Behavioral, metabolic, and lipidomic characterization of the 5xFADxTg30 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is associated with both extracellular amyloid-ß (Aß) plaques and intracellular tau-containing neurofibrillary tangles (NFT). We characterized the behavioral, metabolic and lipidomic phenotype of the 5xFADxTg30 mouse model which contains overexpression of both Aß and tau. Our results independently reproduce several phenotypic traits described previously for this model, while providing additional characterization. This model develops many aspects associated with AD including frailty, decreased survival, initiation of aspects of cognitive decline and alterations to specific lipid classes and molecular lipid species in the plasma and brain. Notably, some sex-specific differences exist in this model and motor impairment with aging in this model does compromise the utility of the model for some movement-based behavioral assessments of cognitive function. These findings provide a reference for individuals interested in using this model to understand the pathology associated with elevated Aß and tau or for testing potential therapeutics for the treatment of AD.

Desiccation induces varied responses within a soil bacterial genus.

Desiccation impacts a suite of physiological processes in microbes by elevating levels of damaging reactive oxygen species and inducing DNA strand breaks. In response to desiccation-induced stress, microbes have evolved specialized mechanisms to help them survive. Here, we performed a 128-day lab desiccation experiment on nine strains from three clades of an abundant soil bacterium, Curtobacterium. We sequenced RNA from each strain at three time points to investigate their response. Curtobacterium was highly resistant to desiccation, outlasting both Escherichia coli and a famously DNA damage-resistant bacterium, Deinococcus radiodurans. However, within the genus, there were also 10-fold differences in survival rates among strains. Transcriptomic profiling revealed responses shared within the genus including up-regulation of genes involved in DNA damage repair, osmolyte production, and efflux pumps, but also up-regulation of pathways and genes unique to the three clades. For example, trehalose synthesis gene otsB, the chaperone groEL, and the oxygen scavenger katA were all found in either one or two clades but not the third. Here, we provide evidence of considerable variation in closely related strains, and further elucidation of the phylogenetic conservation of desiccation tolerance remains an important goal for microbial ecologists.

Novel imaging techniques in refractory pituitary adenomas.

Accurate localization of the site(s) of active disease is key to informing decision-making in the management of refractory pituitary adenomas when autonomous hormone secretion and/or continued tumor growth challenge conventional therapeutic approaches. In this context, the use of non-standard MR sequences, alternative post-acquisition image processing, or molecular (functional) imaging may provide valuable additional information to inform patient management.

Association between asymptomatic hyperuricemia and knee osteoarthritis in older outpatients.

OBJECTIVE: To investigate the association between asymptomatic hyperuricemia and knee osteoarthritis in older outpatients in Vietnam. PATIENTS AND METHODS: This cross-sectional study included 257 older outpatients (195 in the knee osteoarthritis group and 62 in the non-knee osteoarthritis group) aged ≥60 years (mean age 73.31 ± 7.96 years) attending rheumatologic and geriatric clinics from November 2020 to May 2021. Data were collected for both groups, including demographics, symptoms and signs of knee osteoarthritis, serum uric acid levels, and knee radiographs. The association between asymptomatic hyperuricemia and knee osteoarthritis was assessed using logistic regression. RESULTS: The mean serum uric acid level among patients with knee osteoarthritis was higher than that among patients without knee osteoarthritis (6.3 ± 1.74 mg/dl vs. 5.71 ± 1.45 mg/dl, p = 0.017). Hyperuricemia was more common among older outpatients with knee osteoarthritis than among those without knee osteoarthritis (39% vs. 19%, p = 0.005). After adjusting for age, sex, body mass index (BMI), and other comorbidities, the association between asymptomatic hyperuricemia and knee osteoarthritis remained significant (odds ratio [OR] 2.61, 95% confidence interval [CI] 1.22-5.60, p = 0.013). Subgroup analyses were performed according to sex and BMI groups. Significant associations between asymptomatic hyperuricemia and knee osteoarthritis were observed among women (p = 0.017) and among individuals who were underweight-normal-weight according to BMI (p = 0.009). CONCLUSIONS: Asymptomatic hyperuricemia is a common comorbidity among older outpatients with knee osteoarthritis. An independent association was identified between asymptomatic hyperuricemia and knee osteoarthritis among older Vietnamese outpatients, although sex and BMI may be confounding factors that impact this association.

Development of a LC-MS/MS method to measure serum 3-sulfate and 3-glucuronide 25-hydroxyvitamin D3 metabolites; comparisons to unconjugated 25OHD in pregnancy and polycystic ovary syndrome.

Vitamin D status is routinely assessed by measuring circulating concentrations of 25-hydroxyvitamin D (25OHD2 or 25OHD3). However as deconjugation is not routinely incorporated into sample treatment prior to analysis, conjugated forms of 25OHD (particularly the more abundant 25OHD3) are often not considered in determining serum concentrations of total 25OHD. Two major circulating conjugated forms of 25OHD3 are 25-hydroxyvitamin D3-3-sulfate (25OHD3-S) and 25-hydroxyvitamin D3-3-glucuronide (25OHD3-G). Incorporating these two conjugated metabolites into the measurement of vitamin D status could improve our understanding of vitamin D status in health, particularly if there are changes in sulfation and glucuronidation activities. The aim of this study was to develop a liquid chromatography tandem-mass spectrometry (LC-MS/MS) targeted method for measurement of 25OHD3-S and 25OHD3-G in serum to enable comparisons with circulating levels of the free 25OHD3 form. We developed and validated a new LC-MS/MS method that measured both 25OHD3-S and 25OHD3-G following a solid phase extraction sample preparation method. Partial separation of analytes by LC, and the separation of analytes by the optimized multiple reaction monitoring transitions enabled the quantitation of both 25OHD3-S and 25OHD3-G in the single method. Serum concentrations of 25OHD3-S (24.7 ± 11.8 ng/mL) and 25OHD3-G (2.4 ± 1.2 ng/mL) were shown to be a significant proportion of circulating vitamin D metabolites in healthy donor serums. These levels of 25OHD3-S and 25OHD3-G closely associated with 25OHD3 concentrations, r = 0.728, p = 0.001 and r = 0.632, p = 0.006 respectively. However in serum from pregnant women and non-pregnant women with polycystic ovary syndrome (PCOS) significant differences in the ratios between conjugated and free 25OHD3 were observed between pregnancy groups (25OHD3/25OHD3-S and 25OHD3/25OHD3-G p < 0.001), and between healthy and PCOS subjects (25OHD3/25OHD3-G p < 0.050). Development of this novel high-throughput LC-MS/MS method indicates that 25OHD3-S and 25OHD3-G are substantial components of circulating vitamin D metabolites. The concentrations of these metabolites relative to conventional 25OHD3 may vary in different physiological and pathophysiological settings, and may therefore play an unrecognized but important role in the actions of vitamin D.

Exceptional human longevity is associated with a specific plasma phenotype of ether lipids.

A lipid profile resistant to oxidative damage is an inherent trait associated with animal lifespan. However, there is a lack of lipidomic studies on human longevity. Here we use mass spectrometry based technologies to detect and quantify 137 ether lipids to define a phenotype of healthy humans with exceptional lifespan. Ether lipids were chosen because of their antioxidant properties and ability to modulate oxidative stress. Our results demonstrate that a specific ether lipid signature can be obtained to define the centenarian state. This profile comprises higher level of alkyl forms derived from phosphatidylcholine with shorter number of carbon atoms and double bonds; and decreased content in alkenyl forms from phosphatidylethanolamine with longer chain length and higher double bonds. This compositional pattern suggests that ether lipids from centenarians are more resistant to lipid peroxidation, and that ether lipid signature expresses an optimized feature associated with exceptional human longevity. These results are in keeping with the free radical theory of aging.

Assessment of Cognitive Impairment in Patients with Chronic Obstructive Pulmonary Disease Using the Rapid Cognitive Screen.

OBJECTIVES: To determine the prevalence of cognitive impairment in chronic obstructive pulmonary disease (COPD) patients using the Rapid Cognitive Screen (RCS). DESIGN: Cross sectional study. PARTICIPANTS: A total of 106 men and women with COPD 50 years of age or older. SETTING: Pulmonary ambulatory clinic in a tertiary academic center in St. Louis, Missouri, USA. MEASUREMENTS: We enrolled patients with COPD who completed the RCS tool. Patients were classified as having normal cognition, mild cognitive impairment (MCI), or dementia if they scored ≥ 8, 6-7, or ≤ 5, respectively. The prevalence of each category was measured, and patients' baseline characteristics were compared. RESULTS: One hundred and six patients completed the RCS. Of those, 36 (33.9%) patients scored ≤ 5 on the RCS, 33 (31.1%) scored 6 or 7, and 37 (34.9%) scored ≥ 8. The prevalence of dementia, MCI, and normal cognition were 33.9%, 31.1%, and 34.9%, respectively. Baseline lung function was not different between patients with normal cognition and those with abnormal cognition (FEV1 1.53 ± 0.39 vs 1.45 ± 0.32 L). In patients with abnormal cognition, there was no difference in patients' characteristics between patients with MCI or dementia. CONCLUSION: Cognitive impairment is highly prevalent in patients with COPD. RCS successfully identified cognitive impairment in COPD. We recommend performing RCS in patients with COPD routinely.

The lipidome in major depressive disorder: Shared genetic influence for ether-phosphatidylcholines, a plasma-based phenotype related to inflammation, and disease risk.

BACKGROUND: The lipidome is rapidly garnering interest in the field of psychiatry. Recent studies have implicated lipidomic changes across numerous psychiatric disorders. In particular, there is growing evidence that the concentrations of several classes of lipids are altered in those diagnosed with MDD. However, for lipidomic abnormalities to be considered potential treatment targets for MDD (rather than secondary manifestations of the disease), a shared etiology between lipid concentrations and MDD should be demonstrated. METHODS: In a sample of 567 individuals from 37 extended pedigrees (average size 13.57 people, range=3-80), we used mass spectrometry lipidomic measures to evaluate the genetic overlap between twenty-three biologically distinct lipid classes and a dimensional scale of MDD. RESULTS: We found that the lipid class with the largest endophenotype ranking value (ERV, a standardized parametric measure of pleiotropy) were ether-phosphodatidylcholines (alkylphosphatidylcholine, PC(O) and alkenylphosphatidylcholine, PC(P) subclasses). Furthermore, we examined the cluster structure of the twenty-five species within the top-ranked lipid class, and the relationship of those clusters with MDD. This analysis revealed that species containing arachidonic acid generally exhibited the greatest degree of genetic overlap with MDD. CONCLUSIONS: This study is the first to demonstrate a shared genetic etiology between MDD and ether-phosphatidylcholine species containing arachidonic acid, an omega-6 fatty acid that is a precursor to inflammatory mediators, such as prostaglandins. The study highlights the potential utility of the well-characterized linoleic/arachidonic acid inflammation pathway as a diagnostic marker and/or treatment target for MDD.

Development and validation of a discretised multi-body spine model in LifeMOD for biodynamic behaviour simulation.

This paper presents a discretised musculoskeletal multi-body spine model using the LifeMOD Biomechanics Modeller. This was obtained by refining spine segments in cervical, thoracic and lumbar regions into individual vertebra segments, using rotational joints representing the intervertebral discs, building various ligaments between vertebrae and implementing necessary lumbar muscles. To validate the model, two comparison studies were made with in vivo intradiscal pressure measurements of the L4-L5 disc as well as extension moments, axial force and shear force around L5-S1 obtained from spine models available in the literature. The results indicated that the present model is in good correlation with both cases and matches well with experimental data which found that the axial forces are in the range of 3929-4688 N and shear forces up to 650 N. This study provides a preliminary overview of our ongoing work towards building bio-fidelity discretised multi-body spine models for investigating various medical applications. These models can be useful for incorporation into design tools for wheelchairs or other seating systems which may require attention to ergonomics as well as assessing biomechanical behaviour between natural spines and spinal arthroplasty or spinal arthrodesis. Furthermore, these models can be combined with haptic-integrated graphic environments to help surgeons to examine kinematic behaviours of scoliotic spines and to propose possible surgical plans before spine correction operations.

A genome-wide survey and functional brain imaging study identify CTNNBL1 as a memory-related gene.

Unbiased genome-wide screens combined with imaging data on brain function may identify novel molecular pathways related to human cognition. Here we performed a dense genome-wide screen to identify episodic memory-related gene variants. A genomic locus encoding the brain-expressed beta-catenin-like protein 1 (CTNNBL1) was significantly (P=7 × 10(-8)) associated with verbal memory performance in a cognitively healthy cohort from Switzerland (n=1073) and was replicated in a second cohort from Serbia (n=524; P=0.003). Gene expression studies showed CTNNBL1 genotype-dependent differences in beta-catenin-like protein 1 mRNA levels in the human cortex. Functional magnetic resonance imaging in 322 subjects detected CTNNBL1 genotype-dependent differences in memory-related brain activations. Converging evidence from independent experiments and different methodological approaches suggests a role for CTNNBL1 in human memory.

Enhanced phosphoinositide 3-kinase(p110α) activity prevents diabetes-induced cardiomyopathy and superoxide generation in a mouse model of diabetes.

AIMS/HYPOTHESIS: Diabetic cardiomyopathy is characterised by diastolic dysfunction, oxidative stress, fibrosis, apoptosis and pathological cardiomyocyte hypertrophy. Phosphoinositide 3-kinase (PI3K)(p110α) is a cardioprotective kinase, but its role in the diabetic heart is unknown. The aim of this study was to assess whether PI3K(p110α) plays a critical role in the induction of diabetic cardiomyopathy, and whether increasing PI3K(p110α) activity in the heart can prevent the development of cardiac dysfunction in a setting of diabetes. METHODS: Type 1 diabetes was induced with streptozotocin in adult male cardiac-specific transgenic mice with increased PI3K(p110α) activity (constitutively active PI3K [p110α], caPI3K] or decreased PI3K(p110α) activity (dominant-negative PI3K [p110α], dnPI3K) and non-transgenic (Ntg) mice for 12 weeks. Cardiac function, histological and molecular analyses were performed. RESULTS: Diabetic Ntg mice displayed diastolic dysfunction and increased cardiomyocyte size, expression of atrial and B-type natriuretic peptides (Anp, Bnp), fibrosis and apoptosis, as well as increased superoxide generation and increased protein kinase C ß2 (PKCß2), p22 ( phox ) and apoptosis signal-regulating kinase 1 (Ask1) expression. Diabetic dnPI3K mice displayed an exaggerated cardiomyopathy phenotype compared with diabetic Ntg mice. In contrast, diabetic caPI3K mice were protected against diastolic dysfunction, pathological cardiomyocyte hypertrophy, fibrosis and apoptosis. Protection in diabetic caPI3K mice was associated with attenuation of left ventricular superoxide generation, attenuated Anp, Bnp, PKCß2, Ask1 and p22 ( phox ) expression, and elevated AKT. Further, in cardiomyocyte-like cells, increased PI3K(p110α) activity suppressed high glucose-induced superoxide generation and enhanced mitochondrial function. CONCLUSIONS/INTERPRETATION: These results demonstrate that reduced PI3K activity accelerates the development of diabetic cardiomyopathy, and that enhanced PI3K(p110α) activity can prevent adverse cardiac remodelling and dysfunction in a setting of diabetes.

Coenzyme Q10 attenuates diastolic dysfunction, cardiomyocyte hypertrophy and cardiac fibrosis in the db/db mouse model of type 2 diabetes.

AIMS/HYPOTHESIS: An increase in the production of reactive oxygen species is commonly thought to contribute to the development of diabetic cardiomyopathy. This study aimed to assess whether administration of the antioxidant coenzyme Q(10) would protect the diabetic heart against dysfunction and remodelling, using the db/db mouse model of type 2 diabetes. Furthermore, we aimed to compare the efficacy of coenzyme Q(10) to that of the ACE inhibitor ramipril. METHODS: Six-week-old non-diabetic db/+ mice and diabetic db/db mice received either normal drinking water or water supplemented with coenzyme Q(10) for 10 weeks. Endpoint cardiac function was assessed by echocardiography and catheterisation. Ventricular tissue was collected for histology, gene expression and protein analysis. RESULTS: Untreated db/db diabetic mice exhibited hyperglycaemia, accompanied by diastolic dysfunction and adverse structural remodelling, including cardiomyocyte hypertrophy, myocardial fibrosis and increased apoptosis. Systemic lipid peroxidation and myocardial superoxide generation were also elevated in db/db mice. Coenzyme Q(10) and ramipril treatment reduced superoxide generation, ameliorated diastolic dysfunction and reduced cardiomyocyte hypertrophy and fibrosis in db/db mice. Phosphorylation of Akt, although depressed in untreated db/db mice, was restored with coenzyme Q(10) administration. We postulate that preservation of cardioprotective Akt signalling may be a mechanism by which coenzyme Q(10)-treated db/db mice are protected from pathological cardiac hypertrophy. CONCLUSIONS/INTERPRETATION: These data demonstrate that coenzyme Q(10) attenuates oxidative stress and left ventricular diastolic dysfunction and remodelling in the diabetic heart. Addition of coenzyme Q(10) to the current therapy used in diabetic patients with diastolic dysfunction warrants further investigation.

Aberrant hypermethylation in primary tumours and sentinel lymph node metastases in paediatric patients with cutaneous melanoma.

BACKGROUND: Debate on how to manage paediatric patients with cutaneous melanoma continues, particularly in those with sentinel lymph node (SLN) metastases who are at higher risk of poor outcomes. Management is often based on adult algorithms, although differences in clinical outcomes between paediatric and adult patients suggest that melanoma in paediatric patients differs biologically. Yet, there are no molecular prognostic studies identifying these differences. OBJECTIVES: We investigated the epigenetic (methylation) regulation of several tumour-related genes (TRGs) known to be significant in adult melanoma progression in histopathology(+) SLN metastases (n = 17) and primary tumours (n = 20) of paediatric patients with melanoma to determine their clinical relevance. METHODS: Paediatric patients (n = 37; ≤ 21 years at diagnosis) with American Joint Committee on Cancer stage I-III cutaneous melanoma were analysed. Gene promoter methylation of the TRGs RASSF1A, RARß2, WIF1 and APC was evaluated. RESULTS: Hypermethylation of RASSF1A, RARß2, WIF1 and APC was found in 29% (5/17), 25% (4/16), 25% (4/16) and 19% (3/16) of histopathology(+) SLNs, respectively. When matched to adult cutaneous melanomas by Breslow thickness and ulceration, hypermethylation of all four TRGs in SLN(+) paediatric patients with melanoma was equivalent to or less than in adults. With a median follow-up of 55 months, SLN(+) paediatric patients with melanoma with hypermethylation of > 1 TRG vs. ≤ 1 TRG had worse disease-free (P = 0·02) and overall survival (P = 0·02). CONCLUSIONS: Differences in the methylation status of these TRGs in SLN(+) paediatric and adult patients with melanoma may account for why SLN(+) paediatric patients have different clinical outcomes. SLN biopsy should continue to be performed; within SLN(+) paediatric patients with melanoma, hypermethylation of TRGs can be used to identify a subpopulation at highest risk for poor outcomes who warrant vigilant clinical follow-up.

A genome-wide survey of human short-term memory.

Recent advances in the development of high-throughput genotyping platforms allow for the unbiased identification of genes and genomic sequences related to heritable traits. In this study, we analyzed human short-term memory, which refers to the ability to remember information over a brief period of time and which has been found disturbed in many neuropsychiatric conditions, including schizophrenia and depression. We performed a genome-wide survey at 909 622 polymorphic loci and report six genetic variations significantly associated with human short-term memory performance after genome-wide correction for multiple comparisons. A polymorphism within SCN1A (encoding the α subunit of the type I voltage-gated sodium channel) was replicated in three independent populations of 1699 individuals. Functional magnetic resonance imaging during an n-back working memory task detected SCN1A allele-dependent activation differences in brain regions typically involved in working memory processes. These results suggest an important role for SCN1A in human short-term memory.

Complete intracellular pH protection during extracellular pH depression is associated with hypercarbia tolerance in white sturgeon, Acipenser transmontanus.

Sturgeons are among the most CO2 tolerant of fishes investigated to date. However, the basis of this exceptional CO2 tolerance is unknown. Here, white sturgeon, Acipenser transmontanus, were exposed to elevated CO2 to investigate the mechanisms associated with short-term hypercarbia tolerance. During exposure to 1.5 kPa Pco2, transient blood pH [extracellular pH (pHe)] depression was compensated within 24 h and associated with net plasma HCO3- accumulation and equimolar Cl- loss, and changes in gill morphology, such as a decrease in apical surface area of mitochondrial-rich cells. These findings indicate that pHe recovery at this level of hypercarbia is accomplished in a manner similar to most freshwater teleost species studied to date, although branchial mechanisms involved may differ. White sturgeon exposed to more severe hypercarbia (3 and 6 kPa Pco2) for 48 h exhibited incomplete pH compensation in blood and red blood cells. Despite pHe depression, intracellular pH (pHi) of white muscle, heart, brain, and liver did not decrease during a transient (6 h of 1.5 kPa Pco2) or prolonged (48 h at 3 and 6 kPa Pco2 blood acidosis. This pHi protection was not due to high intrinsic buffering in tissues. Such tight active cellular regulation of pHi in the absence of pHe compensation represents a unique pattern for non-air-breathing fishes, and we hypothesize that it is the basis for the exceptional CO2 tolerance of white sturgeon and, likely, other CO2 tolerant fishes. Further research to elucidate the specific mechanisms responsible for this tremendous pH regulatory capacity in tissues of white sturgeon is warranted.

Phagosomal acidification: measurement, manipulation and functional consequences.

Phagocytosis holds a central position in the development of a successful innate immune response and in the initiation of the corresponding adaptive response. The destruction of invading pathogens and the presentation of their antigens to lymphoid cells require acidification of the phagosomal lumen. The present review discusses the mechanism of phagosome acidification, with particular reference to the two components of the protonmotive force: the chemical (pH) gradient and the electrical potential across the phagosomal membrane. A method for the in situ measurement of the electrical potential across the phagosomal membrane is described. In addition, we discuss the finding that acidification is not only a consequence, but also a critical determinant of phagosome maturation. Luminal acidification appears to function as a timing device controlling the transition between early and late phagosomes.

Evaluation of an independent prestin mouse model derived from the 129S1 strain.

Studies using the prestin knockout mouse indicate that removal of the outer hair cell (OHC) motor protein is associated with loss of sensitivity, frequency selectivity and somatic electromotility. Here we provide data obtained from another prestin mouse model that was produced commercially. In vivo electrical recordings from the round window indicate that the phenotype is similar to that of the original knockout generated by the Zuo group at St. Jude Children's Research Hospital. Hence, compound action potential (CAP) thresholds are shifted in a frequency-dependent manner and CAP tuning curves at 12 kHz are flat for masker frequencies between 3 and 18 kHz. Although CAP input-output functions at 6 kHz show a shift in sensitivity at low levels, responses approach wild-type magnitudes at high levels where the cochlear amplifier has less influence. In order to confirm that the loss of sensitivity and frequency selectivity is due to loss of prestin, we performed immunohistochemistry using a prestin antibody. Cochlear segments from homozygous mutant mice showed no fluorescence, while wild-type mice displayed a fluorescent signal targeted to the OHC's lateral membrane. Absence of prestin protein was confirmed using LDS-PAGE/Western blot analysis. These results indicate that the loss of function phenotype is associated with loss of prestin protein. Lack of prestin protein also results in a shortening of OHC length to approximately 60% of wild-type, similar to that reported previously by Liberman's group. The linkage shown between the loss of prestin protein and abnormal cochlear function validates the original knockout and attests to the importance of OHC motor function in the auditory periphery.

Cochlear function in mice with only one copy of the prestin gene.

Targeted deletion of the prestin gene reduces cochlear sensitivity and eliminates both frequency selectivity and outer hair cell (OHC) somatic electromotility. In addition, it has been reported by Liberman and colleagues that F2 generation heterozygotes exhibit a 6 dB reduction in sensitivity, as well as a decrease in protein and electromotility. Considering that the active process is non-linear, a halving of somatic electromotility would be expected to produce a much larger change in sensitivity. We therefore re-evaluated comparisons between heterozygotes and wildtype mice using both in vivo and in vitro electrophysiology, as well as molecular biology. Data reported here for F3-F5 generation mice indicate that compound action potential thresholds and tuning curves, as well as the cochlear microphonic, are similar in heterozygotes and wildtype controls. Measurements of non-linear capacitance in isolated OHCs demonstrate that charge density, as well as the voltage dependence and sensitivity of motor function, is indistinguishable in the two genotypes, as is somatic electromotility. In addition, both immunocytochemistry and western blot analysis in young adult mice suggest that prestin protein in heterozygotes is near normal. In contrast, prestin mRNA is always less than in wildtype mice at all ages tested. Results from F3-F5 generation mice suggest that one copy of the prestin gene is capable of compensating for the deleted copy and that heterozygous mice do not suffer peripheral hearing impairment.