Pharmacological consequence of the A118G μ opioid receptor polymorphism on morphine-and fentanyl-mediated modulation of Ca2+ channels in humanized mouse sensory neurons

Background: The most common functional single nucleotide polymorphism of the human OPRM1 gene, A118G, has been shown to be associated with interindividual differences in opioid analgesic requirements, particularly with morphine, in patients with acute postoperative pain. The purpose of this study was to examine whether this polymorphism would modulate the morphine and fentanyl pharmacological profile of sensory neurons isolated from a humanized mouse model homozygous for either the 118A or 118G allele. Methods: The coupling of wild-type and mutant μ opioid receptors to voltage-gated Ca channels after exposure to either ligand was examined by employing the whole cell variant of the patch-clamp technique in acutely dissociated trigeminal ganglion neurons. Morphine-mediated antinociception was measured in mice carrying either the 118AA or 118GG allele. RESULTS:: The biophysical parameters (cell size, current density, and peak current amplitude potential) measured from both groups of sensory neurons were not significantly different. In 118GG neurons, morphine was approximately fivefold less potent and 26% less efficacious than that observed in 118AA neurons. On the other hand, the potency and efficacy of fentanyl were similar for both groups of neurons. Morphine-mediated analgesia in 118GG mice was significantly reduced compared with the 118AA mice. Conclusions: This study provides evidence to suggest that the diminished clinical effect observed with morphine in 118G carriers results from an alteration of the receptor's pharmacology in sensory neurons. In addition, the impaired analgesic response with morphine may explain why carriers of this receptor variant have an increased susceptibility to become addicted to opioids. © 2011 the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins. Anesthesiology.

Navigational traffic conflict technique: a proactive approach to quantitative measurement of collision risks in port waters

Navigational safety analysis relying on collision statistics is often hampered because of low number of observations. A promising alternative approach that overcomes this problem is proposed in this paper. By analyzing critical vessel interactions this approach proactively measures collision risk in port waters. The proposed method is illustrated for quantitative measurement of collision risks in Singapore port fairways, and validated by examining correlations between the measured risks with those perceived by pilots. This method is an ethically appealing alternative to the collision-based analysis for fast, reliable and effective safety assessment, thus possesses great potential for managing collision risks in port waters.

Proactive management of collision risks in port waters using navigational traffic conflicts

Navigational collisions are one of the major safety concerns in many seaports. To address this safety concern, a comprehensive and structured method of collision risk management is necessary. Traditionally management of port water collision risks has been relied on historical collision data. However, this collision-data-based approach is hampered by several shortcomings, such as randomness and rarity of collision occurrence leading to obtaining insufficient number of samples for a sound statistical analysis, insufficiency in explaining collision causation, and reactive approach to safety. A promising alternative approach that overcomes these shortcomings is the navigational traffic conflict technique that uses traffic conflicts as an alternative to the collision data. This paper proposes a collision risk management method by utilizing the principles of this technique. This risk management method allows safety analysts to diagnose safety deficiencies in a proactive manner, which, consequently, has great potential for managing collision risks in a fast, reliable and efficient manner.

Navigational safety in port waters: Techniques for modeling collision risk

Navigational collisions are one of the major safety concerns for many seaports. Continuing growth of shipping traffic in number and sizes is likely to result in increased number of traffic movements, which consequently could result higher risk of collisions in these restricted waters. This continually increasing safety concern warrants a comprehensive technique for modeling collision risk in port waters, particularly for modeling the probability of collision events and the associated consequences (i.e., injuries and fatalities). A number of techniques have been utilized for modeling the risk qualitatively, semi-quantitatively and quantitatively. These traditional techniques mostly rely on historical collision data, often in conjunction with expert judgments. However, these techniques are hampered by several shortcomings, such as randomness and rarity of collision occurrence leading to obtaining insufficient number of collision counts for a sound statistical analysis, insufficiency in explaining collision causation, and reactive approach to safety. A promising alternative approach that overcomes these shortcomings is the navigational traffic conflict technique (NTCT), which uses traffic conflicts as an alternative to the collisions for modeling the probability of collision events quantitatively. This article explores the existing techniques for modeling collision risk in port waters. In particular, it identifies the advantages and limitations of the traditional techniques and highlights the potentials of the NTCT in overcoming the limitations. In view of the principles of the NTCT, a structured method for managing collision risk is proposed. This risk management method allows safety analysts to diagnose safety deficiencies in a proactive manner, which consequently has great potential for managing collision risk in a fast, reliable and efficient manner.

Navigational Safety in Port Waters : Proactive Management of Collision Risk Using Traffic Conflicts

Traditionally navigational safety analyses rely on historical collision data which is often hampered because of low collision counts, insufficiency in explaining collision causation, and reactive approach to safety. A promising alternative approach that overcomes these problems is using navigational traffic conflicts or near-misses as an alternative to the collision data. This book discusses how traffic conflicts can effectively be used in modeling of port water collision risks. Techniques for measuring and predicting collision risks in fairways, intersections, and anchorages are discussed by utilizing advanced statistical models. Risk measurement models, which quantitatively measure collision risks in waterways, are discussed. To predict risks, a hierarchical statistical modeling technique is discussed which identifies the factors influencing the risks. The modeling techniques are illustrated for Singapore port data. Results showed that traffic conflicts are an ethically appealing alternative to collision data for fast, reliable and effective safety assessment, thus possessing great potential for managing collision risks in port waters.

Multi-device key management using visual side channels in pervasive computing environments

In the modern connected world, pervasive computing has become reality. Thanks to the ubiquity of mobile computing devices and emerging cloud-based services, the users permanently stay connected to their data. This introduces a slew of new security challenges, including the problem of multi-device key management and single-sign-on architectures. One solution to this problem is the utilization of secure side-channels for authentication, including the visual channel as vicinity proof. However, existing approaches often assume confidentiality of the visual channel, or provide only insufficient means of mitigating a man-in-the-middle attack. In this work, we introduce QR-Auth, a two-step, 2D barcode based authentication scheme for mobile devices which aims specifically at key management and key sharing across devices in a pervasive environment. It requires minimal user interaction and therefore provides better usability than most existing schemes, without compromising its security. We show how our approach fits in existing authorization delegation and one-time-password generation schemes, and that it is resilient to man-in-the-middle attacks.

Spectroscopic investigation of the polymerisation of pyrrole and thiophene within zeolite channels

The reaction of pyrrole and thiophene monomers with copper- or nickel-exchanged mordenite has been investigated using X-ray photoelectron (XPS) and photoacoustic infrared (PAIRS) spectroscopies. Because of the differing oxidising powers of the cations studied, polymerisation occurred only with copper-exchanged mordenite. PAIRS and XPS data indicated that both polypyrrole and polythiophene were partially oxidised when synthesised within the zeolite structure. IR spectra of polythiophene and polythiophene and polypyrrole showed intense bands typical of ring vibrations which could couple to the large dipole change induced by charges moving along the polythiophene chain. In addition it was noted that only vibrations typical of oxidised polymer structures were recorded, suggesting that the charge carrier was located within these segments. Furthermore, N 1s spectra contained a high binding energy peak at 402.5 eV which was attributed to a positively charged nitrogen species, in agreement with IR data. Significantly, C 1s spectra confirmed that molecular wires were formed within the confines of the zeolite lattice. Depth-profiling experiments suggested that polypyrrole was distributed throughout the entire zeolite host. By contrast, polythiophene may have been restricted to the uppermost zeolite channels owing to the ability of sulfur species to bond to CuI sites [produced by reduction of copper(II) ions during the polymerisation process], thus obstructing movement along the channels.

Fluorescence detection of plant extracts that affect neuronal voltage-gated Ca2+ channels

Structurally novel compounds able to block voltage-gated Ca2+ channels (VGCCs) are currently being sought for the development of new drugs directed at neurological disorders. Fluorescence techniques have recently been developed to facilitate the analysis of VGCC blockers in a multi-well format. By utilising the small cell lung carcinoma cell line, NCI-H146, we were able to detect changes in intracellular Ca2+ concentration ([Ca2+]i) using a fluorescence microplate reader. NCI-H146 cells have characteristics resembling those of neuronal cells and express multiple VGCC subtypes, including those of the L-, N- and P-type. We found that K+-depolarisation of fluo-3 loaded NCI-H146 cells causes a rapid and transient increase in fluorescence, which was readily detected in a 96-well plate. Extracts of Australian plants, including those used traditionally as headache or pain treatments, were tested in this study to identify those affecting Ca2+ influx following membrane depolarisation of NCI-H146 cells. We found that E. bignoniiflora, A. symphyocarpa and E. vespertilio caused dose-dependent inhibition of K+-depolarised Ca2+ influx, with IC50 values calculated to be 234, 548 and 209 μg/ml, respectively. This data suggests an effect of these extracts on the function of VGCCs in these cells. Furthermore, we found similar effects using a fluorescence laser imaging plate reader (FLIPR) that allows simultaneous measurement of real-time fluorescence in a multi-well plate. Our results indicate that the dichloromethane extract of E. bignoniiflora and the methanolic extract of E. vespertilio show considerable promise as antagonists of neuronal VGCCs. Further analysis is required to characterise the function of the bioactive constituents in these extracts and determine their selectivity on VGCC subtypes.

Nedd4-2(NEDD4L) controls intracellular Na(+)-mediated activity of voltage-gated sodium channels in primary cortical neurons.

Nedd4-2, a HECT (homologous with E6-associated protein C-terminus)-type ubiquitin protein ligase, has been implicated in regulating several ion channels, including Navs (voltage-gated sodium channels). In Xenopus oocytes Nedd4-2 strongly inhibits the activity of multiple Navs. However, the conditions under which Nedd4-2 mediates native Nav regulation remain uncharacterized. Using Nedd4-2-deficient mice, we demonstrate in the present study that in foetal cortical neurons Nedd4-2 regulates Navs specifically in response to elevated intracellular Na(+), but does not affect steady-state Nav activity. In dorsal root ganglia neurons from the same mice, however, Nedd4-2 does not control Nav activities. The results of the present study provide the first physiological evidence for an essential function of Nedd4-2 in regulating Navs in the central nervous system.

Structures of μOconotoxins from Conusmarmoreus. Inhibitors of tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels in mammalian sensory neurons

The μO-conotoxins are an intriguing class of conotoxins targeting various voltage-dependent sodium channels and molluscan calcium channels. In the current study, we have shown MrVIA and MrVIB to be the first known peptidic inhibitors of the transient tetrodotoxin-resistant (TTX-R) Na+ current in rat dorsal root ganglion neurons, in addition to inhibiting tetrodotoxin-sensitive Na+ currents. Human TTX-R sodium channels are a therapeutic target for indications such as pain, highlighting the importance of the μO-conotoxins as potential leads for drug development. Furthermore, we have used NMR spectroscopy to provide the first structural information on this class of conotoxins. MrVIA and MrVIB are hydrophobic peptides that aggregate in aqueous solution but were solubilized in 50% acetonitrile/water. The three-dimensional structure of MrVIB consists of a small β-sheet and a cystine knot arrangement of the three-disulfide bonds. It contains four backbone “loops” between successive cysteine residues that are exposed to the solvent to varying degrees. The largest of these, loop 2, is the most disordered part of the molecule, most likely due to flexibility in solution. This disorder is the most striking difference between the structures of MrVIB and the known δ- and ω-conotoxins, which along with the μO-conotoxins are members of the O superfamily. Loop 2 of ω-conotoxins has previously been shown to contain residues critical for binding to voltage-gated calcium channels, and it is interesting to speculate that the flexibility observed in MrVIB may accommodate binding to both sodium and molluscan calcium channels.

Assessment of gene expression of intracellular calcium channels, pumps and exchangers with epidermal growth factor-induced epithelial-mesenchymal transition in a breast cancer cell line

Background Epithelial-mesenchymal transition (EMT) is a process implicated in cancer metastasis that involves the conversion of epithelial cells to a more mesenchymal and invasive cell phenotype. In breast cancer cells EMT is associated with altered store-operated calcium influx and changes in calcium signalling mediated by activation of cell surface purinergic receptors. In this study, we investigated whether MDA-MB-468 breast cancer cells induced to undergo EMT exhibit changes in mRNA levels of calcium channels, pumps and exchangers located on intracellular calcium storing organelles, including the Golgi, mitochondria and endoplasmic reticulum (ER). Methods Epidermal growth factor (EGF) was used to induce EMT in MDA-MB-468 breast cancer cells. Serum-deprived cells were treated with EGF (50 ng/mL) for 12 h and gene expression was assessed using quantitative RT-PCR. Results and conclusions These data reveal no significant alterations in mRNA levels of the Golgi calcium pump secretory pathway calcium ATPases (SPCA1 and SPCA2), or the mitochondrial calcium uniporter (MCU) or Na+/Ca2+ exchanger (NCLX). However, EGF-induced EMT was associated with significant alterations in mRNA levels of specific ER calcium channels and pumps, including (sarco)-endoplasmic reticulum calcium ATPases (SERCAs), and inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RYR) calcium channel isoforms. The most prominent change in gene expression between the epithelial and mesenchymal-like states was RYR2, which was enriched 45-fold in EGF-treated MDA-MB-468 cells. These findings indicate that EGF-induced EMT in breast cancer cells may be associated with major alterations in ER calcium homeostasis.

Authenticating Multicast Streams in Lossy Channels Using Threshold Techniques

We first classify the state-of-the-art stream authentication problem in the multicast environment and group them into Signing and MAC approaches. A new approach for authenticating digital streams using Threshold Techniques is introduced. The new approach main advantages are in tolerating packet loss, up to a threshold number, and having a minimum space overhead. It is most suitable for multicast applications running over lossy, unreliable communication channels while, in same time, are pertain the security requirements. We use linear equations based on Lagrange polynomial interpolation and Combinatorial Design methods.