Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis,Axonal Conduction and Presynaptic Release

Human genetic studies show that the voltage gated sodium channel 1.7 (Na_v1.7) is a key molecular determinant of pain sensation. However, defining the Na_v1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Na_v1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Na_v1.7’s role in nociceptor physiology. We report that Na_v1.7 is the predominant functional TTX-sensitive Na_v in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Na_v1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Na_v1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.     

Statin Safety in Chinese: A Population-Based Study of Older Adults

Background

Compared to Caucasians, Chinese achieve a higher blood concentration of statin for a given dose. It remains unknown whether this translates to increased risk of serious statin-associated adverse events amongst Chinese patients.

Methods

We conducted a population-based retrospective cohort study of older adults (mean age, 74 years) newly prescribed a statin in Ontario, Canada between 2002 and 2013, where 19,033 Chinese (assessed through a validated surname algorithm) were matched (1:3) by propensity score to 57,099 non-Chinese. This study used linked healthcare databases.

Findings

The follow-up observation period (mean 1.1, maximum 10.8 years) was similar between groups, as were the reasons for censoring the observation period (end of follow-up, death, or statin discontinuation). Forty-seven percent (47%) of Chinese were initiated on a higher than recommended statin dose. Compared to non-Chinese, Chinese ethnicity did not associate with any of the four serious statin-associated adverse events assessed in this study [rhabdomyolysis hazard ratio (HR) 0.61 (95% CI 0.28 to 1.34), incident diabetes HR 1.02 (95% CI 0.80 to 1.30), acute kidney injury HR 0.90 (95% CI 0.72 to 1.13), or all-cause mortality HR 0.88 (95% CI 0.74 to 1.05)]. Similar results were observed in subgroups defined by statin type and dose.

Conclusions

We observed no higher risk of serious statin toxicity in Chinese than matched non-Chinese older adults with similar indicators of baseline health. Regulatory agencies should review available data, including findings from our study, to decide if a change in their statin dosing recommendations for people of Chinese ethnicity is warranted.     

Identification and characterization of a bacterial cytochrome P450 for the metabolism of diclofenac

The bacterium Actinoplanes sp. ATCC 53771 is known to perform drug metabolism of several xenobiotics similarly to humans. We identified a cytochrome P450 enzyme from this strain, CYP107E4, and expressed it in Escherichia coli using the pET101 vector. The purified enzyme showed the characteristic reduced-CO difference spectra with a peak at 450 nm, indicating the protein is produced in the active form with proper heme incorporation. The CYP107E4 enzyme was found to bind the drug diclofenac. Using redox enzymes from spinach, the reconstituted system is able to produce hydroxylated metabolites of diclofenac. Production of the human 4′-hydroxydiclofenac metabolite by CYP107E4 was confirmed, and a second hydroxylated metabolite was also produced.     

Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels

The intracellular domains of many ion channels are important for fine-tuning their gating kinetics. In Kv11.1 channels, the slow kinetics of channel deactivation, which are critical for their function in the heart, are largely regulated by the N-terminal N-Cap and Per-Arnt-Sim (PAS) domains, as well as the C-terminal cyclic nucleotide-binding homology (cNBH) domain. Here, we use mutant cycle analysis to probe for functional interactions between the N-Cap/PAS domains and the cNBH domain. We identified a specific and stable charge-charge interaction between Arg⁵⁶ of the PAS domain and Asp⁸⁰³ of the cNBH domain, as well an additional interaction between the cNBH domain and the N-Cap, both of which are critical for maintaining slow deactivation kinetics. Furthermore, we found that positively charged arginine residues within the disordered region of the N-Cap interact with negatively charged residues of the C-linker domain. Although this interaction is likely more transient than the PAS-cNBD interaction, it is strong enough to stabilize the open conformation of the channel and thus slow deactivation. These findings provide novel insights into the slow deactivation mechanism of Kv11.1 channels.     

Antibody engineering and therapeutics conference: The annual meeting of the antibody society,Huntington Beach,CA, December 7–11, 2014

The 25^th anniversary of the Antibody Engineering & Therapeutics Conference, the Annual Meeting of The Antibody Society, will be held in Huntington Beach, CA, December 7–11, 2014. Organized by IBC Life Sciences, the event will celebrate past successes, educate participants on current activities and offer a vision of future progress in the field. Keynote addresses will be given by academic and industry experts Douglas Lauffenburger (Massachusetts Institute of Technology), Ira Pastan (National Cancer Institute), James Wells (University of California, San Francisco), Ian Tomlinson (GlaxoSmithKline) and Anthony Rees (Rees Consulting AB and Emeritus Professor, University of Bath). These speakers will provide updates of their work, placed in the context of the substantial growth of the industry over the past 25 years.     

A Preclinical Physiological Assay to Test Modulation of Knee Joint Pain in the Spinal Cord: Effects of Oxycodone and Naproxen

Sensory processing in the spinal cord during disease states can reveal mechanisms for novel treatments, yet very little is known about pain processing at this level in the most commonly used animal models of articular pain. Here we report a test of the prediction that two clinically effective compounds, naproxen (an NSAID) and oxycodone (an opiate), are efficacious in reducing the response of spinal dorsal horn neurons to noxious knee joint rotation in the monosodium iodoacetate (MIA) sensitized rat. The overall objective for these experiments was to develop a high quality in vivo electrophysiology assay to confidently test novel compounds for efficacy against pain. Given the recent calls for improved preclinical experimental quality we also developed and implemented an Assay Capability Tool to determine the quality of our assay and ensure the quality of our results. Spinal dorsal horn neurons receiving input from the hind limb knee joint were recorded in anesthetized rats 14 days after they were sensitized with 1 mg of MIA. Intravenous administered oxycodone and naproxen were each tested separately for their effects on phasic, tonic, ongoing and afterdischarge action potential counts in response to innocuous and noxious knee joint rotation. Oxycodone reduced tonic spike counts more than the other measures, doing so by up to 85%. Tonic counts were therefore designated the primary endpoint when testing naproxen which reduced counts by up to 81%. Both reductions occurred at doses consistent with clinically effective doses for osteoarthritis. These results demonstrate that clinically effective doses of standard treatments for osteoarthritis reduce pain processing measured at the level of the spinal cord for two different mechanisms. The Assay Capability Tool helped to guide experimental design leading to a high quality and robust preclinical assay to use in discovering novel treatments for pain.     

Branching and intercellular communication in the Section V cyanobacterium Mastigocladus laminosus,a complex multicellular prokaryote

The filamentous Section V cyanobacterium Mastigocladus laminosus is one of the most morphologically complex prokaryotes. It exhibits cellular division in multiple planes, resulting in the formation of true branches, and cell differentiation into heterocysts, hormogonia and necridia. Here, we investigate branch formation and intercellular communication in M. laminosus. Monitoring of membrane rearrangement suggests that branch formation results from a randomized direction of cell growth. Transmission electron microscopy reveals cell junction structures likely to be involved in intercellular communication. We identify a sepJ gene, coding for a potential key protein in intercellular communication, and show that SepJ is localized at the septa. To directly investigate intercellular communication, we loaded the fluorescent tracer 5‐carboxyfluorescein diacetate into the cytoplasm, and quantified its intercellular exchange by fluorescence recovery after photobleaching. Results demonstrate connectivity of the main trichome and branches, enabling molecular exchange throughout the filament network. Necridia formation inhibits further molecular exchange, determining the fate of a branch likely to become a hormogonium. Cells in young, narrow trichomes and hormogonia exhibited faster exchange rates than cells in older, wider trichomes. Signal transduction to co‐ordinate movement of hormogonia might be accelerated by reducing cell volume.     

Application of monoclonal antibodies in quantifying fungal growth dynamics during aerobic spoilage of silage

Proliferation of filamentous fungi following ingress of oxygen to silage is an important cause of dry matter losses, resulting in significant waste. In addition, the production of mycotoxins by some filamentous fungi poses a risk to animal health through mycotoxicosis. Quantitative assessment of fungal growth in silage, through measurement of ergosterol content, colony-forming units or temperature increase is limiting in representing fungal growth dynamics during aerobic spoilage due to being deficient in either representing fungal biomass or being able to identify specific genera. Here, we conducted a controlled environment aerobic exposure experiment to test the efficacy of a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) to detect the proliferation of fungal biomass in six silage samples. We compared this to temperature which has been traditionally deployed in such experiments and on-farm to detect aerobic deterioration. In addition, we quantified ergosterol, a second marker of fungal biomass. After 8 days post-aerobic exposure, the ergosterol and ELISA methods indicated an increase in fungal biomass in one of the samples with a temperature increase observed after 16 days. A comparison of the methods with Pearson's correlation coefficient showed a positive association between temperature and ergosterol and both markers of fungal biomass. This work indicates that the technology has potential to be used as an indicator of microbial degradation in preserved forage. Consequently, if it developed as an on-farm technique, this could inform forage management decisions made by farmers, with the goal of decreasing dry matter losses, improving resource and nutrient efficiency and reducing risks to animal health.     

Comparative phylogeography of West African amphibians and reptiles

Comparative phylogeographic studies often support shared divergence times for co-distributed species with similar life histories and habitat specializations. During the late Holocene, West Africa experienced aridification and the turnover of rain forest habitats into savannas. These fragmented rain forests harbor impressive numbers of endemic and threatened species. In this setting, populations of co-distributed rain forest species are expected to have diverged simultaneously, whereas divergence events for species adapted to savanna and forest-edge habitats should be absent or idiosyncratic. We conducted a Bayesian analysis of shared evolutionary events to test models of population divergence for 20 species of anurans (frogs) and squamates (lizards and snakes) that are distributed across the Dahomey Gap, a climate change-induced savanna barrier responsible for fragmenting previously contiguous rain forests of Ghana into two regions: the Togo-Volta Hills and the Southwestern Forests. A model of asynchronous diversification is supported for anurans and squamates, suggesting that drivers of diversification are not specifically related to ecological and life history associations with habitat types. Instead, the wide variability of genetic divergence histories exhibited by these species suggests that biodiversity in this region has been shaped by diversification events that extend beyond the Holocene. Comparisons of the genealogical divergence index, a measure of the genetic divergence between populations due to the combined effects of genetic isolation and gene flow, illustrate that these populations represent a broad sampling of the speciation continuum.