Isolation and Culture of Larval Cells from C. elegans

Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×104 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81%) of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology

How does the tobacco industry attempt to influence marketing regulations? A systematic review

BACKGROUND: The Framework Convention on Tobacco Control makes a number of recommendations aimed at restricting the marketing of tobacco products. Tobacco industry political activity has been identified as an obstacle to Parties' development and implementation of these provisions. This study systematically reviews the existing literature on tobacco industry efforts to influence marketing regulations and develops taxonomies of 1) industry strategies and tactics and 2) industry frames and arguments. METHODS: Searches were conducted between April-July 2011, and updated in March 2013. Articles were included if they made reference to tobacco industry efforts to influence marketing regulations; supported claims with verifiable evidence; were written in English; and concerned the period 1990-2013. 48 articles met the review criteria. Narrative synthesis was used to combine the evidence. RESULTS: 56% of articles focused on activity in North America, Europe or Australasia, the rest focusing on Asia (17%), South America, Africa or transnational activity. Six main political strategies and four main frames were identified. The tobacco industry frequently claims that the proposed policy will have negative unintended consequences, that there are legal barriers to regulation, and that the regulation is unnecessary because, for example, industry does not market to youth or adheres to a voluntary code. The industry primarily conveys these arguments through direct and indirect lobbying, the promotion of voluntary codes and alternative policies, and the formation of alliances with other industrial sectors. The majority of tactics and arguments were used in multiple jurisdictions. CONCLUSIONS: Tobacco industry political activity is far more diverse than suggested by existing taxonomies of corporate political activity. Tactics and arguments are repeated across jurisdictions, suggesting that the taxonomies of industry tactics and arguments developed in this paper are generalisable to multiple jurisdictions and can be used to predict industry activity

Stochastic Ion Channel Gating in Dendritic Neurons: Morphology Dependence and Probabilistic Synaptic Activation of Dendritic Spikes

Neuronal activity is mediated through changes in the probability of stochastic transitions between open and closed states of ion channels. While differences in morphology define neuronal cell types and may underlie neurological disorders, very little is known about influences of stochastic ion channel gating in neurons with complex morphology. We introduce and validate new computational tools that enable efficient generation and simulation of models containing stochastic ion channels distributed across dendritic and axonal membranes. Comparison of five morphologically distinct neuronal cell types reveals that when all simulated neurons contain identical densities of stochastic ion channels, the amplitude of stochastic membrane potential fluctuations differs between cell types and depends on sub-cellular location. For typical neurons, the amplitude of membrane potential fluctuations depends on channel kinetics as well as open probability. Using a detailed model of a hippocampal CA1 pyramidal neuron, we show that when intrinsic ion channels gate stochastically, the probability of initiation of dendritic or somatic spikes by dendritic synaptic input varies continuously between zero and one, whereas when ion channels gate deterministically, the probability is either zero or one. At physiological firing rates, stochastic gating of dendritic ion channels almost completely accounts for probabilistic somatic and dendritic spikes generated by the fully stochastic model. These results suggest that the consequences of stochastic ion channel gating differ globally between neuronal cell-types and locally between neuronal compartments. Whereas dendritic neurons are often assumed to behave deterministically, our simulations suggest that a direct consequence of stochastic gating of intrinsic ion channels is that spike output may instead be a probabilistic function of patterns of synaptic input to dendrites

Conditioned Pain Modulation Is Associated with Common Polymorphisms in the Serotonin Transporter Gene

BACKGROUND: Variation in the serotonin transporter (5-HTT) gene (SLC6A4) has been shown to influence a wide range of affective processes. Low 5-HTT gene-expression has also been suggested to increase the risk of chronic pain. Conditioned pain modulation (CPM)--i.e. 'pain inhibits pain'--is impaired in chronic pain states and, reciprocally, aberrations of CPM may predict the development of chronic pain. Therefore we hypothesized that a common variation in the SLC6A4 is associated with inter-individual variation in CPM. Forty-five healthy subjects recruited on the basis of tri-allelic 5-HTTLPR genotype, with inferred high or low 5-HTT-expression, were included in a double-blind study. A submaximal-effort tourniquet test was used to provide a standardized degree of conditioning ischemic pain. Individualized noxious heat and pressure pain thresholds (PPTs) were used as subjective test-modalities and the nociceptive flexion reflex (NFR) was used to provide an objective neurophysiological window into spinal processing. RESULTS: The low, as compared to the high, 5-HTT-expressing group exhibited significantly reduced CPM-mediated pain inhibition for PPTs (p = 0.02) and heat-pain (p = 0.02). The CPM-mediated inhibition of the NFR, gauged by increases in NFR-threshold, did not differ significantly between groups (p = 0.75). Inhibition of PPTs and heat-pain were correlated (Spearman's rho = 0.35, p = 0.02), whereas the NFR-threshold increase was not significantly correlated with degree of inhibition of these subjectively reported modalities. CONCLUSIONS: Our results demonstrate the involvement of the tri-allelic 5-HTTLPR genotype in explaining clinically relevant inter-individual differences in pain perception and regulation. Our results also illustrate that shifts in NFR-thresholds do not necessarily correlate to the modulation of experienced pain. We discuss various possible mechanisms underlying these findings and suggest a role of regulation of 5-HT receptors along the neuraxis as a function of differential 5-HTT-expression

Exploring new physics frontiers through numerical relativity

The demand to obtain answers to highly complex problems within strong-field gravity has been met with significant progress in the numerical solution of Einstein's equations - along with some spectacular results - in various setups. We review techniques for solving Einstein's equations in generic spacetimes, focusing on fully nonlinear evolutions but also on how to benchmark those results with perturbative approaches. The results address problems in high-energy physics, holography, mathematical physics, fundamental physics, astrophysics and cosmology