Tmc1 point mutation affects Ca2+ sensitivity and block by dihydrostreptomycin of the mechanoelectrical transducer current of mouse outer hair cells

The transduction of sound into electrical signals depends on mechanically sensitive ion channels in the stereociliary bundle. The molecular composition of this mechanoelectrical transducer (MET) channel is not yet known. Transmembrane channel-like protein isoforms 1 (TMC1) and 2 (TMC2) have been proposed to form part of the MET channel, although their exact roles are still unclear. Using Beethoven (Tmc1Bth/Bth) mice, which have an M412K point mutation in TMC1 that adds a positive charge, we found that Ca2+ permeability and conductance of the MET channel of outer hair cells (OHCs) were reduced. Tmc1Bth/Bth OHCs were also less sensitive to block by the permeant MET channel blocker dihydrostreptomycin, whether applied extracellularly or intracellularly. These findings suggest that the amino acid that is mutated in Bth is situated at or near the negatively charged binding site for dihydrostreptomycin within the permeation pore of the channel. We also found that the Ca2+ dependence of the operating range of the MET channel was altered by the M412K mutation. Depolarization did not increase the resting open probability of the MET current of Tmc1Bth/Bth OHCs, whereas raising the intracellular concentration of the Ca2+ chelator BAPTA caused smaller increases in resting open probability in Bth mutant OHCs than in wild-type control cells. We propose that these observations can be explained by the reduced Ca2+ permeability of the mutated MET channel indirectly causing the Ca2+ sensor for adaptation, at or near the intracellular face of the MET channel, to become more sensitive to Ca2+ influx as a compensatory mechanism

Calcium entry into stereocilia drives adaptation of the mechanoelectrical transducer current of mammalian cochlear hair cells

Mechanotransduction in the auditory and vestibular systems depends on mechanosensitive ion channels in the stereociliary bundles that project from the apical surface of the sensory hair cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the excitatory direction, Ca2+ entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca2+-dependent adaptation is also present in mammalian hair cells. Hair bundles of both outer and inner hair cells from mice were deflected by using sinewave or step mechanical stimuli applied using a piezo-driven fluid jet. We found that when cochlear hair cells were depolarized near the Ca2+ reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca2+ concentration (40 µM), all manifestations of adaptation, including the rapid decline of the MET current and the reduction of the available resting MET current, were abolished. MET channel adaptation was also reduced or removed when the intracellular Ca2+ buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) was increased from a concentration of 0.1 to 10 mM. The findings show that MET current adaptation in mouse auditory hair cells is modulated similarly by extracellular Ca2+, intracellular Ca2+ buffering, and membrane potential, by their common effect on intracellular free Ca2+. Hearing and balance depend on the transduction of mechanical stimuli into electrical signals. This process depends on the opening of mechanoelectrical transducer (MET) channels located at the tips of the shorter of pairs of adjacent stereocilia (1), which are specialized microvilli-like structures that form the hair bundles that project from the upper surface of hair cells (2,3). Deflection of hair bundles in the excitatory direction (i.e., toward the taller stereocilia) stretches specialized linkages, the tip-links, present between adjacent stereocilia (3⇓–5), opening the MET channels. In hair cells from lower vertebrates, open MET channels reclose during constant stimuli via an initial fast adaptation mechanism followed by a much slower, myosin-based motor process, both of which are driven by Ca2+ entry through the channel itself (6⇓⇓⇓⇓⇓⇓–13). In mammalian auditory hair cells, MET current adaptation seems to be mainly driven by the fast mechanism (14⇓–16), although the exact process by which it occurs is still largely unknown. The submillisecond speed associated with the adaptation kinetics of the MET channels in rat and mouse cochlear hair cells (17, 18) indicates that Ca2+, to cause adaptation, has to interact directly with a binding site on the channel or via an accessory protein (16). However, a recent investigation on rat auditory hair cells has challenged the view that Ca2+ entry is required for fast adaptation, and instead proposed an as-yet-undefined mechanism involving a Ca2+-independent reduction in the viscoelastic force of elements in series with the MET channels (19). In the present study, we further investigated the role of Ca2+ in MET channel adaptation in mouse cochlear hair cells by deflecting their hair bundles using a piezo-driven fluid jet, which is believed to produce a more uniform deflection of the hair bundles (20⇓⇓–23) compared with the piezo-driven glass rod (19, 24)

Automated cold vapour flow-injection analysis of mercury at high concentrations

Continuous-flow cold vapour- atomic fluorescence spectrometry is shown to be an extremely sensitive technique for the determination of mercury with detection limits typically below 0.01 μg l-1. Linear calibration ranges were found to be at least four orders of magnitude (i.e. up to 0.1 mg l-1). Samples with concentrations exceeding the linear range are susceptible to self-absorption, and may, in severe cases, cause carry-over problems between samples. The flow-injection approach has been utilized to extend the upper limit of the linear calibration range allowing determinations up to 10 mg l-1 of mercury. A range of certified reference materials and zinc battery anodes have been successfully analysed with a minimal number of sample dilutions

Generation of otic lineages from integration-free human-induced pluripotent stem cells reprogrammed by mRNAs

Damage to the sensory hair cells and the spiral ganglion neurons of the cochlea leads to deafness. Induced pluripotent stem cells (iPSCs) are a promising tool to regenerate the cells in the inner ear that have been affected by pathology or have been lost. To facilitate the clinical application of iPSCs, the reprogramming process should minimize the risk of introducing undesired genetic alterations while conferring the cells the capacity to differentiate into the desired cell type. Currently, reprogramming induced by synthetic mRNAs is considered to be one of the safest ways of inducing pluripotency, as the transgenes are transiently delivered into the cells without integrating into the genome. In this study, we explore the ability of integration-free human-induced pluripotent cell lines that were reprogrammed by mRNAs, to differentiate into otic progenitors and, subsequently, into hair cell and neuronal lineages. hiPSC lines were induced to differentiate by culturing them in the presence of fibroblast growth factors 3 and 10 (FGF3 and FGF10). Progenitors were identified by quantitative microscopy, based on the coexpression of otic markers PAX8, PAX2, FOXG1, and SOX2. Otic epithelial progenitors (OEPs) and otic neuroprogenitors (ONPs) were purified and allowed to differentiate further into hair cell-like cells and neurons. Lineages were characterised by immunocytochemistry and electrophysiology. Neuronal cells showed inward Na+ () currents and outward () and inward K+ () currents while hair cell-like cells had inward and outward delayed rectifier K+ currents, characteristic of developing hair cells. We conclude that human-induced pluripotent cell lines that have been reprogrammed using nonintegrating mRNAs are capable to differentiate into otic cell types

Developing Historic Building Information Modelling Guidelines and Procedures for Architectural Heritage in Ireland

Cultural heritage researchers have recently begun applying Building Information Modelling (BIM) to historic buildings. The model is comprised of intelligent objects with semantic attributes which represent the elements of a building structure and are organised within a 3D virtual environment. Case studies in Ireland are used to test and develop the suitable systems for (a) data capture/digital surveying/processing (b) developing library of architectural components and (c) mapping these architectural components onto the laser scan or digital survey to relate the intelligent virtual representation of a historic structure (HBIM). While BIM platforms have the potential to create a virtual and intelligent representation of a building, its full exploitation and use is restricted to narrow set of expert users with access to costly hardware, software and skills. The testing of open BIM approaches in particular IFCs and the use of game engine platforms is a fundamental component for developing much wider dissemination. The semantically enriched model can be transferred into a WEB based game engine platform

Do young children get the message? The effects of repeated video viewing on explicit and implicit information

The aim of this study was to explore the effects of repeat viewing on comprehension of explicitly and implicitly presented information in an animated movie. Seventy-three pre-school children watched an animated film and were tested for comprehension after either their single or fifth viewing. Only children&rsquo;s comprehension of explicitly presented information was facilitated by repeat viewing. However, post hoc analyses revealed that children&rsquo;s explicit and implicit comprehension of a central character Thunderbolt significantly increased across viewing conditions, whereas, repeat viewing only facilitated children&rsquo;s explicit comprehension of the central character Patch. The theoretical and practical implications of these findings are discussed.<br /

MET currents and otoacoustic emissions from mice with a detached tectorial membrane indicate the extracellular matrix regulates Ca2+ near stereocilia

The tectorial membrane (TM) is an acellular structure of the cochlea that is attached to the stereociliary bundles of the outer hair cells (OHCs), electromotile cells that amplify motion of the cochlear partition and sharpen its frequency selectivity. Although the TM is essential for hearing, its role is still not fully understood. In Tecta/Tectb−/− double knockout mice, in which the TM is not coupled to the OHC stereocilia, hearing sensitivity is considerably reduced compared with that of wild‐type animals. In vivo, the OHC receptor potentials, assessed using cochlear microphonics, are symmetrical in both wild‐type and Tecta/Tectb−/− mice, indicating that the TM does not bias the hair bundle resting position. The functional maturation of hair cells is also unaffected in Tecta/Tectb−/− mice, and the resting open probability of the mechanoelectrical transducer (MET) channel reaches values of ∼50% when the hair bundles of mature OHCs are bathed in an endolymphatic‐like Ca2+ concentration (40 μM) in vitro. The resultant large MET current depolarizes OHCs to near –40 mV, a value that would allow optimal activation of the motor protein prestin and normal cochlear amplification. Although the set point of the OHC receptor potential transfer function in vivo may therefore be determined primarily by endolymphatic Ca2+ concentration, repetitive acoustic stimulation fails to produce adaptation of MET‐dependent otoacoustic emissions in vivo in the Tecta/Tectb−/− mice. Therefore, the TM is likely to contribute to the regulation of Ca2+ levels around the stereocilia, and thus adaptation of the OHC MET channel during prolonged sound stimulation

Aging of the mammalian gastrointestinal tract: a complex organ system

Gastrointestinal disorders are a major cause of morbidity in the elderly population. The gastrointestinal tract is the most complex organ system; its diverse cells perform a range of functions essential to life, not only secretion, digestion, absorption and excretion, but also, very importantly, defence. The gastrointestinal tract acts not only as a barrier to harmful materials and pathogens but also contains the vast number of beneficial bacterial populations that make up the microbiota. Communication between the cells of the gastrointestinal tract and the central nervous and endocrine systems modifies behaviour; the organisms of the microbiota also contribute to this brain–gut–enteric microbiota axis. Age-related physiological changes in the gut are not only common, but also variable, and likely to be influenced by external factors as well as intrinsic aging of the cells involved. The cellular and molecular changes exhibited by the aging gut cells also vary. Aging intestinal smooth muscle cells exhibit a number of changes in the signalling pathways that regulate contraction. There is some evidence for age-associated degeneration of neurons and glia of the enteric nervous system, although enteric neuronal losses are likely not to be nearly as extensive as previously believed. Aging enteric neurons have been shown to exhibit a senescence-associated phenotype. Epithelial stem cells exhibit increased mitochondrial mutation in aging that affects their progeny in the mucosal epithelium. Changes to the microbiota and intestinal immune system during aging are likely to contribute to wider aging of the organism and are increasingly important areas of analysis. How changes of the different cell types of the gut during aging affect the numerous cellular interactions that are essential for normal gut functions will be important areas for future aging research

Neurogenic mechanisms in bladder and bowel ageing

The prevalence of both urinary and faecal incontinence, and also chronic constipation, increases with ageing and these conditions have a major impact on the quality of life of the elderly. Management of bladder and bowel dysfunction in the elderly is currently far from ideal and also carries a significant financial burden. Understanding how these changes occur is thus a major priority in biogerontology. The functions of the bladder and terminal bowel are regulated by complex neuronal networks. In particular neurons of the spinal cord and peripheral ganglia play a key role in regulating micturition and defaecation reflexes as well as promoting continence. In this review we discuss the evidence for ageing-induced neuronal dysfunction that might predispose to neurogenic forms of incontinence in the elderly

Dopamine Receptor Antagonists Enhance Proliferation and Neurogenesis of Midbrain Lmx1a-expressing Progenitors

Degeneration of dopamine neurons in the midbrain causes symptoms of the movement disorder, Parkinson disease. Dopamine neurons are generated from proliferating progenitor cells localized in the embryonic ventral midbrain. However, it remains unclear for how long cells with dopamine progenitor character are retained and if there is any potential for reactivation of such cells after cessation of normal dopamine neurogenesis. We show here that cells expressing Lmx1a and other progenitor markers remain in the midbrain aqueductal zone beyond the major dopamine neurogenic period. These cells express dopamine receptors, are located in regions heavily innervated by midbrain dopamine fibres and their proliferation can be stimulated by antagonizing dopamine receptors, ultimately leading to increased neurogenesis in vivo. Furthermore, treatment with dopamine receptor antagonists enhances neurogenesis in vitro, both from embryonic midbrain progenitors as well as from embryonic stem cells. Altogether our results indicate a potential for reactivation of resident midbrain cells with dopamine progenitor potential beyond the normal period of dopamine neurogenesis