derived from the three texts. In the pilot study, the participants were instructed to write down the spatial descriptions after listening to the texts. The mean recall rate of the three texts was 70.27  as a between-participants factor, and the requirements of the three concurrent task conditions  as a within-participant factor.Each participant was tested individually for approximately 30 min. The primary task consisted of the participants listening to the texts only once, with the expectation that they should later write down the descriptions on a sheet of paper. The participants were told before the listening task to imagine they were walking in a city as the protagonist when memorizing the descriptions, based on studies showing that imagery instructions help participants to form a spatial situation model , and the other 20 participants with a score ≤ 3.50 were placed in the low VWM capacity group . Student’s t-test showed a significant difference between both groups  = 6.88, p < 0.001, r = 0.74). Both groups had the same mean scores in the Corsi block-tapping task .The initial score was 1.0 and the total score was set to 5.0. The mean score of the 40 participants was 3.88. Twenty participants with a score ≥ 4.00 were placed in the high VWM capacity group (t-test indicated that there was no difference in the degree of interference due to the complex tapping task between both groups during the listening task (t(38) < 0.1, p = 0.486, r = 0.01).In the simple task condition, the completion of the task by the participants was confirmed by the experimenter. In the complex task condition, the mean number of correct taps in each text was calculated. Comparisons between the means of the two VWM capacity groups by Student’s The score of the free recall test was based on the idea unit , the analyses were carried out on the scores given by the first judge (the experimenter).The scores were determined by two independent judges. According to the high consistency between the scores of both judges as determined with Pearson's correlation coefficient  = 17.11, p < 0.001, η2 = 0.31; in this study, partial η2 is reflected as η2), indicating a higher recall rate in the high VWM capacity group  compared to the low VWM capacity group . The main effect of concurrent task condition was also significant;  = 16.95, p < 0.001, η2 = 0.31). A comparison among pairs of means, using Bonferroni’s method, confirmed that the recall rate was higher in the control condition than in the simple and complex tapping conditions, which showed no difference between each other . A significant interaction effect was observed  = 9.10, p = 0.001, η2 = 0.33). Multiple comparisons with Bonferroni’s method showed no difference in the main effect of concurrent task conditions in the high VWM capacity group (p = 0.076), indicating that the recall rate was not significantly different among the three conditions . In addition, a significant difference was observed in the low VWM capacity group (p < 0.001), confirming that the recall rate was higher in the control condition  than in the simple  and complex  tapping conditions, which were not significantly different between each other.Figure The results showed that the high and low VWM capacity groups were influenced differently by the two tapping tasks, i.e., with no significant difference between the tasks in the high VWM capacity group, while significantly higher recall rates were observed in the control condition than in both tapping conditions in the low VWM capacity group, which are partially in accordance with our hypothesis. Combined with the observed main effect of VWM capacity, it was shown that verbal and imagery representations are constructed in parallel to a certain degree in both groups, but the degree of imagery representation formation, i.e., the degree of involvement of VSWM was affected by VWM capacity when verbal memory for spatial descriptions was required.To recall a listened description, it is assumed that a certain amount of processing resources are assigned to retain verbal representations. The lack of a significant difference among the concurrent tasks in the high VWM capacity group suggests that an increased ability to construct verbal representations allows more resources to be distributed to the processing of imagery representations in parallel. Conversely, in the low VWM capacity group, the decreased recall rate during the concurrent tapping tasks indicates that the required body movements deplete the processing resources available for the construction of imagery representations in VSWM. Since a certain amount of resources must be distributed preferentially to the processing and retention of sequential language information input, interference with the generation of imagery representations and further bindings in the episodic buffer occur. This results in the maintenance of more complete bound verbal and imagery representations in the control condition, but only verbal representations and incomplete imagery representations are maintained in the simple and complex tapping conditions.From the results of Experiment 1, it is inferred that in the same time frame, listeners with high VWM capacity construct more complete verbal representations, allowing more efficient binding with imagery representations than individuals with low VWM capacity when verbal memory is recalled.In Experiment 2, we investigated whether the degree of involvement of VSWM depends on VWM capacity when imagery memory for spatial descriptions is required. The recall of linguistic information as a map requires the retrieval of formed imagery representations transferred from verbal representations. Therefore, participants tend to retain imagery representations rather than verbal representations in the map drawing test. In other words, the bound representations updated along the time axis in the episodic buffer are ultimately retained and produced in the form of imagery representations. Since verbal representations do not need to be retained, which leads to the reduction of required VWM processing resources, it is plausible that more resources could be distributed to VSWM for the construction and retention of imagery representations. Therefore, the high and low VWM capacity groups should be able to construct imagery representations through VSWM in the control condition, and correspondingly, interference due to the spatial tapping tasks is likely to be observed in both groups in the tapping conditions, but the degree of interference is not expected to differ clearly between both groups. In addition, from the point of view that the formation of imagery representations is temporally based on linguistic processing during the construction of a spatial situation model, better performance should be observed in the high VWM group compared to the low VWM group.SD = 4.23). There were three males and 17 females in both groups. All of the participants were paid 500 yen as a reward.A total of 40 undergraduates and graduates  from Hiroshima University, who did not take part in Experiment 1, participated in Experiment 2. Their mean age was 25.5 years (SD = 15.50), and one-way ANOVA confirmed a comparable completion level of the spatial situation model  = 0.03, p = 0.975). After the map drawing task, a survey on the difficulty of the texts with a five-point evaluation method was conducted. The mean score of the three texts was 3.44 (SD = 0.51), and one-way ANOVA confirmed a comparable level of difficulty  = 0.14, p = 0.869).The texts and listening materials are the same as in Experiment 1. A pilot study, with six Chinese L1 speakers who did not take part in Experiment 2, is conducted as in Experiment 1. In the pilot study, the participants were told to draw a map after listening to the texts. The mean recall rate of the three texts was 76.04 , and the other 20 participants with a score ≤ 3.50 were placed in the low VWM capacity group . Student’s t-test showed a significant difference between both groups  = 6.02, p < 0.001, r = 0.70). The two groups had the same mean scores in the Corsi block-tapping task .The mean score was 3.84. Twenty participants with a score ≥ 4.00 were placed in the high VWM capacity group  < 0.1, p = 0.457, r = 0.01).As in Experiment 1, in the simple task condition, the participants were confirmed to have completed the task by the experimenter. In the complex tapping task condition, comparisons between the mean number of the correct tap between the two VWM capacity groups by Student’s Map drawing scoring evaluated landmark recall and relative landmark locations, which assesses the correspondence with landmark-to-landmark comparisons derived from the texts. If the landmark and relative locations were recalled correctly, two points were given, i.e., the precise locations of the landmarks were recalled and the correct relationship with other nearby landmarks was maintained, for example, when the participants put the cafe on the right side of the square with a fountain on the left side. One point was assigned when the participants partially recalled the landmark positions within the environment, for example, when the participants put the cafe on the left side of the square with a fountain on the right side. One point was also assigned when the relative locations were described correctly but without the correct names of the landmarks, for example, when the participants put a blank instead of “cafe” or with other landmarks on the left side of the square with a fountain on the right side.r = 0.90, p < 0.001), the analyses were carried out on the scores assigned by the first judge (the experimenter).The scores were determined by two independent judges. According to the high consistency between the scores of both judges as determined with Pearson's correlation coefficient  = 56.87, p < 0.001, η2 = 0.60), indicating an overall higher recall rate in the high VWM capacity group  than in the low VWM capacity group . The main effect of concurrent task condition was also significant  = 10.17, p < 0.001, η2 = 0.21). A comparison among pairs of means, using Bonferroni’s method, confirmed that the recall rate was higher in the control and simple tapping condition, which showed no difference between each other, than in the complex tapping condition . A significant interaction effect was observed  = 4.69, p = 0.012, η2 = 0.11). Multiple comparisons with Bonferroni’s method showed a significant difference in the high VWM capacity group with concurrent task conditions (p = 0.001), indicating that the recall rate was higher in the control  and simple tapping conditions , which showed no difference between each other, than in the complex tapping condition . In addition, a significant difference was observed in the low VWM capacity group (p = 0.002), confirming that the recall rate was higher in the control condition  than in the simple  and complex tapping conditions , which showed no difference between each other.Figure The results showed that the high and low VWM capacity groups were influenced differently by the two tapping tasks, i.e., with significantly higher recall rates in the control and simple tapping conditions than in complex tapping condition in the high VWM capacity group, while significantly higher recall rates were observed in the control condition than in the simple and complex tapping conditions in the low VWM capacity group. These results do not support our hypothesis, in which an interaction was not predicted. Combined with the observed main effect of VWM capacity and concurrent task condition, it was clarified that the degree of imagery representation formation differed depending on VWM capacity when imagery memory for spatial descriptions was required.When drawing a map of spatial descriptions, more processing resources are distributed to the manipulation of imagery representations due to the non-retention of verbal representations. Listeners with high VWM capacity can construct verbal representations more efficiently, i.e., they utilize less processing resources so that more resources can be distributed to the construction and retention of imagery representations. In the simple tapping condition, though the simple physical movements required some processing resources of VSWM, the capacity gap could be filled rapidly using processing resources from VWM to maintain the imagery representations translated from verbal information. Therefore, there was no significant difference between the simple tapping and control condition. However, in the complex tapping condition, which requires not only physical movements but also attentional control through visual perception, processing resources have to be redistributed to prioritize continuous information processing of the input language. Consequently, the updating of prior formed imagery representations is interfered with. This means that more complete imagery representations could be retained in the control and simple tapping conditions, but only incomplete imagery representations and former verbal ones could be retained in the complex tapping condition. Conversely, listeners with low VWM capacity showed a decreased recall rate in both tapping conditions. Although the necessary processing resources for retaining verbal representations were reduced to some degree, the resources that could be distributed to VSWM were limited by the efficiency of constructing verbal representations. Combined with the observed main effect of VWM capacity, it can be inferred that in the tapping conditions, the updating of verbal representations and subsequent generation of imagery are disrupted by the reduction of processing resources due to spatial tapping. This causes more complete imagery representations to be retained in the control condition, but only incomplete imagery representations and former verbal ones are retained in the simple and complex tapping conditions.From the results of Experiment 2, it is inferred that in the same time frame, the ability of the high VWM capacity group to construct verbal representations efficiently allows more processing resources to be distributed for the construction and maintenance of imagery representations, thereby resulting in more complete imagery representations of the spatial situation model than in those with low VWM capacity when imagery memory recall is required.Several studies have revealed the significant roles of VWM and VSWM in spatial text processing in the general population  and map drawing test (Experiment 2) were used as dependent variables. The results of Experiment 1 showed no differences in performance in the high VWM capacity group between the three tapping conditions; in the low VWM capacity group, a lower performance with both types of spatial tapping was observed in comparison to the control condition. The results of Experiment 2 showed that complex spatial tapping impaired performance in the high VWM capacity group in comparison to the simple spatial tapping and control conditions; in the low VWM capacity group, both spatial tapping tasks impaired performance. In addition, the participants with high VWM capacity demonstrated better performance than those in the low VWM capacity group. It could be concluded that the low VWM capacity group needs more verbal and visuospatial processing resources to construct a spatial situation model derived from verbal descriptions in comparison to the high VWM capacity group.The differences in the interference effects between the high and low VWM capacity groups were consistent with those reported by Meneghetti et al. , who sugWhen spatial descriptions are included in listening comprehension, in which linguistic information is input sequentially along a time axis, VWM and VSWM function in parallel to construct a spatial situation model consisting of verbal and imagery representations. Verbal representations are constructed dependent on VWM and LTM in the episodic buffer. While these former verbal representations are retained temporarily in this buffer, the corresponding imagery representations are constructed in a VSWM and LTM-dependent manner. The imagery representations are then transferred to the episodic buffer and bound to the next incoming verbal representations.The lack of an inference effect of both tapping tasks in the performance of the free recall test in the high VWM capacity group confirmed that increased VWM capacity enables the processing and retention of verbal information when verbal memory is required; rather, the impairment observed in the complex tapping task in the map drawing test confirms the need for VSWM function when imagery memory is required. In addition, the impairment caused by both tapping tasks in the free recall and map drawing tests in the low VWM capacity group reveals that the interference in the formation and binding of verbal and imagery representations is due to the need for more verbal resources. These observations demonstrate that the spatial situation model derived from spatial text is constructed through the same process, regardless of VWM capacity. However, the formation of VWM-dependent verbal representations and VSWM-dependent imagery representations and the binding of both in the episodic buffer, i.e., the abundance of modalities of the representations constituting the spatial situation model, varied between both groups with the requirements to the production of different representations. Accordingly, it can be concluded that the involvement of VSWM is constrained by the capacity of VWM in the construction of spatial situation models.The present study characterized the construction of the spatial situation model in listening comprehension and addressed the involvement of VWM and VSWM in this process. However, a degree of caution is required in the interpretation of our findings in view of the small number of participants and the generalizability of the results. In addition, the interaction of high and low VSWM capacity with visuospatial ability and VSWM processing in spatial texts is an interesting issue that needs to be examined in future.


Solute carriers (SLCs) are important transmembrane transporters with members organized into 65 families. They play crucial roles in transporting many important molecules, such as ions and some metabolites, across the membrane, maintaining cellular homeostasis. SLCs also play important roles in hearing. It has been found that mutations in some SLC members are associated with hearing loss. In this review, we summarize SLC family genes related with hearing dysfunction to reveal the vital roles of these transporters in auditory function. This summary could help us understand the auditory physiology and the mechanisms of hearing loss and further guide future studies of deafness gene identification. Most of the SLC proteins have 12 transmembrane domains , with more than 400 members . Among these genes, five of them belong to the SLC family. Besides, another 13 SLC members were reported to be associated with hearing loss or involved in auditory organ development. These SLC genes, although most of them act as transporters, have different expression patterns in auditory organs and distinct function in hearing.Hearing is one of the most important sensory functions, and hearing loss would cause great inconvenience to the daily life of deaf people. The causes of hearing loss vary, from congenital to acquired impairments, but the defects of genes account for the majority. More than 200 hearing loss genes, including the syndromic and the nonsyndromic hearing loss genes, were identified in the last decades  gene, encoding the Na–K–2Cl cotransporter-1 (NKCC1), is mainly expressed in the stria marginal cell of the cochlea  gene, which encodes electroneutral cation-coupled chloride cotransporters, is very important in some physiological processes, such as cell volume regulation, modulation of intraneuronal chloride concentration, transepithelial ion movement, and blood pressure regulation . There a hearing . The sol cochlea , which i cochlea . In mice cochlea , and knobehavior . Besidesn humans . Therefon humans , which pslc17a8 gene, is exclusively expressed in hair cells and localized to the basal end of hair cells , encoded by the ir cells , and theebrafish . Similar stimuli , and alsn humans . In addiing loss . A recening loss .SLC22A4, also named as OCTN1, a 551-amino acid–long protein, is a pH-dependent organic cation transporter  and inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α)  . It was  disease . Mice wion model . All of SLC22A4 was found to be associated with hereditary hearing loss in humans. In mammalian auditory organs, SLC22A4 is expressed in all of the inner ear epithelia at the early stage, including the hair cells and spiral ganglion neurons; however, the expression is restricted at the apical surface of stria vascularis (SV) endothelial cells in the mature cochlea  gene, encoding the protein pendrin, is the causal gene of Pendred syndrome, which is a recessively inherited disorder with hearing loss as the obvious feature  (SLC26A4 mutation are likely to be resistant to high blood pressure (The  feature . In addie (EVAS) . It was pressure .SLC26A4 gene was found to be expressed in the thyroid at high levels, and it can help the thyroid follicular cells transport iodine  gene, is the most well-studied solute carrier in hearing-related research. In the mammalian inner ear, it is specifically expressed in the basolateral membrane of outer hair cells  protein, encoded by the ir cells , and defing loss . In nonmy organs . The proy organs . Unlike y organs , which wy organs . Indeed,y organs . Nonmammy organs  and a wey organs . Prestiny organs  and intey organs . Moreovey organs . Recently organs , providiExcept for the SLC members discussed above, which have been identified as hearing loss genes, some other SLCs, waiting to be verified as deafness genes, were reported to function in hearing-related processes.SLC4 family members are bicarbonate transporters and play vital roles in acid–base homeostasis . Among tSLC4A2 gene, encoding HCO3−/Cl− anion exchangers, was reported to be expressed in mammalian inner ear cells, including hair cells and supporting cells , which is important in maintaining intracellular pH homeostasis by exchanging one intracellular H+ for one extracellular Na+ genes was found in a boy with nonsyndromic hearing loss; however, no hearing impairment occurred in his brother, father, and grandfather who have the same translocation , without hearing deficits  and SLC16A10 , which share similarities with each other, prefer to transport the iodothyronines (T4 and T3)  . Distinc and T3) . As know and T3) . Unsurpr tissues , and the tissues .SLC19A1 transports folates but not thiamine, and the other two transport thiamine but not folates , were reported to be associated with thiamine-responsive megaloblastic anemia (TRMA) , of which  folates . Mutatioa (TRMA) , which iSLC26A4 and SLC26A5, discussed above, another gene, SLC26A2, encoding for a sulfate/chloride transporter, may be associated with hearing loss. In zebrafish, the slc26a2 gene was proven to be critical for otic development and hair cell survival, and loss of function of slc26a2 led to defective auditory organ development and impaired hearing  are included in this family . In addi hearing .The SLC44 family, with five members (SLC44A1–5), is a choline-like transporter family .SLC44A2 and blocking its transporter function , is a transmembrane glycoprotein. It was identified as the target of the Kresge Hearing Research Institute-3 (KHRI-3) antibody, which can lead to autoimmune hearing loss by binding to function . Moreoveing loss .SLC44A4 encodes the choline transport protein CTL4. Mutations in this gene were found in a Chinese family with postlingual nonsyndromic mid-frequency sensorineural hearing loss, and knockdown of the slc44a4 gene in zebrafish led to significant defects in the otic vesicle and lateral line neuromast development, accompanied by defective hearing , was found in the BVVL syndrome  syndrome is a rare neurodegenerative disease characterized by sensorineural hearing loss and a variety of cranial nerve palsies . Mutatiosyndrome , and a hsyndrome , indicatAccording to the World Health Organization (WHO), one in four, about 2.5 billion, people worldwide will be living with some degree of hearing loss by 2050, and nearly 60% of hearing loss is caused by genetic factors. Therefore, hereditary hearing loss is a serious problem that needs attention. In the last decades, more than 200 human hearing loss genes have been identified by scientists all over the world.The SLC family genes, with more than 400 members, encode different types of transporters that function in solute transporting. Deficiency of some of these genes would cause human diseases. Until now, 18 members, as listed in this review, in the SLC family were reported to be associated with the occurrence of hereditary deafness, including five known hearing loss genes, which indicated the crucial roles of solute carriers in hearing function. These SLCs have different expression patterns in the inner ear and have distinct function, and each of them is essential for normal hearing. Mutations in any of these genes would result in inner ear cell dysfunction or even cell death, leading to hearing impairment or hearing loss.In this review, we systematically summarized all of the SLCs involved in hearing, from their expression to function to the underlying mechanisms. This study would be helpful in understanding the roles of solute transporting for normal hearing, and it can also provide ideas for deafness gene identification.


Beauveria bassiana, as a well-studied entomopathogenic fungus, has a great potential for the biological control of insect pests. Lipid metabolism has been linked to the life cycle of B. bassiana; however, the underlying mechanisms remain unknown. In this study, a homolog of yeast steryl acetyl hydrolase 1 (Say1) was functionally characterized. The loss of B. bassianaSAY1 (BbSAY1) impaired the lipid homeostasis in conidia, with a significant reduction in oleic acid content. The ΔBbsay1 mutant strain displayed anelevated accumulation of lipid bodies and aweakened membrane permeability. As for phenotypic aspects, gene loss resulted in significant defects in germination, conidiation, and virulence. Our findings highlight that Say1, involved in lipid homeostasis, contributes to the cytomembrane integrity, development, and virulence in B. bassiana. Beauveria bassiana is one of the most prevalent insect pathogens in the eco-system and has great potential for the biological control of pests [of pests ,2. Conidof pests ,4. Afterof pests .B. bassiana, conidia form on the ‘zig-zag’-shaped conidiophores and accumulate a plethora of fatty acids (FAs) and lipids [B. bassiana, the OLE pathway also contributes to conidial storage of OA and is transcriptionally regulated by HapX (BbHapX). In addition, BbHapX has a greater influence on lipid homeostasis than BbOle1 [B. bassiana.In d lipids ,7. The hd lipids  and can d lipids . Oleic ad lipids . HapX red lipids . In B. bn BbOle1 . We suppB. bassiana by constructing the gene disruption and complementation mutant strains. Our data revealed that BbSAY1 contributes to conidial lipid reserves, formation, and virulence.In this study, we functionally characterized Say1 in B. bassiana ARSEF2860 was cultured on Sabouraud dextrose agar  for routine maintenance. Escherichia coli DH5α  for plasmid proliferation was cultured in Luria-Bertani (LB) medium with appropriate antibiotics. A yeast extract broth  was used to culture Agrobacterium tumefaciens AGL-1, which was used in fungal transformation. Czapek-Dox agar (CZA)  was used as a chemical-defined medium.The wild-type strain of S. cerevisiae Say1 (GenBank No.: DAA08353) was used as a query to search the potential ortholog in the B. bassiana genome [http://smart.embl-heidelberg.de/) (last accessed on: 9 September 2020). The ortholog of yeast Say1 in B. bassiana was determined with the method of reciprocal best alignment between two species [B. bassiana, their homologs in other fungi were retrieved via BLAST analyses and proofed via domain analysis. Tested fungal species included Aspergillus nidulans FGSC A4, A. niger CBS 513.88, A. fumigatus Af293, Candida albicans SC5314, Cordyceps militaris CM01, Magnaporthe grisea 70-15, MetarhiziumacridumCQMa 102, M. robertsii ARSEF 23, S. cerevisiae S288C, and Yarrowialipolytica CLIB122.The protein sequence of a genome . After m species . After sTM Plus Reagent , and cDNA was reverse transcribed using the PrimeScript® RT reagent Kit (TaKaRa). The resultant cDNA was used as templates to perform the qRT-PCR reaction on a Mastercycler® EP Realplex  cycler. All primers are listed in −ΔΔCt method [The transcriptional analyses of the Say1-domain protein genes were performed as the methods documented . Conidiat method .GFP) by ligating into the NcoI/EcoRI sites of pBMGB. The resulting vector was transformed into the wild-type strain. The transformant was screened on CZA plates with 200 µg/mL glufosinate and cultured on an SDAY plate at 25 °C. Fungal cells were sampled, and the fluorescent signals were observed under a laser scanning confocal microscope .Localization of BbSay1 was performed as described previously . PrimersBbSAY1 open reading frame (ORF) were amplified by PCR with the paired primers P1/P2 and P3/P4, respectively, using genomic DNA as the template. The amplified PCR fragments were digested with EcoRⅠ/BamHⅠ and XbaⅠ/HpaⅠ, respectively, and then successively cloned into the EcoRI/BamHI and XbaⅠ/HpaⅠ sites of p0380-bar [BbSAY1 ORF plus 1.85 kb of upstream and 0.34 kb of downstream sequences was amplified with primers P7/P8, and the resultant fragment was recombined into the vector p0380-sur-gateway with a sulfonylurea resistance cassette [B. bassiana strain using an Agrobacterium-mediated transformation method. Putative disruption mutants were screened on CZA supplemented with phosphinothricin (200 μg/mL), and the complemented strains were screened on CZA plates with 10 mg/mL chlorimuron ethyl.The methods for gene disruption and complementation were the same as those described previously . All pri0380-bar . The rescassette , generatSacⅠ/NcoⅠ were resolved on a 0.7% agarose gel and then electronically transferred to a Biodyne B nylon membrane . Target fragments were hybridized with the probes prepared from the templates amplified with the primer pair P9/P10 and visualized with the DIG-High Prime DNA Labeling and Detection Starter Kit II .Southern blotting analyses were used to further confirm the transformants. Fungal genomic DNA (30 μg) digested by Bbsay1, and complementation mutant strains with three parallel experiments [The indicated strain was cultured on SDAY plates at 25 °C for 7 days, and the resultant conidia were suspended in a 0.02% Tween-80 solution. All phenotypes were compared among the wild-type, Δeriments .v/v/v) and placed at −20 °C for 2 h. Then, 3 mLCM mixture  was added and the conidial suspension stood for 1 h. The bottom layer was carefully collected and dried. FAs were methylated and analyzed on a Focus series gas chromatograph , using a standard mixture of fatty acid methyl esters (47885U)  as the internal references.Conidia (100 mg) were suspended in 10 mLCMW mixture  was inoculated on the plates. After 7 d of incubation at 25 °C, the colony diameter was examined. A feeding assay was conducted by adding oleic acid (0.3%) in medium. Oxidative stress was caused by including manedione (0.02 mM) into CZA plates. Conidial suspension was point inoculated on the plates and cultured at 25 °C.The colony diameter was determined at 7 d. Relative growth inhibition was calculated using the diameter on the CZA plate as a control.Fungal radial growth was determined on CZA and SDAY plates. Conidial suspension  was uniformly smeared on SDAY plates and incubated at 25 °C for 9 d. From the sixth day after incubation, a mycelial disc (5 mm in diameter) was sampled, and conidia on the disc were washed into a 0.02% Tween-80 solution by vigorous vortexing. The conidial concentration of the suspension was quantified and used to calculate the conidial yield as the number of conidia per square centimeter. In the feeding assay, 0.3% oleic acid was added to the SDAY plates, and conidial production was also quantified as described above. Additionally, a transformant for the sub-cellular localization of BbSay1 was used to evaluate the effect of the over-expression of BbSAY1 on conidiation.An aliquot of conidial suspension  was sprayed on the locust hindwings. After an incubation of 24 h at 25 °C, the conidial morphology was observed under a microscope.To simulate conidial germination under an oligotrophic condition, the conidial suspension was inoculated on a water agar plate (WA) (1.2% agarose). After an incubation of 24 h at 25 °C, the germination percentage was calculated. For each sample, at least 100 conidia were counted. A chemical feeding test was performed by adding oleic acid (0.3%) into the WA plates. To simulate conidial germination on the host cuticle, a conidial suspension  were used in this study [7 conidia/mL) for 15 s. All infected insects were reared at 25 °C, and the mortality was recorded daily. The median lethal time (LT50) was calculated by a Probit analysis for three replicates of each bioassay. In the feeding test, oleic acid (0.3%) was added into the conidial suspension.To determine fungal virulence, the larvae of is study . In the Bbsay1 mutant strains.The total lipid sample was prepared with the methods used in FA extraction as described previously . Lipids To view the variation of total lipids in mycelia, a conidial suspension was grown on the SDAY for 3 d at 25 °C. The resulting mycelia were stained with observed hyphae with the lipid-specific dye BODIPY493/503 .Nucleus staining with SYTOX Green was applied as previously described . The conAll measurements from biochemical and phenotypic assays were subjected to a one-way ANOVA. The significant difference was determined by a Tukey’s honest significance test (Tukey’s-HSD).S. cerevisiae (GenBank No.: DAA08353)  and was designated as BbSay1. The open rearing frame of BbSAY1 was 1453 bp in length with 3 introns, encoding a 386-amino acid protein. The domain analysis indicated that BbSay1 contained a domain of Say1_Mug180 (PF10340.9), and the domain annotation revealed that there were 10 Say1-domain-containing proteins in the B. bassiana genome. Their orthologs in other fungi were revealed via sequence alignment and domain annotation analyses  and 2.23 ± 0.05 cm (SDAY)) (mean ± standard deviation). Feeding oleic acid could increase the colony sizes on both the CZA and SDAY plates. This indicated that exogenous oleic acid could restore vegetative growth under an aerial condition. The colony morphologies on the CZA and SDAY plates areshown in As shown in Bbsay1 mutant strain was 23.3 ± 2.1%; however, the wide-type and complementation strains displayed a germination percentage of approximately 35%. After adding oleic acid, there was no significant difference in the germination percentage among the wild-type, ΔBbsay1, and complementation strains. On the hindwing of a locust, the conidia of the wild-type germinated well and developed into a germling; however, the ΔBbsay1 mutant displayed aretarded germination (F). Under oxidative stress by menadione, the gene disruption mutant displayed a significant defect in vegetative growth (G). The conidial yield was evaluated on SDAY plates , with a slight delay, when compared with that of the wild-type (5.43 ± 0.11 d)  C. When a strains B,D.BbSAY1 resulted in an enhanced accumulation of diacylglycerols (DG) and a decrease in the content of triacylglycerols (TG). Four kinds of phospholipids were detected in the B. bassiana conidia, including lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), and phosphatidylethanolamine (PE), in their cationic or anionic forms. Gene disruption resulted in a significant decrease in the content of phospholipids   B. The log., LPC) C. Intere strains D.B. bassiana.FA/lipid metabolism plays an important role in the interaction between entomopathogenic fungi and insect hosts . This stS. cerevisiae, Say1 catalyzes the reaction of sterol deacetylation, which is required for sterol homeostasis [B. bassiana, BbSay1 contributes to the homeostasis of lipid metabolism. Its loss leads to a comprehensive fluctuation in the cellular lipidome, although more investigations are needed to reveal the direct effects of BbSay1 in B. bassiana. This finding suggests the conservative role of Say1 in lipid metabolism. It is well known that triacylglycerol is synthesized by the conjugation of acyl coenzyme A (acyl-CoA) to diacylglycerol, which is catalyzed by acyl-CoA:diacylglycerol acyltransferase [BbSAY1, B. bassiana accumulated a high level of diacylglycerol and a low level of triacylglycerol. Additionally, BbSay1 contributes to maintaining the morphologies of lipid bodies and the conidial reserve of oleic acid. Lipid droplets are organelles that store neutral lipids  and maintain the homeostasis of stored lipids and free fatty acids [Lipid homeostasis is maintained by a series of biochemical reactions of esterification and transesterification, as well as the interaction among different catalytic pathways . In S. ceostasis ,23. In Bnsferase . After dty acids . These rB. bassiana, including the biosynthesis of unsaturated FA  [The conidial capacity to germinate and penetrate through the host cuticle is determinant for successful infection by a fungal entomopathogen . Particu BbOle1) ), lipid  BbOle1) , and thi BbOle1) . In addi BbOle1) . These fA. nidulans, two Δ9-stearic acid desaturases catalyze the biosynthesis of oleic acid, which is required for fungal development [Blumeriagraminis), the oxidation of unsaturated fatty acids is involved in the conidiation process [Magnaportheoryzea, the elevated accumulation of lipid bodies leads to severe conidiation defects [B. bassiana conidiation. In addition, BbSNF1, an AMPK protein kinase, mediates the amino-acid homeostasis during conidiation in B. bassiana [Conidia, generated through asexual development, are essential for entomopathogenic fungi to survive and disperse in the environment ,30.BbSayelopment . In fila process . In Magn defects . ConsideB. bassiana contains 10 Say1-domain-containing proteins. BbSay1, the ortholog of yeast Say1, maintains lipid homeostasis, which contributes to the cytomembrane integrity, development, and virulence in B. bassiana. In future, more investigations are needed to functionally identify other Say1 proteins, which should provide novel insights into the lipid metabolisms involved in the differentiation and virulence of entomopathogenic fungi.In conclusion,