Multidisciplinary rehabilitation including occupational therapy after COVID-19 is recommended. However, evidence on how COVID-19 affects the ability to perform activities of daily living (ADL) is sparse.  The aim of this study was to explore the ability to perform ADL and cognitive status in patients with COVID-19 at time of discharge and three months postdischarge.  This prospective multiple case study included adults with COVID-19, who at time of discharge had decreased ADL performance compared to habitual functional level. Data collection included Assessment of Motor and Process Skills (AMPS) and Montreal Cognitive Assessment (MoCA) at discharge and three-month follow-up. Exploratory analysis was used to identify similarities and trends within and across cases.  Eleven patients were included. 75% had a significant increase in motor ability measures, and 27% had a significant increase in process ability measures at follow-up. 67% of follow-up cases showed mild cognitive impairment, where executive functioning and memory were most predominant. Significance. Occupational therapists can apply performance-based assessments to identify the need for rehabilitation of ADL in patients with COVID-19 during and posthospitalization. The ability to perform ADL was affected at discharge and at three-month follow-up. Furthermore, mild cognitive impairment was present at both hospital discharge and follow-up in most cases.  The coronavirus disease 2019 (COVID-19) global pandemic has resulted in over 200 million confirmed cases and over four million reported deaths worldwide . CognitiRecently, two single-case reports describing OT evaluation and treatment approaches for a client with COVID-19 have emerged , 14. BotWe did not have guidelines for OT practice when patients with COVID-19 were initially referred to OT at Copenhagen University Hospital, Rigshospitalet (RH), Denmark. Thus emerged, the need for gaining knowledge and evidence of patients' abilities to participate in ADL, their cognitive status and rehabilitation needs.The aim of this study was to explore similarities and trends in ability to perform ADL and cognitive status at discharge and three months postdischarge in patients diagnosed with COVID-19, who at discharge had diminished ADL performance compared to habitual functional level. Furthermore, we aimed to explore rehabilitation needs and the implications for OT practice.A prospective multiple case study design was chosen . This derehabilitation plan as referral for further community rehabilitation [All cases were enrolled from RH, a highly specialized hospital where medical care is provided in an up-front tax paid public healthcare system. Patients can be referred to the OT department from all other departments when the need for rehabilitation is identified. In-patient rehabilitation is initially provided by occupational therapists during hospital admission. Upon discharge, a physician, in collaboration with a multidisciplinary team, is responsible for assessment of rehabilitation needs of patients and where relevant complete a litation . During The organization of departments treating patients with COVID-19 varied across the data collection period. During periods with increased COVID-19 hospitalization (spring 2020 and winter 2020-2021), cohort isolation wards specific for COVID-19 patients were established. During periods with less COVID-19 admissions, the patients were admitted to general wards, including the wards of the Department of Infectious Diseases. Cases were recruited from the cohort isolation wards as well as wards on the Department of Infectious Diseases between May 2020 and January 2021.Data collection included Assessment of Motor and Process Skills (AMPS) , 24 as wThe first author called patients four weeks prior to the three-month follow-up to confirm participation and plan activities for AMPS. After case 1 declined follow-up due to low functional level and lengthy transportation to the hospital, a collaboration with occupational therapists in the community was initiated. If participants declined to participate in the follow-up due to lengthy transportation, the first author, with permission of the patient, contacted the multidisciplinary community rehabilitation units to ask the local occupational therapists if they could perform follow-up testing. The three community occupational therapists who were contacted accepted.Patients were included if they were diagnosed with COVID-19, admitted to RH and at discharge had lower ability to perform ADL compared with abilities prehospitalization. For example, if the patients needed assistance in activities like toileting, bathing, or dressing, which they would usually perform independently at home, patients were considered eligible for inclusion if they were adults ≥ 18 years and able to understand and follow instructions in Danish during testing. Patients were excluded if they were in isolation and sufficiently challenging ADL tasks could not be performed on the ward due to the COVID-19 restrictions, e.g., if the task required observation of kitchen activities.Information of changes in ADL ability was primarily obtained through the OT intervention via interview and/or observation of the patient. Three cases were identified during multidisciplinary conferences where clinical handover by nurses and physiotherapists reviled ability discrepancies in current and prior hospitalization ADL performance.effort, efficiency, safety, and independence [AMPS was used to investigate and describe ability to perform ADL , 24, andpendence . This obsafety and independence. The motor ability measure indicates how much effort  the patient experiences during performance of ADL, and the process ability measure how efficient (inefficiency and/or disorganization) the patient is during performance [questionable, mild, moderate, or marked clumsiness and/or increased physical effort or fatigue or inefficiency and/or disorganization.In collaboration, the patient and occupational therapist choose two ADL tasks, that the patient wants or needs to perform and find challenging. The patient performs the two tasks after standardized instruction by the occupational therapist. The quality of 16 ADL motor skills  and 20 ADL process skills  are scored by a calibrated assessor on a 4-point ordinal scale. The scores of the 16 motor and 20 process skills are analyzed using the Rasch-based computer AMPS-software , Center for Innovative OT Solutions, Fort Collins, Colorado, USA), which converts raw skill scores into logistically transformed probability units (logits), that are presented in two graphic rapports of ADL motor and ADL process skills. The logits are adjusted for rater severity and difficulty of the ADL tasks observed. Both rapports reflect formance , 24, 26.efficient , safe, and independent ADL task performance [A motor skill logit of 2.0 is a criterion referenced cut-off, where a person is likely to begin showing slight clumsiness or physical effort. There is a risk zone of 0.3 logits, and logits ≥ 2.3 reflect skilled (no clumsiness or effort), safe, and independent ADL task performance. The cut-off for process skills is 1.0 logit, where a person is likely to begin showing inefficiency, and logits ≥ 1.3 reflect formance .MoCA is a cognitive screening tool. A one-page, 30-point, pen, and paper assessment administered by clinicians in approximately 10 minutes . The scrInspired by Yin , we alloCase descriptions, as well as data on non-participators, will be presented in text and tabular forms presenting median, as well as range for continuous data, frequencies, and percentages for all categorical and dichotomized data.The data presentation for each individual case includes the performed AMPS tasks and difficulty, AMPS results in logits, AMPS age and sex adjusted percentile rank, and the MoCA results.When interpreting AMPS at follow-up, a higher logit for the second observation indicates improved ADL ability. The ADL motor ability measures must differ by at least 0.5 and/or the ADL process ability measures by at least 0.4 to indicate that the assessed person's ADL ability has a high likelihood to have changed significantly between two AMPS observations . It is nTrends across MoCA domain scores will be presented, and trends between AMPS process logits and MoCA total score will be analyzed. Individual scores, logits, and changes over time between discharge and three-month follow-up will be presented visually using a spaghetti plot for both AMPS and MoCA.The study complied with ethical principles for medical research as described in the Helsinki Declaration . EligiblFourteen patients were assessed for eligibility, and eleven patients were included in this study. A flow diagram in Six of the seven cases referred with dysphagia had been admitted to the ICU. The OT interventions ranged from reassessment of swallowing, eating and drinking, and assessment of ADL which involved the facilitation to participate in personal ADL such as washing the face and lifting a glass to the mouth, as well as more complex activities of showering and dressing. Full case descriptions are presented in At discharge assessment, nine participants completed AMPS and MoCA the same day. Due to fatigue, two patients asked to be tested on separate days with one and four days apart (cases 4 and 8). Due to general weakness and geographical distance, it was not possible to complete follow-up assessments at the hospital in four cases . In three of these cases, follow-up assessments were conducted by an occupational therapist in the local community .Follow-up AMPS were performed on eight participants (73%): follow-up MoCA on six participants (55%). The AMPS three-month follow-up assessments were conducted with a median of +1.5 day (range -15 to +25 days), to the exact day, where three months had passed since discharge testing. One participant declined MoCA due to rehospitalization (case 8), however, participated in an AMPS during standard OT intervention during rehospitalization at the scheduled time for follow-up. Five of the MoCA follow-up assessments were performed on the same day as AMPS, and one was performed 60 days later (case 3). No trends were identified in loss to follow-up and lower or higher test results at discharge.Analysis for trends in the data from AMPS showed that at discharge, all cases had a motor ability measure below the cut-off of the criterion referenced 2.0 logits, ranging from -1.3 logits (marked) to 1.9 logits (questionable to mild), indicating clumsiness and/or increased physical effort or fatigue during ADL task performance, see At follow-up, all cases had improved motor ability measures with a median change of 1.1 logits (range 0.1 to 1.5). Six cases (75%) showed significant improvements, with increased logits by at least 0.5 .Contrary to the trends of motor ability measures, trends and patterns across process ability measures showed greater variation between patients at both discharge and follow-up see . At discAt follow-up, seven cases (88%) had improved process ability measures, the median change being 0.25 logits (range -0.4 to 0.9), but only three (38%) showing significant changes with process ability measures improving by at least 0.4 logit . Cases 4, 7, and 8 displayed efficient process ability measures during ADL task performance at follow-up.endures, was reported as problematic, and nine cases had especially difficulty in reaches. Flows was described in six cases and grips in five. No patterns nor similarities were found across cases in the process performance skills (raw scores) highlighted as predominant.At discharge testing, occupational therapists reported similar predominant problematic motor performance skills (raw scores) across cases in the electronic patient journals. In 10 out of 11 cases, the motor performance skill, motor and process ability measures and another group of three patients (27%) showing significant change in motor ability measures only.When searching for patterns across significant changes in AMPS results between discharge and follow-up, two groups were identified: one group of three patients (27%) with significant changes in both motor and process ability measures. When comparing all cases, exploratory analysis showed similarities across these three cases. All three cases had been referred to OT during admission and were all discharged to inpatient community rehabilitation units. These were the only participants to complete follow-up, whom at discharge testing had motor ability measures indicating marked or moderate-marked clumsiness and/or increased physical effort or fatigue during ADL task performance.The AMPS result of cases 3, 4, and 7 indicated that their ability to perform ADL had a high likelihood to have changed significantly in both mild to moderate clumsiness and/or increased physical effort or fatigue during ADL task performance at follow-up. In contrast, two of the patients showed process ability measures of efficient  ADL task performance at follow-up (cases 4 and 7 in Two of the three cases (cases 3 and 7) needed assistance for personal ADL prior to admission and had several coexisting illnesses. These two patients did not have the energy levels to attend the follow-up assessment at the hospital, and these were conducted in a community rehabilitation facility. As illustrated in motor ability measures at follow-up. Their process ability measures did not show significant change. When looking for trends, patterns, and similarities across these cases, all were independent prior to admission; two were employed (case 2 and 5). All three cases had process ability measures at cut-off or in the risk zone, where questionable inefficiency or disorganization during ADL task performance could be observed at discharge as well as follow-up. At follow-up, AMPS age and sex adjusted percentile rank indicated that 90.5% of healthy people of the same age likely had a higher ADL process ability measure. At follow-up, case 5 had started work part time, and case 2 was able to carry out personal ADL but not complex ADL like heavy household or work. Case 6 was retired, and she felt close to habitual functional level, although did experience fatigue. Cases 5 and 6 were not referred to OT during admission.Three cases  had significant changes in ADL Cases 5 and 6 were the only cases, of all, showing skilled (no clumsiness or effort), safe, and independent motor abilities during ADL task performance at follow-up with motor logits above 2.3.As presented in orientation in time and place, with 73% of cases achieving the highest score at discharge and 83% at follow-up. Furthermore, memory and executive functioning appeared to be the dominant domains most difficult across cases. At discharge, the median total MoCA score was 24 (range 8-30). Eight cases (73%) had a total MoCA score indicating mild cognitive impairment with a cut-off of <26. The median of the six follow-up MoCA scores  was 24.5 (range 21-30); the MoCA score of four follow-up cases (67%) indicated mild cognitive impairment with <26. Trends across cases were an increase in MoCA score between discharge and follow-up: a tendency to high scores in Across cases, it was found that for the patients independent in ADL prior to admission , 50% had a total MoCA score indicating normal cognitive functioning at discharge . The three other cases were below cut-off <26 . Cases 2, 5, and 6 completed follow-up MoCA: two having MoCA total scores ≥ 26; case 5 remained <26 indicating mild cognitive impairment.Of the cases, whom at habitual functional level needed assistance for personal or instrumental ADL , all had a MoCA score below 26. We were not able to find similarities in MoCA scores at discharge among the groups of cases needing help for personal ADL  and cases needing help for instrumental ADL only (cases 9 and 11).Across all cases, no pattern could be found between the MoCA results and AMPS results. For example, the three cases showing significant changes in motor ability measures  had very similar process ability measures, however, very different MoCA scores. Likewise, case 7 showed a rise in efficient process ability measures at three-month follow-up, but at the same time having a fall in MoCA from 24/30 to 21/30 at follow-up. Unfortunately, the MoCA of case 4 is missing at follow-up and therefore trends between MoCA and an efficient process ability measure that are not possible to comment on.All cases received a multidisciplinary rehabilitation plan at discharge from hospital for further rehabilitation in the community. Occupational therapists participated in the completion of 10 rehabilitation plans. The rehabilitation plans all described further need for facilitation of independence in habitual ADL level, including return to work and complex instrumental daily activities, where it was relevant. They also focused on fatigue and endurance during activity performance, some specifically naming the use of principles of energy conservations. Examples included education in adapting activity performance and/or the environment to be able to carry out ADL. The majority of rehabilitation plans described a need for an increase in global muscle strength. Only few rehabilitation plans mentioned the need for further swallowing assessments and/or treatment. (cases 2 and 9) .Eleven patients with COVID-19 and decreased ADL performance at hospital discharge were included in this study. Three-month follow-up included AMPS data from eight cases and MoCA data from six cases. The cases represented a diverse sample across age, sex, preexisting illness, and habitual functional level; some needing assistance in ADL prehospitalization, and some working full time and/or being independent in all ADL. The course of admission was heterogenic in terms of length of stay, ICU treatment, and referrals for OT. Across cases, very similar P-ADL tasks were performed at discharge, no matter the prior level of functional ability. At follow-up, the difficulty of ADL tasks performed varied, where cases being independent prehospitalization performed more challenging ADL tasks.significant increase in motor ability measures. Furthermore, most cases still showed signs of clumsiness and/or increased physical effort or fatigue at follow-up. In contrast, the cases showed larger variation in process ability measures. Only 27% had a significant increase in process ability measures at follow-up, and no patterns were identified across process skills. Three of the cases habitually independent in ADL (50%) did not have significant changes in process ability measures at three-month follow-up. This revealed questionable inefficiency or disorganization during ADL task performance. We could not find any patterns across cases between MoCA and AMPS scores.Ability to perform ADL was affected at discharge as well as three-month postdischarge across all cases. This included the following: low endurance and a trend of low motor ability measures; all cases being below the criterion referenced cut-off at discharge and all cases showing increased motor skills during ADL task performance with 75% of cases having a Mild cognitive impairment was detected in the majority of cases at discharge (73%). Executive functioning and memory appeared the most difficult across cases and orientation the least difficult. Attention and memory scores showed a diverse heterogenic pattern at follow-up, e.g., some scores being lower at follow-up, than discharge. Four out of the six follow-up cases had mild cognitive impairment, including one case, who had returned to work part time.Interestingly, the majority of patients with COVID-19 referred to OT were referred for assessment of dysphagia, but only two still needed rehabilitation for dysphagia after hospital discharge. Rehabilitation needs postdischarge focused on interventions regarding endurance, low mental/physical energy, and performing ADL, more specifically focusing on motor skills and for some, also on process and cognitive skills.with lower ability to perform ADL at discharge compared to prehospitalization, whereas Halpin et al. [all hospital admitted patients diagnosed with COVID-19.Currently, no other research using AMPS for assessing ADL performance in patients with COVID-19 has been identified. In a study from the United Kingdom in 2020 [Interestingly, two of the three cases in our study showing significant improvement in ADL performance at follow-up in both motor and process skills were patients needing assistance for personal ADL prior to admission and having coexisting illnesses. Patients with preexisting disabling conditions are more prone to experience physical or psychological effects after COVID-19 . These tStudies have shown that the cut-off score in MoCA of 26 to determine cognitive impairment may be inadequate , and somIn people diagnosed with stroke, cancer, or cardiac disease, problems of mild cognitive functioning and executive functioning are often not detected on the ward during hospital admission, however, can be detected through ADL testing, including AMPS , 36. TypThe present multiple case study adds to a growing body of evidence on the role of occupational therapists in a hospital setting as part of the initial rehabilitation of patients with COVID-19, as well as postdischarge. Our data describe extensive difficulties in carrying out ADL for this patient population. Occupational therapists can apply performance-based assessments to identify rehabilitation needs of ADL, including possible cognitive rehabilitation needs that may present during ADL performance . This maOur findings underline the importance for a comprehensive assessment of COVID-19 patients to identify OT rehabilitation needs. The same methods of testing may not necessarily be relevant to all COVID-19 patients but should include assessment of cognitive functioning and ability to perform ADL.It is relevant to assess ADL ability of patients with COVID-19, using AMPS in a larger population, preferably in a collaboration with communities for long-term follow-up. Further research could also benefit from investigating which OT assessments best apply to COVID-19 patients during hospitalization and postdischarge, as well as the appropriate timing of testing. Future studies could investigate interventions regarding fatigue and energy management in rehabilitation and the possible connection between fatigue, physical exercise, and cognitive function for this population. The lack of clear patterns between MoCA and AMPS results could indicate a need for further research in adequate assessment for identifying cognitive impairment in COVID-19 patients, including the optimal cut-off in MoCA.The design of the study is a case series, which inherently has some strength and limitations , 20. CasCommon to healthcare systems worldwide in 2020, we were learning how to assess and treat patients admitted with COVID-19, on the go. And as such, this study was conducted due to the need for more clinical experience and knowledge about this patient population and how OT can best be provided. There are, however, some limitations due to the nature of planning a study with an unknown long-term trajectory. These limitations include a nonconsecutive and convenience sample. Due to the rapid changes in environment and organization of the new COVID-19 wards, patients were not systematically included or assessed for eligibility. Patients with other primary diagnosis were also included, however, in future research excluding patients not admitted with COVID-19 as their primary diagnoses will make comparisons more adequate. Several patients were lost to follow-up, which reduced the number of participants significantly. It was a limitation that follow-up MoCA was also being used by testers in a separate nonrelated study with a parallel time frame of this current study. A lack of coordination between projects resulted in missing one case at three-month follow-up, and another case had a 60-day gap between AMPS and MoCA.In addition, we did not know the AMPS ability measures of the cases before admission to hospital, and therefore, we were unable to report exactly how close the patients were to their actual habitual functional AMPS score at follow-up. It was a strength using a comprehensive objective measure of ADL performance in AMPS. This, however, could have been supported by subjective assessments such as the Canadian Occupational Performance Measure (COPM), gaining knowledge on self-perception and satisfaction of occupational performance. As follow-up was performed in different settings with different journal practice, predominant problematic motor and process performance skills were not consequently described in patient journals by the occupational therapist at follow-up.In conclusion, in a series of eleven hospitalized patients diagnosed with COVID-19 with heterogenic prehospital ADL abilities and various admission trajectories including admission to ICU, the ability to perform ADL was affected at discharge and for the majority at three-month follow-up. Most cases also showed mild cognitive impairment. The comprehensive description of cases and the investigation of similarities and trends adds to the growing academic research on patients with COVID-19 and on how to plan OT intervention during hospitalization and, furthermore, in community rehabilitation units. It also highlights the need for further evidence of the impact of COVID-19 on the ability to perform ADL as well as using occupation-based assessment and treatment in COVID-19 rehabilitation.


Recent data has shown a consistent but modest association between hearing impairment and poor mobility; both are strongly associated with cognition. Cognitive function may moderate the relationship between hearing and mobility. We analyzed 601 cognitively normal older participants from the Baltimore Longitudinal Study of Aging who had concurrent data on cognition , hearing , and mobility . We performed multivariable-adjusted linear regression to test two-way interactions between each cognitive measure and PTA. There were significant PTA interactions with all cognitive measures on 400-meter time. There was a significant interaction between PTA and sensorimotor function on 6-meter gait speed. Among cognitively normal older adults, poorer hearing is more strongly associated with poor mobility in those with low cognition, especially sensorimotor function. Future studies are needed to understand how cognition may moderate the relationship of hearing impairment with mobility decline over time.


Bone conduction is an efficient pathway of sound transmission which can be harnessed to provide hearing amplification. Bone conduction hearing devices may be indicated when ear canal pathology precludes the use of a conventional hearing aid, as well as in cases of single-sided deafness. Several different technologies exist which transmit sound via bone conduction. Here, we will review the physiology of bone conduction, the indications for bone conduction amplification, and the specifics of currently available devices. The concept of bone conduction hearing, the phenomenon through which a vibrating object can transmit sound, was first described in writing in the 1500s and credited to Girolamo Cardano . The conMultiple physiologic mechanisms contribute to bone conduction hearing. Put simply, sound energy is transmitted from vibrations in the skull to the cochlea, which ultimately results in wave propagation along the basilar membrane and stimulation of the cochlear nerve—the same endpoint as air conduction hearing . There iBone conduction hearing aids take advantage of the above mechanisms by converting sound energy into skull vibrations. Since the initial work by Tjellström  and his When choosing a bone conduction device, many factors must be considered. Each patient has unique needs which are related to their degree and type of hearing impairment, anatomy, vocational or educational needs, and personal preferences. Finding this information in a consolidated location can be challenging for patients and providers. The goal of this review is to provide an overview of the current device landscape including the hearing losses best treated by each device, surgical and nonsurgical advantages and disadvantages for each class of devices, magnetic resonance imaging (MRI) compatibility, processor characteristics, wireless connectivity, and available accessories. The following description of devices includes products currently available and utilized in the United States. While meant to be inclusive of all manufacturers and products, devices in the ever-evolving landscape may have been inadvertently excluded or developed following the preparation of this review.Surgically implanted bone conduction devices convert acoustic sound waves into mechanical vibration, which is conducted to the inner ear via direct contact with the skull. These can be classified broadly into percutaneous and transcutaneous devices based on the presence or absence of a skin-penetrating abutment. The transcutaneous devices can be further classified into active and passive implants. Passive transcutaneous devices have an implanted portion of the device in direct connection with the skull and a separate, external portion held in place magnetically which drives vibration through the skin to the implanted device. In a passive system, vibration occurs at the level of the external processor, and vibrations are transmitted transcutaneously to the implanted device. Active transcutaneous devices contain an external microphone and processor which send electronic signals to an implanted, vibrating device in direct contact with the skull. With an active system, the external processor is static and transmits electronic signals. Vibration occurs at the level of the implanted device only. Currently available devices including indications for the selection of each, benefits, and drawbacks will be discussed.Direct contact with the skull affords a meaningful advantage for percutaneous devices over passive transcutaneous devices. Passive transcutaneous devices rely on vibratory signal delivery through the skin and are subject to signal attenuation up to 20 dB, especially at high frequencies . The dirThe most significant disadvantage of percutaneous implants is the potential for adverse skin reactions, device extrusion, and the need for revision surgery. The reported complication rate varies widely and appears to be influenced by the surgical technique, surgeon experience, patient age, and patient factors predisposing to infection or poor wound healing. Surgery for the placement of a percutaneous abutment was often performed with skin grafting in the past; however, skin grafting is no longer performed regularly which has resulted in overall improved cosmesis with fewer graft complications. Adverse skin reactions continue to be the most common complication of percutaneous devices, and can be categorized using the Holgers classification, a scale from zero to four described in TM Baha® Connect System  . . TM  is also available . This deTM consists of an in-the-mouth (ITM) piezoelectric transducer placed on the buccal surface of the maxillary molars and a device worn on the poorer hearing ear which consists of a behind-the-ear (BTE) transducer and a microphone in the ear canal [TM for military personnel [The SoundBite  is a dental appliance designed to transmit vibratory signals to the skull via the teeth ,51. The ar canal . Productersonnel .® and BonebridgeTM, take advantage of new electronic signal transmission, optimize bone conduction efficiency, and reduce the incidence of skin complications. The current landscape of devices is described here and includes fitting criteria, patient selection, and benefits and drawbacks of each device. This condensed information is intended to be a resource for patients and providers alike to assist with proper device selection for each situation.Since the introduction of bone conduction hearing technology, numerous devices have been developed to optimize signal transmission, limit skin and wound complications, and rehabilitate hearing for patients with conductive and mixed hearing loss and single-sided deafness. The recently introduced active transcutaneous devices, the Osia


To examine the associations of specific dietary fats with the risk of disabling hearing impairment in the UK Biobank study.This cohort study investigated 105,592 participants  aged ≥ 40 years. Participants completed a minimum of one valid 24-h recall (Oxford Web-Q). Dietary intake of total fatty acids, polyunsaturated fatty acids (PUFA), saturated fatty acids (SFA), and monounsaturated fatty acids (MUFA) was assessed at baseline. Functional auditory capacity was measured with a digit triplet test (DTT), and disabling hearing impairment was defined as a speech reception threshold in noise > − 3.5 dB in any physical exam performed during the follow-up.Over a median follow-up of 3.2 (SD: 2.1) years, 832 men and 872 women developed disabling hearing impairment. After adjustment for potential confounders, including lifestyles, exposure to high-intensity sounds, ototoxic medication and comorbidity, the hazard ratios (HRs), and 95% confidence interval (CI) of disabling hearing function, comparing extreme quintiles of intakes were 0.91 (0.71–1.17) for total fat, 1.09 (0.83–1.44) for PUFA, 0.85 (0.64–1.13) for SFA and 1.01 (0.74–1.36) for MUFA among men. Among women, HRs comparing extreme intakes were 0.98 (0.78–1.24) for total fat, 0.69 (0.53–0.91) for PUFA, 1.26 (0.96–1.65) for SFA, and 0.91 (0.68–1.23) for MUFA. Replacing 5% of energy intake from SFA with an equivalent energy from PUFA was associated with 25% risk reduction  among women.PUFA intake was associated with decreased risk of disabling hearing function in women, but not in men.The online version contains supplementary material available at 10.1007/s00394-021-02644-7. Hearing loss is one of the main leading causes of years lived with disability; moreover, it has been considered an “invisible disability”, since it is usually underestimated in comparison with other health problems . In oldeBesides age-associated biological degeneration and noise exposure, one mechanism of hearing loss is impaired vascular function. The cochlea of the inner ear is highly vascularized and supported by a single artery; thus, dietary exposures that are able to improve vascular function may have a protective role on hearing capacity. However, the evidence on the effect of habitual diet on hearing loss is limited , 11. HigThere is compelling evidence that different types of dietary fats have opposed effects on cardiovascular disease by modifying serum lipid profiles, endothelial function, chronic inflammation, and blood clotting, and that the type of fat is more important than the total amount ingested . SimilarThe UK Biobank study is a large population-based cohort study established in 2006–2010 throughout the United Kingdom . The stuFood consumption was collected with five 24-h recalls (Oxford Web-Q), a detailed computerized questionnaire on the intake of 200 commonly consumed foods and beverages in the previous 24 h . Unlike r = 0.77), which suggests that about 60% of the performance on DTT is explained by standardized audiometric data . Among men, in fully adjusted models, no significant associations were found between fatty acids intake and hearing impairment: HR (95% CI) for quintile 5 vs. 1 were 0.91 (0.71–1.17) for total fat; 1.09 (0.83–1.44) for PUFA; 0.85 (0.64–1.13) for SFA; and 1.01 (0.74–1.36) for MUFA , compared with those with the lowest intake and none of these characteristics. For SFA, we observed a higher risk for those women with the highest intake and ≥ 60 y, with tinnitus, BMI < 25 kg/m2 or chronic diseases (p for interaction non-significant). For MUFA, no association was found in these analyses . In stratified analyses, women with the highest PUFA intake and < 60 y, without tinnitus, with BMI ≥ 25 kg/mIn this large population-based cohort study, we found that energy intake > 15% from PUFA was associated with lower risk of disabling hearing impairment among women. Replacing 5% of energy intake from SFA for PUFA was associated with a significant reduced risk. By contrast, no association was observed among men.Gopinath et al. , using dThe causes of hearing function impairment are multiple. The main ones include the exposure to environmental risk factors, age-associated degenerative processes, and chronic diseases that alter blood supply . If therWe found a statistically significant interaction between the PUFA intake and sex on the risk of disabling hearing impairment. Some studies suggest that estrogens could protect hearing function , 48. TheStratified analyses were robust showing a reduced risk associated with higher intake of PUFA in women. We observed stronger associations in women younger than 60 years; this is biologically plausible, since at increasing age, the ability to identify speech stimuli decreases greatly and beneficial dietary effects may be insufficient to reverse this situation . We alsoRegarding our findings on the intake of SFA, we observed a higher risk of hearing impairment associated to consumptions above 10% of energy intake. Although we could not find a dose–response association, substitution analyses showed that replacing PUFA for SFA decreased the risk of disabling hearing loss; more research is needed to understand the role of SFA in hearing function. In addition, replacing PUFA for MUFA was also associated with a slight reduction of risk. A recent paper has suggested that MUFA from sources other than olive oil  may have a detrimental effect on health .The strengths of our study lie in its prospective design and large sample size. Besides, the hearing measurement test includes not only pure tones but also speech in noise that simultaneously scans many frequencies of human speech, especially those close to 1000 Hz. Furthermore, our analyses were adjusted for many potential confounders, such as lifestyle, exposure to high-intensity sounds, ototoxic medication, and morbidity. The study also has several limitations, including the use of 24-h-recall questionnaires to estimate habitual diet. We could not explore the separate association of different PUFA, including n−3 and n−6, or MUFA from plant vs. animal sources with hearing function due to the lack of this information in the database analyzed. Specifically, since n−6 PUFA are the main source of PUFA in most populations, including the cohort analyzed here, we cannot discard that most of the effect observed is due to this fatty acid instead of n−3 PUFA, in contrast to the previous studies. Although the role of n−6 PUFA in inflammation has been controversial, our previous findings based on the effect of these fatty acids on plasma concentration of inflammatory biomarkers  and otheIn conclusion, higher consumption of PUFA was associated with decreased risk of disabling hearing function in women. Replacing 5% of total energy intake from SFA by the same energy from dietary PUFA may contribute to delay hearing loss. Further research may help understanding the differences found in men and women and which types of PUFAs are contributing to this association; also, other studies should elucidate if SFA intake increases the risk of hearing loss.Supplementary file1 (DOCX 22 KB)Below is the link to the electronic supplementary material.


Direct drive hearing devices, which deliver a signal directly to themiddle ear by vibrating the tympanic membrane via a lens placed incontact with the umbo, are designed to provide an extension of audiblebandwidth, but there are few studies of the effects of these deviceson preference, speech intelligibility, and loudness. The current studyis the first to compare aided speech understanding between narrow andextended bandwidth conditions for listeners with hearing loss whilefitted with a direct drive hearing aid system. The study also exploredthe effect of bandwidth on loudness perception and investigatedsubjective preference for bandwidth. Fifteen adult hearing aid userswith symmetrical sensorineural hearing loss participated in aprospective, within-subjects, randomized single-blindrepeated-measures study. Participants wore the direct drive hearingaids for 4 to 15 weeks (average 6 weeks) prior to outcome measurement.Outcome measures were completed in various bandwidth conditionsachieved by reducing the gain of the device above 5000 Hz or byfiltering the stimuli. Aided detection thresholds provided evidence ofamplification to 10000 Hz. A significant improvement was found inhigh-frequency consonant detection and recognition, as well as forspeech in noise performance in the full versus narrow bandwidthconditions. Subjective loudness ratings increased with provision ofthe full bandwidth available; however, real-world trials showed mostparticipants were able to wear the full bandwidth hearing aids withonly small adjustments to the prescription method. The majority ofparticipants had either no preference or a preference for the fullbandwidth setting. Direct drive hearing aid technology has been developed that sends an amplified,processed signal into the umbo of the tympanic membrane. This multicomponentsystem by Earlens  is comprised of a lens, asound processor, and an emitter. The emitter, which is housed in a vented ear-tip,couples to the sound processor which rests behind the ear and sends the signalfrom the processor to the lens. Earlier iterations of the device used light as themeans to send the signal; however, since April 2019, commercial products haveincorporated electromagnetic energy which the company reports as providing morestability of sound transference, with the transmission from the processor to thelens being affected less by jaw and facial movement . The lenPrevious studies investigating the influence of bandwidth onsuprathreshold measures of speech understanding and discrimination inquiet and noise in adults with sensorineural hearing loss have shownthat providing extended high-frequency amplification to adults withhearing loss can improve speech understanding in both quiet and noise.HoweverStudies have also shown that degree of hearing loss can influence soundquality and preference for extended bandwidth. Most studies have showneither subjective preference and higher sound quality, or nosignificant aversion to the provision of extended high frequencies inlisteners with normal hearing and less severe hearing losses . HoweverLoudness perception may also vary with audible bandwidth. A recent studyof loudness and bandwidth with acoustic hearing aids fitted to theDesired Sensation Level v5.0 adult targets  foundthPrevious evaluations of conventional air conduction hearing aids haverevealed upper bandwidth limits of 5000–6000 Hz in the early 2000s with morBecause the direct drive hearing device does not produce acoustic output,electroacoustic measures used to determine the bandwidth of aconventional hearing aid are not feasible. This system also allows low-frequency output to be transmitted to theumbo without occluding the ear. The purposes of this study were (a) to assess the functional bandwidth ofthe Earlens direct drive hearing aid by measuring aided sound fieldthresholds; (b) evaluate suprathreshold aided speech and loudnessperception obtained with the Earlens device across bandwidthconditions that compare to past studies with acoustic hearing aids inlisteners who had received a trial with the Earlens system; and (c)determine listener preference for restricted versus narrow bandwidthwhen wearing the Earlens system. In these tasks, we compared outcomeswith the full bandwidth condition compared with narrow bandwidth testconditions. This method is consistent with previous studies ofbandwidth . In a coThis study was approved by the Western University Human Research Ethics Board(109433) and Lawson Health Research Institute (R-18-057).Potential participants were screened for study inclusion and exclusioncriteria  prior ton = 1); visualization of a weakspot on the tympanic membrane at the time of ear cleaning(n = 1); voluntary withdrawal of permissionprior to impression (n = 1); participant could nottolerate the impression process (n = 1); initialimpression was not successful and participant declined to return(n = 2); impressions were taken successfully,but a device could not be fabricated due to size and shaperestrictions (n = 6); and device was fitted but theparticipant withdrew due to autophony when singing(n = 1). The remaining 15 participants completed theentire protocol. Of these, seven participants were female ; eight were male .Twenty-eight participants from London, Ontario and the surrounding areasigned letters of informed consent. Thirteen of the 28 participantsenrolled but did not complete the trial for the following reasons:small exostosis that prevented inclusion and was not apparent untilafter ear cleaning  standards. Participants’audiometric thresholds are shown in M = 9.8 years,SD = 7.4 years). Probe-tube microphone measuresof participants’ own aids were completed to measure the output of thehearing aids at 55, 65, and 75 dB sound pressure level (SPL) using theInternational Speech Test Signal . The fitting software uses the audiometricthresholds to compute modified CAM2 prescriptive targets. In situdetection thresholds for the frequencies 125–10000 Hz are alsomeasured using this software to determine the signal levelstransmitted from the device to the listener. The test platform allowsthe clinician to present pure tones (via the device) and to manuallysearch for and bracket thresholds using 5 dB steps in a one-up,two-down Hughson-Westlake adaptive tracking procedure . The sofBecause the application of oil during lens placement is known to cause adamping effect on hearing that is expected to subside after a fewdays, device programming was monitored and readjusted as follows. Eachparticipant was seen 1–2 weeks postfitting to confirm device functionand use and to address any questions. In situ calibration thresholdswere reassessed at this and each subsequent appointment to ensure thestability of the fitting and to reprogram the device and/or providemodified physical fits to ensure comfortable and stable fittings ifneeded. On average, the final fittings fell within 2.3 dB root meansquare error (RMSE) from the prescribed targets across all frequencies(125–10000 Hz) following fine tuning for preference .ParticiSD = 3.3). The majority of participants(73%) were within the range of 5–7 weeks. During this time,participants could revisit the laboratory for fine tuning ortroubleshooting. Four participants returned and required remakes tothe custom lens and ear-tip, which extended their wear time to10–15 weeks before outcome measurement. One participant with a largeamount of ear canal change with jaw movement required two sets oflenses and three sets of ear-tips. One participant had a new lens andear-tip made due to a lens malfunction at the time of the fitting. Twoparticipants had complaints of “static” that were not resolved withreprogramming and were refit with a new lens and ear-tip monaurally(n = 1) and new ear-tips bilaterally(n = 1). Troubleshooting components resulted inthese four participants being seen for 2–7 additional in-labappointments. One additional participant returned to have a noiseprogram added for real-world use. In situ light calibration on the dayof outcomes measurement confirmed stable fittings for all participantsincluding those who had had the lens and ear-tip remakes.Following the fitting and follow-up procedures mentioned previously,participants were requested to wear the direct drive aids for aminimum of 4 weeks. The mean trial time before outcome measurement was6 weeks  full bandwidth, withthe participants’ real-world trial frequency response and (b) narrowbandwidth with a frequency response programmed to provide no outputabove 5000 Hz. All outcome measures except loudness perception werecompleted in both hearing aid programs. The 5000 Hz cutoff for thenarrow bandwidth condition was chosen to be comparable to narrowbandconditions reported for acoustic hearing aid fittings . ThetesFor the loudness rating task, listeners wore the hearing devices in thefull bandwidth program, and the stimuli were filtered to create fourdifferent bandwidths: (a) 123–10869 Hz; (b) 123–4455 Hz; (c)313–10869 Hz; and (d) 313–4455 Hz. These filter conditions wereselected to match those used by The order of tests and conditions was randomized across listeners andtest sessions. Testing was completed in one or two test sessionsdepending on scheduling and to prevent fatigue. If testing wascompleted on multiple days, repeated tests were scheduled on the sameday to avoid differences in rest periods between conditions. All aidedtesting was completed in a standard 283 cm × 305 cm double-walledsound booth with a reverberation time (RT60) of 0.1 s. The participantwas seated with access to a computer monitor and mouse. Sevenloudspeakers surrounded the listener at horizontal angles of 45°, 90°,135°, 180°, 225°, 270°, and 315° relative to the look direction at adistance of 110 cm and a height of 120 cm (floor to speaker centre).An eighth speaker at 0° was located at a distance of 115 cm and aheight of 125 cm to accommodate conditions that required both speechand noise from 0°. In those conditions, the 315° speaker was moved tobe directly in front of the 0° speaker. Listeners wore the Earlenssystem in both ears while the test battery was completed.Detection thresholds were conducted in a calibrated sound fieldusing frequency-modulated tones from 125 to 10000 Hz produced bya clinical audiometer (GSI-61). All detection thresholds werebracketed using 5 dB steps and one-up, two-down adaptivetracking using the Hughson-Westlake procedure. Detectionthresholds were measured unaided  and aided . Aided thresholds weremeasured twice, once with the hearing aids set to the fullbandwidth condition and also with the hearing aids set to thenarrow bandwidth condition.Speech recognition was assessed using three measures; one usingsentences, one using nonsense syllables, and one measuringword-final /s/ detection, described later. Each test wascompleted twice, with the participant wearing the hearingdevices in each of the full and narrow bandwidth programs.Sentence recognition in noise was measured with the Hearing inSpeech Test (HIST). This test uses sentences and bracketingprocedures from the Hearing In Noise Test , with reConsonant recognition in noise was measured with the University ofWestern Ontario Distinctive Features Differences test . The UniAided fricative detection was measured with the University ofWestern Ontario Plurals Test , which mOcean andWater . With each sentencepresentation, the listeners indicated their perceived loudnesscategory on a computer screen. Seven loudness categoriesincluded the following: Very soft, Soft, Comfortable butslightly soft, Comfortable, Comfortable but slightly loud,Loud, and Uncomfortably loud. Presentation levelsranged from 52 to 80 dB SPL and increased in steps of 4 dB untileither the maximum level was reached or a rating ofUncomfortably loud was given. At thatpoint, the stimulus level reversed (descending run). Loudnessratings were scored from 1 to 7. Ratings from the ascending anddescending run were averaged together to obtain one number foreach stimulus level for a given test condition  the listener selectedno preference on both trials or (b) the listener selected apreference for one condition in one trial and a preference forthe other condition in the other trial. Correlation analyseswere completed to determine any relations between degree andslope of hearing loss with bandwidth preference , 9000 Hz(n = 7), and 10000 Hz (n = 8)in the unaided condition; and at 6000 Hz (n = 2) and8000–10000 Hz (n = 9) in the narrow bandwidthcondition. Thresholds above the limits of the audiometer were coded asthe maximum presentation level for that frequency +1 dB. In the fullbandwidth condition, all participants had measurable aided thresholdsat all frequencies tested (125–10000 Hz).Unaided and aided detection thresholds are shown in F = 86.90, p ≤ .001, ŋ2 = .85, andbandwidth condition, F = 211.31,p < .001, ŋ2 = .94, as well as aninteraction between bandwidth condition and test frequency,F = 76.68,p < .001, ŋ2 = .85. Frequency-specificpairwise comparisons were completed for (a) unaided versus fullbandwidth to measure aided benefit and (b) narrow versus fullbandwidth to test the impact of bandwidth. Aided full bandwidththresholds were better than unaided at 500 Hz and above, through10000 Hz . Narrowband aided thresholds werepoorer than full bandwidth aided thresholds at 6000 Hz and above(p < .001). At all other frequencies, narrowand full bandwidth thresholds did not differ significantly .These results indicate that improved detection was observed in aidedversus unaided conditions and also that the narrow bandwidthprogramming successfully reduced high-frequency audibility, at leastfor the low levels tested in this task.Detection thresholds were analyzed with a repeated-measures analysis ofvariance, using Greenhouse–Geisser corrections to adjust for lack ofsphericity. Aided thresholds were the dependent variable, and testfrequency and bandwidth condition were repeated factors. There was anoverall effect of test frequency, F = 27.34,p ≤ .001, ŋ2 = .66, and bandwidth,F = 4.98, p = .042,ŋ2 = .26. The interaction between bandwidth andspatial configuration was nonsignificant, F =  3.10, p = .063, ŋ2 = .184. Onaverage, participants were able to recognize HIST sentences with1.1 dB more background noise in the full bandwidth condition versusthe narrow bandwidth condition, averaged across spatialconfigurations.Mean reception thresholds for sentences from the HIST are shown in t test wascompleted on the RAU scores. Scores were significantly better by 10.2RAU in the full bandwidth condition comparedwith the narrow bandwidth condition ; t(14) =  –2.83,p = .01.The differences in error patterns for individual consonants between thetwo bandwidth conditions were analyzed on a confusion differencematrix by subtracting the number of correct trials in the narrowbandwidth condition from those in the full bandwidth condition, perconsonant . With fot test comparing bandwidth conditions wascompleted. Results indicated a significant improvement in the fullbandwidth condition  compared with the narrow bandwidthcondition ,t(14) =  –6.2,p = .<001.2(1) = 1087.08, p < .001 and χ2(3) = 27.75, p < .001, respectively, while addingthe interaction did not significantly further improve the model,χ2 (3) = 4.04, p = .26. Thereference bandwidth was 123–10869 Hz. Post hoc pairedt tests with Bonferroni correction were chosento further examine differences between pairs of bandwidth conditions.Results showed that the reference bandwidth condition (123–10869 Hz)was significantly louder than the other three bandwidth conditions(p < .001). There was also a significantdifference between the two narrowest bandwidth conditions , indicating thatproviding the low-frequency band yielded increases in loudnessjudgments. There were no significant differences found between theloudness judgments of the remaining conditions(p > .05).The input level at which the categories “Comfortable, but slightly soft”and “Comfortable, but slightly loud” were reached for each of theconditions was computed to summarize the functional magnitude of thedifferences in loudness judgment across conditions. The largestbandwidth condition (123–10869 Hz) reached these loudness categoriesat input levels that were 2.2 and 3.4 dB lower than in the narrowbandcondition.p = .02). Further investigation was performed todetermine if audiometric pure-tone average (PTA) and/or slope werefactors in preference. PTA did not relate significantly to preference,whether this was defined for four frequency PTA   or threefrequency PTA   or PTA . For slope,we investigated two definitions based on previous studies: (a) thedifference between 8000 Hz thresholds and 4000 Hz thresholds .Paired comparisons testing  indicateresholds  and (b) resholds . NeitherIn the present study, the effects of bandwidth on speech perception, loudness,and preference were evaluated by fitting bilateral Earlens direct drivehearing devices to participants with sensorineural hearing loss.Participants in this study met recommended criteria for use of the Earlensin terms of ear size and characteristics, medical history, and audiometricthresholds. The audibility of the extended bandwidth up to 10000 Hz wasconfirmed with aided sound field audiometric testing, which demonstrated asignificant improvement in high-frequency thresholds between the unaided andaided conditions. In this study, we examined the effects of extendedbandwidth. For speech recognition and preference tasks, the bandwidth of thedirect drive devices was restricted to 5000 Hz, thereby creating two testconditions: (a) the full clinical bandwidth as fitted to the participants’hearing loss using the CAM2 fitting method and (b) a narrow bandwidthfitting. The 5000 Hz cutoff for the narrow bandwidth condition was chosen tobe comparable to narrowband conditions reported for acoustic hearing aidfittings . AlthougAs noted earlier, hearing tests in the sound field confirmed improvedaudibility in the full versus narrow bandwidth condition for threshold ofhearing with use of the direct drive devices. We also examinedsuprathreshold outcomes with full versus narrow bandwidth fittings,including speech sound detection and recognition and speech in noiseunderstanding. Overall performance of speech recognition for nonsensesyllables in noise significantly improved in the full versus narrowbandwidth condition, with improved recognition of consonants withhigh-frequency content such as the phonemes /s/ and /z/. Specifically, thisimprovement in fricative identification is consistent with the resultspublished in the development of the Plurals test, where high-frequency /s/in word-final position was detected with greater accuracy in the broadbandcondition . These rOne of the most common challenges for those with hearing loss is understandingspeech in background noise. There was an overall benefit of extendedbandwidth for sentence recognition in speech noise using the HIST. Theseresults are consistent with findings reported by To explore the interaction between the provision of extended bandwidth andloudness perception, the effect of bandwidth on loudness perception wasmeasured in this study using four bandwidth conditions. Consistent withresults from Van Eeckhoutte et al. (2020), perceived loudness judgments werehigher for the stimuli with the broadest bandwidth and exceeded that ofeither the low- or high-frequency conditions. When low-frequency energy wasadded , asignificant increase in loudness was observed, indicating low-frequencyenergy contributed to the loudness percept. This increased further whenstimuli were extended in both low- and high-frequency bands  which indicates that high-frequency energy also contributes toloudness perception. This result is generally consistent with previous workthat found contribution of energy at both low and high frequencies dependingupon sensation level . We notePreference ratings were consistent with previous literature which found thataccess to extended bandwidth did not degrade the perceived sound quality forspeech stimuli for most participants . ResultsListeners’ preference ratings and their real-world trial settings were within6.3 dB RMSE from the CAM2 fitting targets for most participants. This maysuggest that the direct drive system provided an acceptable fitting using astrategy that aims for broad audible bandwidth. The increase to perceivedloudness from the signal produced by the full bandwidth fittings, althoughaudible, did not make a large change to sound levels associated withfunctional loudness ratings. Coupled with the positive effects on speechrecognition and the significantly higher sound quality ratings in the fullbandwidth condition , the batA possible limitation to this study is that participants had real-worldtrials with only the full bandwidth condition without a correspondingnarrow bandwidth trial. This may have influenced their preference forthe extended bandwidth condition. The goal of this trial was toacclimatize the participants to gain in the extended high frequencies,prior to completing the outcome measures as, on average, their ownacoustic hearing aids provided a narrow bandwidth. Future studiescould incorporate a design where participants have real-world trialswearing devices in both the narrow bandwidth condition and the fullbandwidth condition. In addition, preference ratings were measuredwith speech stimuli in quiet and may not generalize to other listeningsituations.Earlens devices are fitted using vented ear-tips and as such,low-frequency sound presented in the sound field could enter the earcanal through this pathway for at least some of the participants.Measurements of insertion loss are included in supplemental materialS3 and indicate that significant vent-transmitted sound was providedfor the majority of ears to approximately 800 Hz. Although the goal ofthis study was to evaluate outcomes with the devices as fitted, futurestudies could incorporate a design where filtered stimuli are sent tothe aids via streaming or direct audio input to determine whether thelow-frequency perceptual impacts are provided by the direct drivesound or via the vent, and how this may interact with audiometricconfiguration. Finally, we note that our outcome measures werecompleted using the aided conditions only with no unaided comparison.Future studies could include both unaided and aided conditions todemonstrate aided benefit of the direct drive fittings.Participants with mild-to-severe sensorineural hearing loss were fitted withthe Earlens direct drive hearing devices for a clinical trial period.Broadband fittings were achievable without feedback, and devices werewearable with average requested fine-tuning based on user preference under3 dB RMSE across frequencies. Compared with a narrowband condition,provision of full bandwidth was beneficial for high-frequency consonantdetection and recognition and for sentence recognition in multitalkermasking. There was a small but perceivable increase in the loudness with theprovision of the full bandwidth. Preference results suggested that themajority of participants either preferred the full bandwidth setting or hadno preference between the two bandwidth conditions. These results providefurther evidence to support hearing aid fittings which include the provisionof extended bandwidth to those with sensorineural hearing loss in themild-to-severe range.Click here for additional data file.Supplemental material, sj-pdf-1-tia-10.1177_2331216521999139 forDetection, Speech Recognition, Loudness, and Preference Outcomes Witha Direct Drive Hearing Aid: Effects of Bandwidth by Paula Folkeard,Maaike Van Eeckhoutte, Suzanne Levy, Drew Dundas, ParvanehAbbasalipour, Danielle Glista, Sumit Agrawal and Susan Scollie inTrends in Hearing


Drosophila grooming. Grooming confers survival and social benefits. Grooming features of five Drosophila species exposed to a dust irritant were analyzed. Aspects of grooming behavior, such as anterior to posterior progression, were conserved between and within species. However, significant differences in activity levels, proportion of time spent in different cleaning movements, and grooming syntax were identified between species. All species tested showed individual variability in the order and duration of action sequences. Genetic diversity was not found to correlate with grooming variability within a species: melanogaster flies bred to increase or decrease genetic heterogeneity exhibited similar variability in grooming syntax. Individual flies observed on consecutive days also showed grooming sequence variability. Standardization of sensory input using optogenetics reduced but did not eliminate this variability. In aggregate, these data suggest that sequence variability may be a conserved feature of grooming behavior itself. These results also demonstrate that large genetic differences result in distinguishable grooming phenotypes (variation), but that genetic heterogeneity within a population does not necessarily correspond to an increase in the range of grooming behavior (variability).Behavioral differences can be observed between species or populations (variation) or between individuals in a genetically similar population (variability). Here, we investigate genetic differences as a possible source of variation and variability in  Differences in phenotype arise from differences in genotype. Changes in DNA account for variation in traits among species, and differences between individuals of the same species. Animal behavior contains phenotypes partially under genetic control, and specific genes associated with observable differences in behavior between and within species have been uncovered . DiffereDrosophila melanogaster demonstrate that the degree of behavioral variability in locomotion is partially controlled by genetic expression of proteins such as teneurin-α, a cell adhesion molecule , which are genetically distinct—separated by millions of years of evolution—and inhabit different ecological niches. We also examined four common melanogaster lab stocks , and several isogenic lines derived from these parent stocks in our laboratory.In this work, min each . We analTo analyze this large data set, we employed tools from computational ethology . An autoDrosophila species tested here, five grooming actions were observed consistently, indicating a conserved behavioral response  (Across the response . Previouethods”) . Here, tethods”) , indicatethods”) .p < 0.05, multiple comparison correction via Holm’s method; N = 18) was used as the representative melanogaster group. Full action proportion distributions are shown in To quantify the behavioral response to dusting, the proportion of time spent grooming  was calculated for each fly . The proBehavior was then examined in more detail by considering all seven actions  and the progression of those actions over time, as in N = 390). With seven behavioral states, 42 transitions were possible, excluding self-transitions. Thus, syntax was represented as a 42-dimensional vector for subsequent classification analysis and visualization. Syntax across all flies exhibited high transition probabilities within the anterior grooming motif  and posterior grooming motif . The average syntax for each species is illustrated as a weighted, directed graph in Syntax (the transition probabilities between discrete behaviors) was calculated from the ethogram of each dusted fly (erecta and santomea) between any species .Finally, continuous grooming action duration distributions  were calculated from ethograms. Distributions of action durations were qualitatively similar across species and had probability peaks between 500 and 750 ms . When comelanogaster and non-melanogaster species were identified. p < 0.05, multiple comparison correction via Holm’s method). Overall, 125 of 420 (30%) of pairwise syntax comparisons revealed differences between species .Several significant differences in behavioral features between melanogaster and non-melanogaster species. In particular, posterior motif grooming transitions  were consistently significantly different, on average, as were transitions between back leg rubbing, standing, and walking. Of these syntactic differences, 71 (60%) occurred between Classification analysis was applied to behavioral features to verify this interpretation and quantify the degree of variation between species. Multinomial logistic regression classified flies by species according to behavioral proportions, progressions, and syntax with >80% accuracy . Notablymelanogaster flies possessed the lowest entropy (highest degree of stereotypy) due to high transition probabilities between head cleaning and front leg rubbing .  rubbing . In summDrosophila melanogaster lab strains  were analyzed for differences in grooming features  differed significantly between any two stocks and, of these, only two within-motif transition (both posterior) differed significantly. Within-motif syntax elements are of particular interest because they represent the most common, most highly stereotyped action transitions observed across flies of all genotypes see .melanogaster was less pronounced than variation between species  yielded results similar to classification using full grooming behavior syntax. This is illustrated by the fact that Canton-S flies’ higher propensity to walk after grooming their wings is reflected both in their syntax and grooming proportions in Classification accuracy was moderate but above chance for all features examined; as expected, variation within  compare , 3A. Varmelanogaster stock lines using syntax, but lower than the same comparison for interspecies data.Within Canton-S, activity levels separated male and female flies, as male flies tended to walk more than females . Male anmelanogaster syntax. The wing grooming to back leg cleaning transition exhibited the largest difference between median values of any syntax element (comparison of Canton-S and Oregon-R yielded this difference), but none of these distributions possessed detectable statistical differences due to their concomitantly large variances.Since all flies examined showed variability in syntax, we wondered whether the extent of this variability differed among species or strains. melanogaster lab strain was compared to lines bred to maximize genetic heterogeneity (MaxVar) or minimize genetic heterogeneity (isogenic lines). These can be considered outbred and inbred strains. If variability in grooming syntax within a population is strongly related to genetic heterogeneity, we would expect populations with larger genetic heterogeneity to also contain flies with more variable syntax.To examine the potential relationship between genetic heterogeneity and behavioral variability, each p < 05 after correction for multiple comparisons via Holm’s method). Moreover, none of these differences corresponded to within-motif transitions, indicating that variability of common transitions is similar regardless of genetic heterogeneity in a population. These findings also held for Oregon-R (8/252), Berlin-K (19/108), and w1118 (2/108) stock and isogenic comparisons. See p values of pairwise action transition distribution variance comparisons.All lines, regardless of genetic heterogeneity, exhibit variable grooming . To quanWe also examined stock lines derived from selected wild isolates  to deterFinally, we analyzed dust-induced grooming in 15 Canton-S flies that were assayed on three consecutive days. Since a given individual’s genome remains constant through the three trials, we could isolate the magnitude of grooming variability that is due to differences in sensory experience  and life history . Ethograms from three example flies are provided in Flies exhibited some longitudinal grooming trends, as the total amount of grooming decreased between the first and third days of the experiment. However, the time to completion of 50% of their total grooming did not decrease, suggesting that flies are not simply grooming quicker, but rather are grooming less consistently over time  . ImportaTo probe the sensory contribution to within-individual variability, we used optogenetic stimulation to induce anterior grooming. 20 Bristle-spGAL4-1 > UAS-CsChrimson flies were tested . Figure p < 0.05, Wilcoxon rank-sum test), indicating a higher degree of stereotypy in grooming , it provides a much stricter definition of stereotypy than Markov entropy, which we use as a measure of stereotypy earlier in our analysis. In addition, it is most useful as a stereotypy measure when it is possible to identify a synchronizing “start” signal between sequences of interest, which is not present in the previously described experiments, but is present for optogenetically-stimulated flies. Therefore, when comparing flies across recording sessions or sequences from different flies, the use of edit distance helps answer the specific question, “Do flies perform repeated, similar sequences or subsequences?”For all dusted flies, we calculated the edit distance within flies across consecutive days to assess whether flies possess stereotyped repeats. For these comparisons, we compared the first continuous anterior motif sequence lasting at least 30 s on consecutive days, shown by the blue boxes in Edit distance calculations were also performed for all optogenetically-stimulated flies. Within-fly comparisons  correspond to identifiable differences in several grooming features, including the rules governing action transitions known as syntax. Within melanogaster, stock lines exhibited smaller variation in grooming syntax, as well as differences in overall activity levels. All flies showed variability in the details of the grooming movement sequence, but increased genetic heterogeneity did not correspond to increased behavioral variability. Analysis of 15 Canton-S flies recorded over consecutive days showed that intra-individual and inter-individual comparisons had similar—high—levels of variability. Optogenetically-stimulated flies also exhibited intra-individual variability in grooming behavior, but less. Taken together, these results demonstrate that large genetic differences result in distinguishable grooming phenotypes, but that genetic heterogeneity within a population does not necessarily correspond to an increase in the range of grooming behavior variability.Here, we analyzed fly grooming behavior in five different Advantageous behavioral phenotypes that are under genetic control can be selected over evolution to produce populations with differing behaviors. Here, we identified significant inter-species variation in grooming syntax, suggesting a genetic basis for group differences in grooming behavior.melanogaster in their propensity to perform anterior grooming actions; the anterior motif actions are significantly less strongly coupled in non-melanogaster drosophilids, suggesting that anterior neuronal circuitry or sensory physiology may differ. We also identified differences in grooming behavior between commonly used melanogaster stock lines and between male and female Canton-S flies; most of these differences relate to overall activity levels.Some species differ from Variability itself is a trait that can also be selected for, but is often overlooked . At the Drosophila grooming action sequences suggests that non-stereotyped grooming may be advantageous, perhaps for removing diverse distributions or kinds of debris. We examined whether greater genetic heterogeneity within a population corresponded to greater behavioral variability but did not detect any significant impact.The prevalence of variability in Drosophila species detected differences in spontaneous grooming between species and among individuals within a species  grooming behavior in nogaster  and in o species . Our res species  and dustThe causes of grooming variability are still under investigation. Differences in developmental processes such as neural wiring or synaptic connectivity may contribute to behavioral differences between flies, but our experiments show that even individual flies exhibit variability in grooming over repeated trials with dust or optogenetic stimulation. This suggests that non-genetic factors such as sensory stimuli, internal state, previous experience, and circuit noise contribute to the variability we observe in grooming action sequences. The reduction of variability when sensory inputs are optogenetically controlled supports diversity of sensory stimulation as a contributor. The persistence of variability within individuals suggests that intrinsic stochasticity or noise within the neurons or circuits themselves may also play a role, which are possibilities which should be explored further.melanogaster stock were generated; note that many attempts to isogenize result in lethality, as anecdotally reported by colleagues. Maximum Variability stocks were obtained by crossing each melanogaster strain to double balancers and then crossing the progeny together and selecting against the balancers. This allowed combination of chromosomes for all four strains. The progeny were allowed to interbreed for several generations to enable recombination in the females.Canton-S, Oregon-R, Berlin-K, w1118, Bristle- spGAL4- 1 , and 20XUAS- CsChrimson. mVenus (attp18) stocks were obtained from the Bloomington Stock Center. Isogenic  stocks were made by crossing single males to double-balanced stocks and then back-crossing males to the double balancer stock to isolate single second and third chromosomes. Single pairs were mated to reduce variability of X and IV. ∼ 2 independent isogenic lines from each Drosophilid species stocks were obtained from Tom Turner, UCSB, and the National Drosophila Species Stock Center1.Grooming was induced and analyzed as described in Transfer chamber was tapped against an empty pipette tip box to remove extra dust. Dusted flies were then immediately tapped into recording chamber for video recording. The entire dusting process was performed in a WS-6 downflow hood. Approximately 10 individuals were recorded for each genotype. 30 Hz videos were recorded for 50,000 frames (27.78 min) with a DALSA Falcon2 color 4 M camera. A white LED ring right was used for illumination.Optogenetic stimulation protocol is replicated from N = 83), melanogaster stocks and isogenic lines = 252), additional male/female Canton-S flies (N = 31), individual Canton-S flies followed for three sessions (N = 45), and optogenetically stimulated flies (N = 10).For each set of experimental comparisons , a single experimenter performed all dusting assays to eliminate experimenter-related differences that may arise. In total, 390 ethograms were recorded. This number includes species data . Features were extracted in three timescales and saved into different channels of ST images: 1. raw frame; 2. difference between two frames; 3. Spectral features extracted from a 0.5 s window. A convolutional network trained by ST images under different light conditions was then used to label the behavior identified in each frame. A different network was trained for classification of each species due to differences in body size and light conditions. All networks achieved >70% validation accuracy within the training protocol, which reserved 20% of frames as test data after training .All ethogram features were extracted using custom-written code in MATLAB 2019a. Grooming progression vectors were generated for each fly by calculating the proportion of each action in 10 non-overlapping windows (2.78 min each), yielding a 70-dimensional vector for each fly . Grooming syntax was defined as the first-order transition probabilities between actions. Syntax for each fly was calculated as described in Bout duration distributions were generated as described in Statistics for comparisons between grooming features were calculated using built-in MATLAB functions. t-SNE, and multinomial logistic regression classification analysis were performed using built-in MATLAB functions .The original data analyzed in the study are included in the article/JS: experimental design. JS and NZ: data collection. JM: data analysis. JM and JS: manuscript writing. JM, JS, and JC: manuscript editing. JS and JC: funding acquisition. 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.


Xanthophyllomyces dendrorhous is an appealing alternative due to its fast growth rate and easy large-scale production. In order to increase X. dendrorhous astaxanthin yields, random mutant strains able to produce from 6 to 10 mg/g dry mass have been generated; nevertheless, they often are unstable. On the other hand, site-directed mutant strains have also been obtained, but they increase only the yield of non-astaxanthin carotenoids. In this review, we insightfully analyze the metabolic carbon flow converging in astaxanthin biosynthesis and, by integrating the biological features of X. dendrorhous with available metabolic, genomic, transcriptomic, and proteomic data, as well as the knowledge gained with random and site-directed mutants that lead to increased carotenoids yield, we propose new metabolic engineering targets to increase astaxanthin biosynthesis.Astaxanthin is a carotenoid with a number of assets useful for the food, cosmetic and pharmaceutical industries. Nowadays, it is mainly produced by chemical synthesis. However, the process leads to an enantiomeric mixture where the biologically assimilable forms  are a minority. Microbial production of  astaxanthin by  Carotenoids have gained biotechnological importance due to their benefits to human health. They are powerful antioxidative and anti-inflammatory agents, as well as efficient UV radiation protectants. In addition, they are precursors of retinol (Vitamin A) and other derivatives that participate in cell regeneration. Therefore, carotenoids are interesting for food, cosmetic and pharmaceutical industries –5. AmongAstaxanthin  is probably one of the most interesting and valuable carotenoids. Its antioxidant activity has been reported to be 10 and 100 times higher than those of β-carotene and α-tocopherol, respectively –10. It iXanthophyllomyces dendrorhous ,  and . Natural astaxanthin is predominantly  or , while synthetic astaxanthin contains a mixture of all three possible enantiomeric forms, , , and , in a 1:2:1 ratio, respectively –18. Becaluvialis , 21.Fig.Meganyctiphanes norvegica, and two different species of salmonids: Atlantic salmon  and Pacific salmon (Oncorhynchus tshawytscha). They observed that -astaxanthin accumulated more (77–85%) than  stereoisomers (11–18%). However, Foos et al. ..crtYB ovNevertheless, the described increase of carotenoids synthesis is not enough to compete against the production costs of chemical synthesis. Therefore, it is necessary to explore other genes/enzymes of the metabolic pathway involved in astaxanthin biosynthesis.crtI encodes phytoene desaturase in X. dendrorhous, the second enzyme of the carotenoid biosynthesis pathway , responsible for the astaxanthin biosynthesis, HDCO and other unwanted byproducts of the astaxanthin biosynthesis pathway  that produced 6.01 mg β-carotene/g dry biomass in 96 h. The transformation was achieved through the simultaneous integration of three copies of crtE, one copy of crtYB and one copy of crtI. All genes were isolated from X. dendrorhous.Similarly, Yamada et al.  construcIn accordance with the discussed results, it is necessary to find metabolic engineering alternatives that do not divert the carbon flow towards the synthesis of torulene, HDCO or unwanted byproducts but that allow the accumulation of astaxanthin as the main product.crtS in X. dendrorhous, together with cytochrome P450 reductase (crtR), catalyzes the last step of the astaxanthin pathway transforming β-carotene into astaxanthin. This is the limiting step in the astaxanthin biosynthesis. As previously mentioned in “crtS has monooxygenase activity  that depends on crtR activity [Astaxanthin synthase, encoded by activity , 86.crtYB and crtS in X. dendrorhous genome to increase astaxanthin production  or two (Xd_2H2S) copies of crtS. While the control strain, UCD 67–385, achieved astaxanthin yields of 128 µg/g dry biomass, Xs_1H1S and Xd_2H2S reached a production of 154 µg/g and 179 µg/g biomass in 96 h, respectively. X. dendrorhous has been also transformed with additional copies of crtS and crtE using a Cre–lox-P system . Since CYP51 and CYP61 are also involved in ergosterol biosynthesis, they have the ability to decrease the flux of available carbon for astaxanthin production  capable of producing astaxanthin in high yields (up to 8.1 mg/g dry biomass). As a first step, they built a β-carotene producing strain by heterologously expressing crtE, crtI and crtYB from X. dendrorhous. Subsequently, they expressed crtZ and bkt from H. pluvialis. crtZ encodes β-carotene hydroxylase, which catalyzes the conversion of canthaxanthin to astaxanthin. bkt encodes β-carotene ketolase, which catalyzes the conversion of β-carotene to canthaxanthin [H. pluvialis genes led to a yield of 8.1 mg astaxanthin/g of biomass, but it turned out to be the  stereoisomer. The graphic representation of this genetic engineering scheme is shown in Fig. S. cerevisiae strain by introducing the carRA (orthologous to crtYB from X. dendrorhous) and crtI genes from Blakeslea trispora, crtZ from Agrobacterium aurantiacum, crtW from Brevundimonas vesicularis, which encodes a β-carotene ketolase (non-encoded in X. dendrorhous genome) and crtE from Taxus media. The transforming S. cerevisiae strain was only capable of producing 20 mg astaxanthin/L, but, additionally, it was subjected to several rounds of physical mutagenesis and adaptation to hydrogen peroxide. After 15 rounds, the best selected strain was able to produce > 65 mg astaxanthin/L in a batch system, while in a fed batch system the maximum concentration obtained was 404.78 mg/L.Astaxanthin biosynthesis has been recreated in u et al.  generateaxanthin . The insX. dendrorhous and S. cerevisiae. The highest astaxanthin yield to date is around 10 mg/g biomass. Since X. dendrorhous is the microorganism that possesses the native enzymatic machinery for the astaxanthin biosynthesis, it could be the option with the greatest potential for metabolic engineering. However, it is still necessary to study in greater detail the carbon flux and the metabolic limitations that exist in this biological system, the goal we are pursuing in this review.In terms of -astaxanthin overproduction, it has not yet been achieved in satisfactory levels. Table X. dendrorhous has not led to competitive astaxanthin  overproducing mutants. In addition to the use of mevalonate, citrate, glutamate, succinate, glycerol and other molecules as inductors of enzymes that participate in the astaxanthin biosynthesis [Nowadays, metabolic engineering of ynthesis , 98, 99,ynthesis , 49. ThiCandida utilis, S. cerevisiae and other yeasts, a strong limitation in the expression levels of HMG-CoA reductase and GGPP synthase has been found and, subsequently, are limited the levels of mevalonate and isoprenoids derivatives available for carotenoid and astaxanthin biosynthesis [X. dendrorhous could be reduced for isoprenoids, lipids and carotenoid biosynthesis  in X. dendrorhous increases HMG1 expression levels from 2 to 5 times [ERG13 and HMG1 activities  [In te, GGPP , 31, 33.te, GGPP , 31, 42. 2 times , 31, 35.ynthesis  demonstr 5 times  and, substrains)  with whiS. cerevisiae, it has been observed that overexpression of stearoyl-CoA 9-desaturase increases the capacity to accumulate lycopene up to 30% [ino2, which encodes a transcriptional factor involved in the response to physiological stress, increases up to 10% the ability to accumulate lipids and derivatives (including lycopene) [Nevertheless, high yields of carotenoids may affect the cell since their intracellular accumulation could be toxic , 52. Howp to 30% , while oycopene) .X. dendrorhous, proteomic and transcriptomic analysis have been performed on overproducing strains [crtE and crtS, and crtI at levels 3 and 2 times above the control strain, respectively. However, expression levels of genes such as idi were variable, while crtYB and crtR expression was even lower than those of the control strain. According to the expression levels of the genes involved in the carotenoids and astaxanthin biosynthesis, crtS action is essential to astaxanthin biosynthesis while crtR could be expressed at non-limiting levels in this overproducer X. dendrorhous strain. Nevertheless, the crtR expression levels may be variable and limiting between different mutant strains due to their polymorphism [To determine which are the metabolic mechanisms that influence the carotenoids and astaxanthin biosynthesis in  strains , 81. Bar strains , using r strains , 106. Gl strains . On the  strains , 108. GP strains , 51. UDP strains , 109. It strains , 81 revemorphism , 86 and morphism .X. dendrorhous UV3-721 mutant strain. After a proteomic analysis, they found 1.5- to 2.5-fold more Grg2, aspartic protease precursor, transaldolase, 3-isopropylmalate dehydrogenase and a disulfide isomerase precursor compared to that observed at low 19:1 C/N ratio. They also observed sixfold overexpression of phospho-methyl-pyridine kinase, 3.3 times more astaxanthin synthase, and overexpression of IPP isomerase and pyruvate decarboxylase. On the other hand, in this study, unlike the results reported by Barbachano et al. [Pan et al.  demonstro et al. , a tenfoo et al. , 59.The strategy of feeding 0.368 g/L glutamate in media culture, preserving a high 76:1 C/N ratio, has been used . GlutamacrtYB (4.2- to 4.5-fold), crtI (1.7- to 1.8-fold) and crtS (up to 1.3-fold) compared to the use of glucose as carbon source [Proteomic analysis has revealed that the oxidative stress generated by the tricarboxylic acid cycle is a key factor in increasing the expression levels of genes involved in astaxanthin biosynthesis, as well as of precursor compounds , 76, 106n source .X. dendrorhous leads to twofold increase of crtYB and crtS expression levels [crtI in the mutant strain was lower than that of the wild-type one (− 2 times). Since crtS and crtR work simultaneously in the astaxanthin biosynthesis  , Pan et X. dendrorhous showed overexpression of astaxanthin biosynthesis genes [HMG1 and idi showed a significant 3.5-fold overexpression, while crtE had a twofold increase. crtS and crtYB had a 1.7 and twofold increase, respectively [crtI, in the middle of the fermentation process, had a 1.35-fold expression increase, but at the end it decreased to its basal level [crtI expression levels close to basal since increasing its activity can divert the carbon flux towards the generation of torulene and HDCO [Transcriptional analysis of 6-BAP treated is genes . HMG1 anectively . crtI, ial level . It is iand HDCO , 91. Regand HDCO .X. dendrorhous.The synthesis of HMGR and NADPH is also important in the mevalonate metabolic pathway for the synthesis of carotenoid precursors , 115–117X. dendrorhous Sre1 functions as a sterol regulatory element-binding protein (SREBP) [S. cerevisiae) that inhibits sterols biosynthesis when the cellular levels are sufficient. When sterol levels drop, the Sre1–Scp1 complex is transported to the Golgi apparatus, leading to Sre1 release, and translocation to the nucleus. Then, transcription of genes involved in sterol biosynthesis is activated [SRE1 deletion inhibits both sterol and carotenoid biosynthesis [SRE1 increases 1.5 and twofold the expression of ERG13 and HMG1. This could be used as an alternative to the isoprenoids overproduction to increase the astaxanthin biosynthesis.The  (SREBP) , 119. Th (SREBP)  and the ctivated , and conctivated . Howeverynthesis , 119. GuS. cerevisiae, mevalonate kinase (ERG12) and farnesyl pyrophosphate synthase (ERG20) have been recently identified as limiting factors for the isoprenoid accumulation in carotenoid biosynthesis [S. cerevisiae strain able to accumulate up to 11 times more isoprenoids was obtained. ERG12 and ERG20, in a similar way to S. cerevisiae, could generate a bottleneck for the astaxanthin biosynthesis in X. dendrorhous.In recombinant carotenoids-producing ynthesis . Using Cynthesis , 122, a HMG1, ERG12, ERG20 and idi genes that encode enzymes that limit the accumulation of FPP, the main substrate for carotenoid biosynthesis. Despite the latter has been performed in S. cerevisiae and Y. lipolytica, this metabolic engineering program has not been carried out in X. dendrorhous. If increasing FPP accumulation is achieved, a second step focused on increasing the expression of genes directly involved in the astaxanthin biosynthesis pathway is necessary [crtYB, crtR and crtS are the main bottlenecks in the synthesis of astaxanthin and their overexpression would also prevent the accumulation of isoprenoid products, thus avoiding the effect of inhibition by products such as sterols.The genomic, transcriptomic and proteomic data described above, indicate it is necessary to increase the carbon flux towards astaxanthin biosynthesis. Knowing that the mevalonate pathway and its isoprenoid products feed carotenoid biosynthesis , 49, 51,ecessary –59, 61. X. dendrorhous activates the entire metabolic machinery for carotenoid/astaxanthin biosynthesis, a process that results from the inactivation of MIG1 and, subsequently, increase of crtYB and crtS expression levels [Among other important considerations, decrease of catabolic repression in n levels , 111.OLE1) has been shown to increase the accumulation of substrates for carotenoid biosynthesis [CYP61 increases the substrates availability for astaxanthin biosynthesis and inhibition by sterols is evaded [Overexpressing stearoyl-CoA 9-desaturase  and glutathione peroxidase, may further induce astaxanthin biosynthesis. In X. dendrorhous, four copies of CTT1 have been found [S. cerevisiae, it has been found a great diversity of glutathione peroxidases encoded by the genes URE2, GPX1, GPX2, GTT1 and/or hyr1p [The presence of oxygen, including reactive oxygen species such as Hl damage , 123–126X. dendrorhous and thereby promote the biosynthesis of carotenoids and astaxanthin is to keep the biological system under constant oxidative stress throughout the fermentation process. Taking advantage of the knowledge gained about the addition of H2O2 as an inducer of the production of astaxanthin [2O2 autonomously. Within the genome of basidiomycota fungi, the superfamily of glucose-methanol-choline (GMC) oxidoreductases includes enzymes that produce extracellular H2O2 involved in the degradation of structural plant polysaccharides such as lignin, cellulose and hemicellulose [GDH), ethanol/methanol oxidase (MOX), aryl-alcohol oxidase (AAO), pyranose 2-oxidase (P2O), pyranose dehydrogenase (PDH) and glyoxal oxidase (GLX). Heterologously expressing some of these genes in X. dendrorhous and, in addition, knocking out the reductases  necessary to avoid oxidative damage, could favor the overproduction of astaxanthin.Another potentially powerful strategy to favor secondary metabolism in axanthin , it is pellulose , 128. WiX. dendrorhous strains for carotenoids production in high yields [As discussed above, there are a number of different strategies to obtain genetically engineered h yields , 61, 66.h yields , 62, 74.X. dendrorhous strains increase astaxanthin production [Random mutagenesis using physical and chemical agents , 71, 78 oduction . NeverthCYP61 and/or MIG1 (the latter involved in catabolic repression) and, in addition, induce protein domains such as SRE1 N-terminal, indirectly related to the metabolism of carotenoids/astaxanthin [X. dendrorhous to increase the -astaxanthin production. The improvement of astaxanthin overproduction is a request.Genes involved in the carotenoids pathway have been induced to increase astaxanthin biosynthesis, but the results have not been enough. It is necessary to inactivate other genes such axanthin , 121. HoX. dendrorhous (such as those proposed in this review and others) could be implemented and studied to obtain more and new specific information of other regulatory mechanisms involved directly or indirectly to astaxanthin biosynthesis and, with this, achieve a genetic modification strategy capable of producing the compound in high yields.New metabolic engineering strategies on