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)

Post-Disaster Supply Chain Interdependent Critical Infrastructure System Restoration: A Review of Data Necessary and Available for Modeling

The majority of restoration strategies in the wake of large-scale disasters have focused on short-term emergency response solutions. Few consider medium- to long-term restoration strategies to reconnect urban areas to national supply chain interdependent critical infrastructure systems (SCICI). These SCICI promote the effective flow of goods, services, and information vital to the economic vitality of an urban environment. To re-establish the connectivity that has been broken during a disaster between the different SCICI, relationships between these systems must be identified, formulated, and added to a common framework to form a system-level restoration plan. To accomplish this goal, a considerable collection of SCICI data is necessary. The aim of this paper is to review what data are required for model construction, the accessibility of these data, and their integration with each other. While a review of publically available data reveals a dearth of real-time data to assist modeling long-term recovery following an extreme event, a significant amount of static data does exist and these data can be used to model the complex interdependencies needed. For the sake of illustration, a particular SCICI (transportation) is used to highlight the challenges of determining the interdependencies and creating models capable of describing the complexity of an urban environment with the data publically available. Integration of such data as is derived from public domain sources is readily achieved in a geospatial environment, after all geospatial infrastructure data are the most abundant data source and while significant quantities of data can be acquired through public sources, a significant effort is still required to gather, develop, and integrate these data from multiple sources to build a complete model. Therefore, while continued availability of high quality, public information is essential for modeling efforts in academic as well as government communities, a more streamlined approach to a real-time acquisition and integration of these data is essential

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

TMC2 modifies permeation properties of the mechanoelectrical transducer channel in early postnatal mouse cochlear outer hair cells

The ability of cochlear hair cells to convert sound into receptor potentials relies on the mechanoelectrical transducer (MET) channels present in their stereociliary bundles. There is strong evidence implying that transmembrane channel-like protein (TMC) 1 contributes to the pore-forming subunit of the mature MET channel, yet its expression is delayed (∼>P5 in apical outer hair cells, OHCs) compared to the onset of mechanotransduction (∼P1). Instead, the temporal expression of TMC2 coincides with this onset, indicating that it could be part of the immature MET channel. We investigated MET channel properties from OHCs of homo- and heterozygous Tmc2 knockout mice. In the presence of TMC2, the MET channel blocker dihydrostreptomycin (DHS) had a lower affinity for the channel, when the aminoglycoside was applied extracellularly or intracellularly, with the latter effect being more pronounced. In Tmc2 knockout mice OHCs were protected from aminoglycoside ototoxicity during the first postnatal week, most likely due to their small MET current and the lower saturation level for aminoglycoside entry into the individual MET channels. DHS entry through the MET channels of Tmc2 knockout OHCs was lower during the first than in the second postnatal week, suggestive of a developmental change in the channel pore properties independent of TMC2. However, the ability of TMC2 to modify the MET channel properties strongly suggests it contributes to the pore-forming subunit of the neonatal channel. Nevertheless, we found that TMC2, different from TMC1, is not necessary for OHC development. While TMC2 is required for mechanotransduction in mature vestibular hair cells, its expression in the immature cochlea may be an evolutionary remnant

‘Subjects and Objects: Material Expressions of Love and Loyalty in Seventeenth-Century England’, in special section on ‘Loyalties and Allegiances in Early Modern England’ in Journal of British Studies Vol. 48: 4 (October, 2009)

This article investigates how and where the emotive relations between subject and state were forged and how these ideas were manifested in early modern England. McShane describes an affective economy of loyalty, embodied in cheap and accessible political commodities: decorated objects made of clay, metals, and paper, on which precious household resources of time, money and emotion were spent. She argues that by engendering, inculcating and insinuating codes of political love into people’s ‘emotional, sensual, representational, and communicative’ lives, ‘loyal’ goods acted as vehicles and texts for what Victoria Kahn describes as ‘the supplementary role of the passions’ in ‘forging political obligation’ and the reformulation of ‘the duty to love’ of both subject and king in 17th-century England. McShane’s research contributes to a growing theme in scholarship, namely the active consumption of politically significant goods. This essay extends the range of objects under examination to include quotidian household items, shedding light on the dissemination and construction of early modern loyalty across a much wider social scale. The research draws on an extensive survey of collections held at the V&A, the Museum of London, Ashmolean Museum, Fitzwilliam Museum and Burrell Collection. Importantly, by putting illustrated print products back together with other political commodities in the early modern home, creating a broad archive of objects and text-objects where each informs the other, McShane’s approach challenges the typical social historical methodology, which uses material culture as merely illustrative of textual sources. This article was part of a special section on loyalty and allegiance in early modern England, co-edited by McShane with Dr Ted Vallance for one of the leading scholarly journals in the field. The material was drawn from a workshop on the topic held at the University of Liverpool funded by the British Academy, University of Liverpool and the Scouloudi Foundation (2007)

Quick and robust method for trace determination of MeHg in rice and rice products without derivatisation

Peer reviewedPostprin

Determination of Arsenic, Mercury and Barium in herbarium mount paper using dynamic ultrasound-assisted extraction prior to atomic fluorescence and absorption spectrometry

A dynamic ultrasound-assisted extraction method using Atomic Absorption and Atomic Flourescence spectrometers as detectors was developed to analyse mercury, arsenic and barium from herbarium mount paper originating from the herbarium collection of the National Museum of Wales. The variables influencing extraction were optimised by a multivariate approach. The optimal conditions were found to be 1% HNO3 extractant solution used at a flow rate of 1 mL min-1. The duty cycle and amplitude of the ultrasonic probe was found to be 50% in both cases with an ultrasound power of 400 W. The optimal distance between the probe and the top face of the extraction chamber was found to be 0 cm. Under these conditions the time required for complete extraction of the three analytes was 25 min. Cold vapour and hydride generation coupled to atomic fluorescence spectrometry was utilized to determine mercury and arsenic, respectively. The chemical and instrumental conditions were optimized to provide detection limits of 0.01ng g-1 and 1.25 ng g-1 for mercury and arsenic, respectively. Barium was determined by graphite-furnace atomic absorption spectrometry, with a detection limit of 25 ng g-1. By using 0.5 g of sample, the concentrations of the target analytes varied for the different types of paper and ranged between 0.4–2.55 µg g-1 for Ba, 0.035–10.47 µg g-1 for As and 0.0046–2.37 µg g-1 for Hg

The acquisition of mechano-electrical transducer current adaptation in auditory hair cells requires myosin VI

Mutations in Myo6, the gene encoding the (F-actin) minus end-directed unconventional myosin, myosin VI, cause hereditary deafness in mice (Snell's waltzer) and humans. In the sensory hair cells of the cochlea, myosin VI is expressed in the cell bodies and along the stereocilia that project from the cells’ apical surface. It is required for maintaining the structural integrity of the mechanosensitive hair bundles formed by the stereocilia. In this study we investigate whether myosin VI contributes to mechano-electrical transduction. We report that Ca²+-dependent adaptation of the mechano-electrical transducer (MET) current, which serves to keep the transduction apparatus operating within its most sensitive range, is absent in outer and inner hair cells from homozygous Snell's waltzer mutant mice, which fail to express myosin VI. The operating range of the MET channels is also abnormal in the mutants, resulting in the absence of a resting MET current. We found that cadherin 23, a component of the hair bundle's transient lateral links, fails to be downregulated along the length of the stereocilia in maturing Myo6 mutant mice. MET currents of heterozygous littermates appear normal. We propose that myosin VI, by removing key molecules from developing hair bundles, is required for the development of the MET apparatus and its Ca²+-dependent adaptation

Standing in a Garden of Forking Paths

According to the Path Principle, it is permissible to expand your set of beliefs iff (and because) the evidence you possess provides adequate support for such beliefs. If there is no path from here to there, you cannot add a belief to your belief set. If some thinker with the same type of evidential support has a path that they can take, so do you. The paths exist because of the evidence you possess and the support it provides. Evidential support grounds propositional justification. The principle is mistaken. There are permissible steps you may take that others may not even if you have the very same evidence. There are permissible steps that you cannot take that others can even if your beliefs receive the same type of evidential support. Because we have to assume almost nothing about the nature of evidential support to establish these results, we should reject evidentialism