Increased activity of Diaphanous homolog 3 (DIAPH3)/diaphanous causes hearing defects in humans with auditory neuropathy and in Drosophila

Auditory neuropathy is a rare form of deafness characterized by an absent or abnormal auditory brainstem response with preservation of outer hair cell function. We have identified Diaphanous homolog 3 (DIAPH3) as the gene responsible for autosomal dominant nonsyndromic auditory neuropathy (AUNA1), which we previously mapped to chromosome 13q21-q24. Genotyping of additional family members narrowed the interval to an 11-Mb, 3.28-cM gene-poor region containing only four genes, including DIAPH3. DNA sequencing of DIAPH3 revealed a c.-172G > A, g. 48G > A mutation in a highly conserved region of the 5′ UTR. The c.-172G > A mutation occurs within a GC box sequence element and was not found in 379 controls. Using genome-wide expression arrays and quantitative RT-PCR, we demonstrate a 2- to 3-fold overexpression of DIAPH3 mRNA in lymphoblastoid cell lines from affected individuals. Likewise, a significant increase (≈1.5-fold) in DIAPH3 protein was found by quantitative immunoblotting of lysates from lymphoblastoid cell lines derived from affected individuals in comparison with controls. In addition, the c.-172G > A mutation is sufficient to drive overexpression of a luciferase reporter. Finally, the expression of a constitutively active form of diaphanous protein in the auditory organ of Drosophila melanogaster recapitulates the phenotype of impaired response to sound. To date, only two genes, the otoferlin gene OTOF and the pejvakin gene PJVK, are known to underlie nonsyndromic auditory neuropathy. Genetic testing for DIAPH3 may be useful for individuals with recessive as well as dominant inheritance of nonsyndromic auditory neuropathy.     

Early-life co-administration of cockroach allergen and endotoxin augments pulmonary and systemic responses

Background Environmental exposures to cockroach allergen and endotoxin are recognized epidemiological risk factors for the early development of allergies and asthma in children. Because of this, it is important to examine the role of early-life concurrent inhalation exposures to cockroach allergen and endotoxin in the pathogenesis of allergic airways disease.
Objective We examined the effects of repeated concomitant endotoxin and cockroach allergen inhalation on the pulmonary and systemic immune responses of newborn and juvenile mice.
Methods C3H/HeBFeJ mice were exposed to inhaled endotoxin and cockroach allergen via intranasal instillation from day 2 to 21 after birth, and systemic and pulmonary responses were examined in serum, bronchoalveolar lavage fluid, and lung tissue.
Results Cockroach allergen exposures induced pulmonary eosinophilic inflammation, total and allergen-specific IgE, IgG₁, and IgG_2a production, and alveolar remodelling. Co-exposures with endotoxin and cockroach allergen significantly increased serum IgE and IgG₁, lung inflammation, and alveolar wall thickness, and decreased airspace volume density. Importantly, compared with exposures with individual substances, the responses to co-exposures were more than additive.
Conclusions Repeated inhalation exposures of neonatal and juvenile mice to endotoxin and cockroach allergen increased the pulmonary inflammatory and systemic immune responses in a synergistic manner and enhanced alveolar remodelling in the developing lung. These data underscore the importance of evaluating the effect of multiple, concurrent environmental exposures, and of using an experimental model that incorporates clinically relevant timing and route of exposures.     

Adenosine 5′-triphosphate (ATP) concentrations in the endolymph and perilymph of the guinea-pig cochlea

The concentration of adenosine 5′-triphosphate (ATP) in endolymph (EL), perilymph (PL) and cerebrospinal fluid (CSF), collected from anesthetized guinea pigs was determined using the luciferase-luciferin reaction. The cochlea was exposed by a ventrolateral approach and the bone overlying scala media of the third turn (EL) or scala vestibuli of the first turn (PL) was shaved to a thin layer and a small fenestrum made. For EL sampling, a double-barrelled pipette was inserted through the spiral ligament-stria vascularis complex. One barrel was filled with 150 mM KCl to record the endocochlear potential (EP) and upon the appearance of the positive EP, 0.12–1.22 μl of fluid was aspirated into the other barrel by gentle negative pressure. For PL sampling, a single-barrelled pipette was advanced into scala vestibuli and 0.3–1.6 μl of fluid was collected by capillarity. CSF (0.36–1.75 μl) was obtained from the cisterna magna. The cochleae were removed and processed for light microscopy to determine the extent of tissue damage from the sampling procedure. ATP concentrations (mean ± SEM, nM) for EL, PL and CSF were 12.95±2.4 (n = 10), 10.5 ± 3.9 (n = 11) and 16.1 ± 5.4 (n =11) respectively. Differences in ATP concentrations among fluids were not statistically significant. To test the effect of hypoxia on ATP levels, a group of guinea pigs was subjected to a90 s period of respiratory anoxia prior to sampling of EL, PL or CSF. ATP concentrations were 14.4 ± 3.5 (n = 11), 20.7 ± 4.1 (n = 10) and 13.5 ± 4.6 (n = 4) for EL, PL and CSF, respectively; only PL ATP concentrations were statistically different (P = 0.018, Wilcoxon rank sum test) to basal conditions. This is the first study which demonstrates the presence of free ATP in cochlear fluids. The results indicate that ATP is present in cochlear fluids at concentrations close to those known to cause hair cell depolarization in vitro.     

Chronic illness experience: insights from a metastudy

Concurrent with the recent enthusiasm for qualitative research in the health fields, an energetic call for methods by which to synthesize the knowledge has been generated on various substantive topics. Although there is an emerging literature on meta-analysis and metasynthesis, many authors overestimate the simplicity of such approaches and erroneously assume that useful knowledge can be synthesized from limited collections of study reports without a thorough analysis of their theoretical, methodological, and contextual foundations and features. In this article, the authors report some of the insights obtained from an extensive and exhaustive metastudy of qualitative studies of chronic illness experience. Their findings reveal the complexities inherent not only in any phenomenon of interest to health researchers but also in the study of how we have come to know what we think we know about it.