Tertiapin-Q blocks recombinant and native large conductance K+ channels in a use-dependent manner

Tertiapin, a short peptide from honey bee venom, has been reported to specifically block the inwardly rectifying K+ (Kir) channels, including G protein-coupled inwardly rectifying potassium channel (GIRK) 1 + GIRK4 heteromultimers and ROMK1 homomultimers. In the present study, the effects of a stable and functionally similar derivative of tertiapin, tertiapin-Q, were examined on recombinant human voltage-dependent Ca2+-activated large conductance K+ channel (BK or MaxiK; alpha-subunit or hSlo1 homomultimers) and mouse inwardly rectifying GIRK1 + GIRK2 (i.e., Kir3.1 and Kir3.2) heteromultimeric K+ channels expressed in Xenopus oocytes and in cultured newborn mouse dorsal root ganglion (DRG) neurons. In two-electrode voltage-clamped oocytes, tertiapin-Q (1-100 nM) inhibited BK-type K+ channels in a use- and concentration-dependent manner. We also confirmed the inhibition of recombinant GIRK1 + GIRK2 heteromultimers by tertiapin-Q, which had no effect on endogenous depolarization- and hyperpolarization-activated currents sensitive to extracellular divalent cations (Ca2+, Mg2+, Zn2+, and Ba2+) in defolliculated oocytes. In voltage-clamped DRG neurons, tertiapin-Q voltage- and use-dependently inhibited outwardly rectifying K+ currents, but Cs+-blocked hyperpolarization-activated inward currents including I-H were insensitive to tertiapin-Q, baclofen, barium, and zinc, suggesting absence of functional GIRK channels in the newborn. Under current-clamp conditions, tertiapin-Q blocked the action potential after hyperpolarization (AHP) and increased action potential duration in DRG neurons. Taken together, these results demonstrate that the blocking actions of tertiapin-Q are not specific to Kir channels and that the blockade of recombinant BK channels and native neuronal AHP currents is use-dependent. Inhibition of specific types of Kir and voltage-dependent Ca2+-activated K+ channels by tertiapin-Q at nanomolar range via different mechanisms may have implications in pain physiology and therapy.

Spin-charge conductance in nanoscale electronic devices

We introduce a spin-charge conductance matrix as a unifying concept underlying charge and spin transport within the framework of the Landauer-Buttiker conductance formula. It turns out that the spin-charge conductance matrix provides a natural and gauge covariant description for electron transport through nanoscale electronic devices. We demonstrate that the charge and spin conductances are gauge invariant observables which characterize transport phenomena arising from spin-dependent scattering. Tunnelling through a single magnetic atom is discussed to illustrate our theory.

Covariation and interference between physiological reflexes: Eye-blink startle cardiac defense, and skin conductance

Comparative studies of autonomic and somatic reflexes, such as cardiac defense and motor startle, are rare. However, examination of the pattern of covariation, independence, or interference among physiological reflexes may help to clarify their functional significance and elucidate their complex modulation by psychological factors. Here we report the results of a study that examined the pattern of interference of eye-blink startle on subsequent cardiac defense. Participants were 63 students (31 women) distributed into three groups according to the sensory modality of the eliciting stimulus during the startle trials: acoustic high intensity (105 dB), acoustic low intensity (65 dB), and visual modality. Startle trials consisted of 12 presentations of the eliciting stimulus with a duration of 50 ms, instantaneous risetime, and a variable inter-stimulus interval of 16 – 20 s.Defense trials began 20 s after the last startle trial and consisted, for all groups, of 3 presentations of the high intensity acoustic stimulus with a duration of 500 ms and an inter-stimulus interval of 215 s. Results showed a clear interference of the startle trials on the subsequent defense trials when both types of trials shared identical sensory modality (acoustic) independently of intensity: the expected pattern of cardiac defense in the first trial only appeared in the visual modality. Similar interference effects were observed in the skin conductance response. Subjective reactivity to the defense stimulus did not detect differences between conditions.

Substance P preferentially inhibits large conductance nicotinic ACh receptor channels in rat intracardiac ganglion neurons

The effects of substance P (SP) on nicotinic acetylcholine (ACh)-evoked currents were investigated in parasympathetic neurons dissociated from neonatal rat intracardiac ganglia using standard whole cell, perforated patch, and outside-out recording configurations of the patch-clamp technique. Focal application of SP onto the soma reversibly decreased the peak amplitude of the ACh-evoked current with half-maximal inhibition occurring at 45 mu M and complete block at 300 mu M SP. Whole cell current-voltage (I-V) relationships obtained in the absence and presence of SP indicate that the block of ACh-evoked currents by SP is voltage independent. The rate of decay of ACh-evoked currents was increased sixfold in the presence of SP (100 mu M), suggesting that SP may increase the rate of receptor desensitization. SP-induced inhibition of ACh-evoked currents was observed following cell dialysis and in the presence of either 1 mM 8-Br-cAMP, a membrane-permeant cAMP analogue, 5 mu M H-7, a protein kinase C inhibitor, or 2 mM intracellular AMP-PNP, a nonhydrolyzable ATP analogue. These data suggest that a diffusible cytosolic second messenger is unlikely to mediate SP inhibition of neuronal nicotinic ACh receptor (nAChR) channels. Activation of nAChR channels in outside-out membrane patches by either ACh (3 mu M) or cytisine (3 mu M) indicates the presence of at least three distinct conductances (20, 35, and 47 pS) in rat intracardiac neurons. In the presence of 3 mu M SP, the large conductance nAChR channels are preferentially inhibited. The open probabilities of the large conductance classes activated by either ACh or cytisine were reversibly decreased by 10- to 30-fold in the presence of SP. The single-channel conductances were unchanged, and mean apparent channel open times for the large conductance nAChR channels only were slightly decreased by SP. Given that individual parasympathetic neurons of rat intracardiac ganglia express a heterogeneous population of nAChR subunits represented by the different conductance levels, SP appears to preferentially inhibit those combinations of nAChR subunits that form the large conductance nAChR channels. Since ACh is the principal neurotransmitter of extrinsic (vagal) innervation of the mammalian heart, SP may play an important role in modulating autonomic control of the heart.

Depolarisation and suppression of burst firing activity in the mouse subthalamic nucleus by dopamine D1/D5 receptor activation of a cyclic-nucleotide gated non-specific cation conductance

Neuronal burst firing in the subthalamic nucleus (STN) is one of the hallmarks of dopamine depletion in Parkinson's disease. Here, we have determined the postsynaptic effects of dopamine in the STN and the functional consequences of dopamine receptor modulation on burst firing in vitro. STN cells displayed regular spiking activity at a rate of 7.9 +/- 0.5 Hz. Application of dopamine (30 mu M) induced membrane depolarisations accompanied by an increase in firing rate of mean 12.0 +/- 0.6 Hz in all 69 cells. The dopamine effect was mimicked by the dopamine D1/D5 receptor agonist SKF38393 (10 mu M, 17 cells) and the dopamine D2-like receptor agonist quinpirole (10 mu M, 35 cells), partly reduced by D1/D5 antagonist SCH23390 (2 mu M, seven cells), but unaffected by the D2 antagonists sulpiride (10 mu M, seven cells) or eticlopride (10 mu M, six cells). Using voltage ramps, dopamine induced an inward current of 69 +/- 9.4 pA at a holding potential of -60 mV (n = 17). This current was accompanied by an increase in input conductance of 1.55 +/- 0.35 nS which reversed at -30.6 +/- 2.3 mV, an effect mimicked by SKF38393 (10 AM, nine cells). Similar responses were observed when measuring instantaneous current evoked by voltage steps and in the presence of the I-h blocker, ZD7288, indicating effects independent of I-h. The increase in conductance was blocked by SCH23390 (2 mu M, n = 4), mimicked by the activator of adenylyl cyclase forskolin (10 mu M, n = 7) and blocked by H-89, an inhibitor of cyclic AMP dependent protein kinase A (10 PM, n = 6). These results indicate that the dopamine depolarisation is in part mediated by D1/D5 receptor mediated activation of a cyclic-nucleotide gated (CNG) non-specific cation conductance. This conductance contributes to the membrane depolarisation that changes STN neuronal bursting to more regular activity by significantly increasing burst duration and number of spikes per burst.

A transient method of measuring thermal contact conductance between metal-plastic surfaces

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Molecular detection using metallic nanowires with quantized conductance

This thesis studies the adsorption of molecules with different binding strengths onto copper nanowires with prestabilized conductance values fabricated by an electrochemical method. Since the diameters of these wires are comparable to the wavelength of conduction electrons the conductance of the nanowires is quantized, and the adsorption of even a few molecules onto atomically thin wires changes the conductance from integer values to fractional ones. These changes are proportional to the binding strength of the adsorbed molecules. The decrease in conductance is hypothesized to be caused by the scattering of the conduction electrons by the adsorbed molecules. The sensitivity of molecular adsorption-induced conductance change can be used for the development of a chemical sensor. The stabilized copper nanowires obtained in this thesis may also be used for other purposes, such as interconnecting conductors between nanodevices and digital switches in functional nanoelectronic circuitry.

Bridging the Scale Gap from Leaf to Canopy in Biosphere-Atmosphere Gas and Particle Exchanges

Terrestrial ecosystems, occupying more than 25% of the Earth's surface, can serve as

`biological valves' in regulating the anthropogenic emissions of atmospheric aerosol

particles and greenhouse gases (GHGs) as responses to their surrounding environments.

While the signicance of quantifying the exchange rates of GHGs and atmospheric

aerosol particles between the terrestrial biosphere and the atmosphere is

hardly questioned in many scientic elds, the progress in improving model predictability,

data interpretation or the combination of the two remains impeded by

the lack of precise framework elucidating their dynamic transport processes over a

wide range of spatiotemporal scales. The diculty in developing prognostic modeling

tools to quantify the source or sink strength of these atmospheric substances

can be further magnied by the fact that the climate system is also sensitive to the

feedback from terrestrial ecosystems forming the so-called `feedback cycle'. Hence,

the emergent need is to reduce uncertainties when assessing this complex and dynamic

feedback cycle that is necessary to support the decisions of mitigation and

adaptation policies associated with human activities (e.g., anthropogenic emission

controls and land use managements) under current and future climate regimes.

With the goal to improve the predictions for the biosphere-atmosphere exchange

of biologically active gases and atmospheric aerosol particles, the main focus of this

dissertation is on revising and up-scaling the biotic and abiotic transport processes

from leaf to canopy scales. The validity of previous modeling studies in determining

iv

the exchange rate of gases and particles is evaluated with detailed descriptions of their

limitations. Mechanistic-based modeling approaches along with empirical studies

across dierent scales are employed to rene the mathematical descriptions of surface

conductance responsible for gas and particle exchanges as commonly adopted by all

operational models. Specically, how variation in horizontal leaf area density within

the vegetated medium, leaf size and leaf microroughness impact the aerodynamic attributes

and thereby the ultrane particle collection eciency at the leaf/branch scale

is explored using wind tunnel experiments with interpretations by a porous media

model and a scaling analysis. A multi-layered and size-resolved second-order closure

model combined with particle

uxes and concentration measurements within and

above a forest is used to explore the particle transport processes within the canopy

sub-layer and the partitioning of particle deposition onto canopy medium and forest

oor. For gases, a modeling framework accounting for the leaf-level boundary layer

eects on the stomatal pathway for gas exchange is proposed and combined with sap

ux measurements in a wind tunnel to assess how leaf-level transpiration varies with

increasing wind speed. How exogenous environmental conditions and endogenous

soil-root-stem-leaf hydraulic and eco-physiological properties impact the above- and

below-ground water dynamics in the soil-plant system and shape plant responses

to droughts is assessed by a porous media model that accommodates the transient

water

ow within the plant vascular system and is coupled with the aforementioned

leaf-level gas exchange model and soil-root interaction model. It should be noted

that tackling all aspects of potential issues causing uncertainties in forecasting the

feedback cycle between terrestrial ecosystem and the climate is unrealistic in a single

dissertation but further research questions and opportunities based on the foundation

derived from this dissertation are also brie

y discussed.

The correlation of pore morphology, interconnectivity and physical properties of 3D ceramic scaffolds with bone ingrowth

In the design of tissue engineering scaffolds, design parameters including pore size, shape and interconnectivity, mechanical properties and transport properties should be optimized to maximize successful inducement of bone ingrowth. In this paper we describe a 3D micro-CT and pore partitioning study to derive pore scale parameters including pore radius distribution, accessible radius, throat radius, and connectivity over the pore space of the tissue engineered constructs. These pore scale descriptors are correlated to bone ingrowth into the scaffolds. Quantitative and visual comparisons show a strong correlation between the local accessible pore radius and bone ingrowth; for well connected samples a cutoff accessible pore radius of approximately 100 microM is observed for ingrowth. The elastic properties of different types of scaffolds are simulated and can be described by standard cellular solids theory: (E/E(0))=(rho/rho(s))(n). Hydraulic conductance and diffusive properties are calculated; results are consistent with the concept of a threshold conductance for bone ingrowth. Simple simulations of local flow velocity and local shear stress show no correlation to in vivo bone ingrowth patterns. These results demonstrate a potential for 3D imaging and analysis to define relevant pore scale morphological and physical properties within scaffolds and to provide evidence for correlations between pore scale descriptors, physical properties and bone ingrowth.

Real-time evaluation of driver’s alertness on highways

Suburbanisation has been internationally a major phenomenon in the last decades. Suburb-to-suburb routes are nowadays the most widespread road journeys; and this resulted in an increment of distances travelled, particularly on faster suburban highways. The design of highways tends to over-simplify the driving task and this can result in decreased alertness. Driving behaviour is consequently impaired and drivers are then more likely to be involved in road crashes. This is particularly dangerous on highways where the speed limit is high. While effective countermeasures to this decrement in alertness do not currently exist, the development of in-vehicle sensors opens avenues for monitoring driving behaviour in real-time. The aim of this study is to evaluate in real-time the level of alertness of the driver through surrogate measures that can be collected from in-vehicle sensors. Slow EEG activity is used as a reference to evaluate driver's alertness. Data are collected in a driving simulator instrumented with an eye tracking system, a heart rate monitor and an electrodermal activity device (N=25 participants). Four different types of highways (driving scenario of 40 minutes each) are implemented through the variation of the road design (amount of curves and hills) and the roadside environment (amount of buildings and traffic). We show with Neural Networks that reduced alertness can be detected in real-time with an accuracy of 92% using lane positioning, steering wheel movement, head rotation, blink frequency, heart rate variability and skin conductance level. Such results show that it is possible to assess driver's alertness with surrogate measures. Such methodology could be used to warn drivers of their alertness level through the development of an in-vehicle device monitoring in real-time drivers' behaviour on highways, and therefore it could result in improved road safety.

PCR bias Toward the wild-type k-ras and p53 sequences: Implications for PCR detection of mutations and cancer diagnosis

PCR-based cancer diagnosis requires detection of rare mutations in k- ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work on factor IX suggests that this assumption is invalid for one case of near- sequence identity. To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For k-ras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerase. Mutant and wild-type segments of the factor V, cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants.

Verbal instruction abolishes fear conditioned to racial out-group faces

Previous research has shown resistance to extinction of fear conditioned to racial out-group faces, suggesting that these stimuli may be subject to prepared fear learning. The current study replicated and extended previous research by using a different racial out-group, and testing the prediction that prepared fear learning is unaffected by verbal instructions. Four groups of Caucasian participants were trained with male in-group (Caucasian) or out-group (Chinese) faces as conditional stimuli; one paired with an electro-tactile shock (CS+) and one presented alone (CS). Before extinction, half the participants were instructed that no more shocks would be presented. Fear conditioning, indexed by larger electrodermal responses to, and blink startle modulation during the CS+, occurred during acquisition in all groups. Resistance to extinction of fear learning was found only in the racial out-group, no instruction condition. Fear conditioned to a racial out-group face was reduced following verbal instructions, contrary to predictions for the nature of prepared fear learning.

On the resistance to extinction of fear conditioned to angry faces

The present study investigated whether, like fear conditioned to pictures of snakes and spiders, fear conditioned to angry faces resists extinction even after verbal instruction and removal of the shock electrode. Participants were trained in a differential Pavlovian fear conditioning procedure with angry face or happy face conditional stimuli (CSs). Prior to extinction, half the participants in each group were informed that no more unconditional stimuli would be presented and the shock electrode was removed. In the absence of this manipulation, participants showed resistance to extinction after training with angry face CSs, but not after training with happy face CSs. Instructed extinction and electrode removal abolished fear conditioning regardless of the emotion expressed by the CS faces. This finding suggests that fear conditioned to angry faces, like fear conditioned to racial out-group faces, is more malleable than fear conditioned to snakes and spiders.