Calcium imaging for analgesic drug discovery

Somatosensation and pain are complex phenomena involving a rangeofspecialised cell types forming different circuits within the peripheral and central nervous systems. In recent decades, advances in the investigation of these networks, as well as their function in sensation, resulted from the constant evolution of electrophysiology and imaging techniques to allow the observation of cellular activity at the population level both in vitro and in vivo. Genetically encoded indicators of neuronal activity, combined with recent advances in DNA engineering and modern microscopy, offer powerful tools to dissect and visualise the activity of specific neuronal subpopulations with high spatial and temporal resolution. In recent years various groups developed in vivo imaging techniques to image calcium transients in the dorsal root ganglia, the spinal cord and the brain of anesthetised and awake, behaving animals to address fundamental questions in both the physiology and pathophysiology of somatosensation and pain. This approach, besides giving unprecedented details on the circuitry of innocuous and painful sensation, can be a very powerful tool for pharmacological research, from the characterisation of new potential drugs to the discovery of new, druggable targets within specific neuronal subpopulations. Here we summarise recent developments in calcium imaging for pain research, discuss technical challenges and advances, and examine the potential positive impact of this technique in early preclinical phases of the analgesic drug discovery process

Histone deacetylase inhibitors induce medulloblastoma cell death independent of HDACs recruited in REST repression complexes

Background Repressor element 1‐silencing transcription factor (REST) acts as a transcriptional repressor by recruiting several chromatin modifiers, including histone deacetylase (HDAC). Elevated REST expression in medulloblastoma has been associated with tumor progression nevertheless, the tumor shows high sensitivity to HDAC inhibitors (HDACi). However, the functional implications of REST and its requirement for HDACi‐induced anti‐cancer effects are not well understood. Methods In this study, the expression of REST was evaluated across the medulloblastoma subgroups and subtypes using published gene expression data. Further, the expression of REST was modulated using the CRISPR/Cas9 knockout and shRNA knockdown in the Daoy medulloblastoma cell line. Results The results of this study showed that the expression of REST is elevated in most medulloblastoma subgroups compared to the non‐cancerous cerebellum. Blocking of REST expression resulted in increasing the expression of REST‐regulated genes, a moderate decrease in the fraction of the cells in the S‐phase, and reducing the cells' migration ability. However, REST deficiency did not lead to a marked decrease in the Daoy cell viability and sensitivity to HDACi. Conclusion The findings of this study indicate that REST is not essential for sustaining the proliferation/viability of the Daoy cells. It also revealed that the anti‐proliferative effect of HDACi is independent of REST expression

High-threshold mechanosensitive ion channels blocked by a novel conopeptide mediate pressure-evoked pain

Little is known about the molecular basis of somatosensory mechanotransduction in mammals. We screened a library of peptide toxins for effects on mechanically activated currents in cultured dorsal root ganglion neurons. One conopeptide analogue, termed NMB-1 for noxious mechanosensation blocker 1, selectively inhibits (IC50 1 µM) sustained mechanically activated currents in a subset of sensory neurons. Biotinylated NMB-1 retains activity and binds selectively to peripherin-positive nociceptive sensory neurons. The selectivity of NMB-1 was confirmed by the fact that it has no inhibitory effects on voltage-gated sodium and calcium channels, or ligand-gated channels such as acid-sensing ion channels or TRPA1 channels. Conversely, the tarantula toxin, GsMTx-4, which inhibits stretch-activated ion channels, had no effects on mechanically activated currents in sensory neurons. In behavioral assays, NMB-1 inhibits responses only to high intensity, painful mechanical stimulation and has no effects on low intensity mechanical stimulation or thermosensation. Unexpectedly, NMB-1 was found to also be an inhibitor of rapid FM1-43 loading (a measure of mechanotransduction) in cochlear hair cells. These data demonstrate that pharmacologically distinct channels respond to distinct types of mechanical stimuli and suggest that mechanically activated sustained currents underlie noxious mechanosensation. NMB-1 thus provides a novel diagnostic tool for the molecular definition of channels involved in hearing and pressure-evoked pain

Probing temperature- and solvent-dependent protein dynamics using terahertz time-domain spectroscopy

The effect of temperature on the terahertz-frequency-range material properties of lyophilized and single-crystal hen egg-white lysozyme has been measured using terahertz time-domain spectroscopy, with the results presented and discussed in the context of protein and solvent dynamical and glass transitions. Lyophilized hen egg-white lysozyme was measured over a temperature range from 4 to 290 K, and a change in the dynamical behaviour of the sample at around 100 K was observed through a change in the terahertz absorption spectrum. Additionally, the effect of cryoprotectants on the temperature-dependent absorption coefficient is studied, and it is demonstrated that terahertz time-domain spectroscopy is capable of resolving the true glass transition temperature of single-crystal hen egg-white lysozyme at 150 K, which is in agreement with literature values measured using differential scanning calorimetry

Accurate Parameter Extraction From Liquids Measured Using On-chip Terahertz Spectroscopy

We introduce a method for estimating the permittivity of liquid samples measured using integrated microfluidic/planar Goubau line terahertz waveguides, in which simulation results are incorporated with measurement data to enable accurate frequency-dependent analysis

Can sacrificial feeding areas protect aquatic plants from herbivore grazing? Using behavioural ecology to inform wildlife management

Effective wildlife management is needed for conservation, economic and human well-being objectives. However, traditional population control methods are frequently ineffective, unpopular with stakeholders, may affect non-target species, and can be both expensive and impractical to implement. New methods which address these issues and offer effective wildlife management are required. We used an individual-based model to predict the efficacy of a sacrificial feeding area in preventing grazing damage by mute swans (Cygnus olor) to adjacent river vegetation of high conservation and economic value. The accuracy of model predictions was assessed by a comparison with observed field data, whilst prediction robustness was evaluated using a sensitivity analysis. We used repeated simulations to evaluate how the efficacy of the sacrificial feeding area was regulated by (i) food quantity, (ii) food quality, and (iii) the functional response of the forager. Our model gave accurate predictions of aquatic plant biomass, carrying capacity, swan mortality, swan foraging effort, and river use. Our model predicted that increased sacrificial feeding area food quantity and quality would prevent the depletion of aquatic plant biomass by swans. When the functional response for vegetation in the sacrificial feeding area was increased, the food quantity and quality in the sacrificial feeding area required to protect adjacent aquatic plants were reduced. Our study demonstrates how the insights of behavioural ecology can be used to inform wildlife management. The principles that underpin our model predictions are likely to be valid across a range of different resource-consumer interactions, emphasising the generality of our approach to the evaluation of strategies for resolving wildlife management problems

A cross-sectional survey of cardiovascular health and lifestyle habits of hospital staff in the UK: Do we look after ourselves?

Background: A high prevalence of stress-related disorders is well known among healthcare professionals. We set out to assess the prevalence of cardiovascular risk factors and compliance with national dietary and physical activity recommendations in NHS staff in the UK with comparison between clinical and non-clinical staff, and national surveys. Design: A multi-centre cross-sectional study. Methods: A web-based questionnaire was developed to include anonymised data on demographics, job role, cardiovascular risk factors and diseases, dietary habits, physical activity and barriers towards healthy lifestyle. This was distributed to staff in four NHS hospitals via emails. Results: A total of 1158 staff completed the survey (response rate 13%) with equal distribution between the clinical and non-clinical groups. Most staff were aged 26–60 years and 79% were women. Half of the staff were either overweight or obese (51%) with no difference between the groups (P = 0.176), but there was a lower prevalence of cardiovascular risk factors compared to the general population. The survey revealed a low compliance (17%) with the recommended intake of five-a-day portions of fruit and vegetables, and that of moderate or vigorous physical activity (56%), with no difference between the clinical and non-clinical staff (P = 0.6). However, more clinical staff were exceeding the alcohol recommendations (P = 0.02). Lack of fitness facilities and managerial support, coupled with long working hours, were the main reported barriers to a healthy lifestyle. Conclusions: In this survey of UK NHS staff, half were found to be overweight or obese with a lower prevalence of cardiovascular risk factors compared to the general population. There was a low compliance with the five-a-day fruit and vegetables recommendation and physical activity guidelines, with no difference between the clinical and non-clinical staff