Na(V)1.5 sodium channel window currents contribute to spontaneous firing in olfactory sensory neurons

Olfactory sensory neurons (OSNs) fire spontaneously as well as in response to odor; both forms of firing are physiologically important. We studied voltage-gated Na+ channels in OSNs to assess their role in spontaneous activity. Whole cell patch-clamp recordings from OSNs demonstrated both tetrodotoxin-sensitive and tetrodotoxin-resistant components of Na+ current. RT-PCR showed mRNAs for five of the nine different Na+ channel α-subunits in olfactory tissue; only one was tetrodotoxin resistant, the so-called cardiac subtype NaV1.5. Immunohistochemical analysis indicated that NaV1.5 is present in the apical knob of OSN dendrites but not in the axon. The NaV1.5 channels in OSNs exhibited two important features: 1) a half-inactivation potential near −100 mV, well below the resting potential, and 2) a window current centered near the resting potential. The negative half-inactivation potential renders most NaV1.5 channels in OSNs inactivated at the resting potential, while the window current indicates that the minor fraction of noninactivated NaV1.5 channels have a small probability of opening spontaneously at the resting potential. When the tetrodotoxin-sensitive Na+ channels were blocked by nanomolar tetrodotoxin at the resting potential, spontaneous firing was suppressed as expected. Furthermore, selectively blocking NaV1.5 channels with Zn2+ in the absence of tetrodotoxin also suppressed spontaneous firing, indicating that NaV1.5 channels are required for spontaneous activity despite resting inactivation. We propose that window currents produced by noninactivated NaV1.5 channels are one source of the generator potentials that trigger spontaneous firing, while the upstroke and propagation of action potentials in OSNs are borne by the tetrodotoxin-sensitive Na+ channel subtypes.This work was aided by support from Boston University, the Rocky Mountain Taste and Smell Center Core for Cellular Visualization and Analysis [National Institute on Deafness and Other Communication Disorders (NIDCD) P30 DC-04657; D. Restrepo, principal investigator], and NIDCD Grants DC-04863 to V. Dionne and DC-006070 to D. Restrepo and T. E. Finger. (Boston University; P30 DC-04657 - Rocky Mountain Taste and Smell Center Core for Cellular Visualization and Analysis [National Institute on Deafness and Other Communication Disorders (NIDCD)]; DC-04863 - Rocky Mountain Taste and Smell Center Core for Cellular Visualization and Analysis [National Institute on Deafness and Other Communication Disorders (NIDCD)]; DC-006070 - Rocky Mountain Taste and Smell Center Core for Cellular Visualization and Analysis [National Institute on Deafness and Other Communication Disorders (NIDCD)])https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4122723/Accepted manuscrip

Technical note: how accurate can stalagmite formation temperatures be determined using vapour bubble radius measurements in fluid inclusions?

Stalagmites are natural archives containing detailed information on continental climate variability of the past. Microthermometric measurements of fluid inclusion homogenisation temperatures allow determination of stalagmite formation temperatures by measuring the radius of stable laser-induced vapour bubbles inside the inclusions. A reliable method for precisely measuring the radius of vapour bubbles is presented. The method is applied to stalagmite samples for which the formation temperature is known. An assessment of the bubble radius measurement accuracy and how this error influences the uncertainty in determining the formation temperature is provided. We demonstrate that the nominal homogenisation temperature of a single inclusion can be determined with an accuracy of ±0.25 °C, if the volume of the inclusion is larger than 105 μm3. With this method, we could measure in a proof-of-principle investigation that the formation temperature of 10–20 yr old inclusions in a stalagmite taken from the Milandre cave is 9.87 ± 0.80 °C, while the mean annual surface temperature, that in the case of the Milandre cave correlates well with the cave temperature, was 9.6 ± 0.15 °C, calculated from actual measurements at that time, showing a very good agreement. Formation temperatures of inclusions formed during the last 450 yr are found in a temperature range between 8.4 and 9.6 °C, which corresponds to the calculated average surface temperature. Paleotemperatures can thus be determined within ±1.0 °C

Experimental in vivo fenestration of guinea pig cochlea using 2.79 Μm laser radiation

Erbium-YSGG laser systems are promising tools in ear, nose and throat (ENT) surgery. The high absorption in biological tissues, resulting in precise tissue ablation with minimal thermal tissue damage, and the possibility to guide the radiation through optical fibres make the 2.79 Μm wavelength a favourite for microsurgery. In order to simulate the fenestration of the human stapes foot plate required for prosthesis implantation when treating otosclerosis, five guinea pig cochleae were irradiated in vivo until perforation was achieved. The laser-induced temperature rise and pressure transients evoke activity in the inner hair cells that was investigated by micro-iontophoresis. Perforation of the cochlea bone (hole diameter of 350 Μm) can be performed with a few laser pulses and high precision with a thermal damage zone of<100 Μm. The bone ablation rate is 10 ± 2 Μm pulse-1 at a radiant exposure of 12 J cm-2. The functionality of the afferent inner hair cells in the guinea pig cochlea was verified before and after laser treatment using glutamate receptor agonists AMPA and NMDA. For the above selected laser parameters, the induced 15-min enhanced activity was blockable with the specific reversible AMPA and NMDA antagonists CNQX and AP-7. Micro-iontophoresis confirms the reversibility of cochlea functionality after its perforation with Er-YSGG laser pulses. A limit of radiant exposure around 12 J cm-2 is found for safe fenestration. It is demonstrated that the Er-YSGG laser is a precise and safe instrument whilst still using adequate laser parameters. On the other hand, this study demonstrates the potential of uncontrollable and unintended induced damage, resulting from vapour channel formation in the perilymph, if a high laser radiant exposure is applie

(±)-2-exo- and endo-Methylamino-1,2,3,4-tetrahydro-1,4-ethanonapthalene Hydrochloride

This is the published version

Giant nuclei is essential in the cell cycle transition from meiosis to mitosis

At the transition from meiosis to cleavage mitoses, Drosophila requires the cell cycle regulators encoded by the genes, giant nuclei (gnu), plutonium (plu) and pan gu (png). Embryos lacking Gnu protein undergo DNA replication and centrosome proliferation without chromosome condensation or mitotic segregation. We have identified the gnu gene encoding a novel phosphoprotein dephosphorylated by Protein phosphatase 1 at egg activation. Gnu is normally expressed in the nurse cells and oocyte of the ovary and is degraded during the embryonic cleavage mitoses. Ovarian death and sterility result from gnu gain of function. gnu function requires the activity of pan gu and plu

Deafness mutation mining using regular expression based pattern matching

<p>Abstract</p> <p>Background</p> <p>While keyword based queries of databases such as Pubmed are frequently of great utility, the ability to use regular expressions in place of a keyword can often improve the results output by such databases. Regular expressions can allow for the identification of element types that cannot be readily specified by a single keyword and can allow for different words with similar character sequences to be distinguished.</p> <p>Results</p> <p>A Perl based utility was developed to allow the use of regular expressions in Pubmed searches, thereby improving the accuracy of the searches.</p> <p>Conclusion</p> <p>This utility was then utilized to create a comprehensive listing of all DFN deafness mutations discussed in Pubmed records containing the keywords "human ear".</p

Improving In Vitro Generated Cartilage-Carrier-Constructs by Optimizing Growth Factor Combination

The presented study is focused on the generation of osteochondral implants for cartilage repair, which consist of bone substitutes covered with in vitro engineered cartilage. Re-differentiation of expanded porcine cells was performed in alginate gel followed by cartilage formation in high-density cell cultures. In this work, different combinations of growth factors for the stimulation of re-differentiation and cartilage formation have been tested to improve the quality of osteochondral implants. It has been demonstrated that supplementation of the medium with growth factors has significant effects on the properties of the matrix. The addition of the growth factors IGF-I (100 ng/mL) and TGF-β1 (10 ng/mL) during the alginate culture and the absence of any growth factors during the high-density cell culture led to significantly higher GAG to DNA ratios and Young’s Moduli of the constructs compared to other combinations. The histological sections showed homogenous tissue and intensive staining for collagen type II

Development of an In Vitro Compartmentalization Screen for High-Throughput Directed Evolution of [FeFe] Hydrogenases

BACKGROUND: [FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library. CONCLUSIONS/SIGNIFICANCE: [FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy

The Interplay Between Post-Critical Beliefs and Anxiety: An Exploratory Study in a Polish Sample

The present research investigates the relationship between anxiety and the religiosity dimensions that Wulff (Psychology of religion: classic and contemporary views, Wiley, New York, 1991; Psychology of religion. Classic and contemporary views, Wiley, New York, 1997; Psychologia religii. Klasyczna i współczesna, Wydawnictwo Szkolne i Pedagogiczne, Warszawa, 1999) described as Exclusion vs. Inclusion of Transcendence and Literal vs. Symbolic. The researchers used the Post-Critical Belief scale (Hutsebaut in J Empir Theol 9(2):48–66, 1996; J Empir Theol 10(1):39–54, 1997) to measure Wulff’s religiosity dimensions and the IPAT scale (Krug et al. 1967) to measure anxiety. Results from an adult sample (N = 83) suggest that three dimensions show significant relations with anxiety. Orthodoxy correlated negatively with suspiciousness (L) and positively with guilt proneness (O) factor—in the whole sample. Among women, Historical Relativism negatively correlated with suspiciousness (L), lack of integration (Q3), general anxiety and covert anxiety. Among men, Historical Relativism positively correlated with tension (Q4) and emotional instability (C), general anxiety, covert anxiety and overt anxiety. External Critique was correlated with suspiciousness (L) by men