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1.  Molecular Mechanisms Underlying the Enhanced Analgesic Effect of Oxycodone Compared to Morphine in Chemotherapy-Induced Neuropathic Pain 
PLoS ONE  2014;9(3):e91297.
Oxycodone is a μ-opioid receptor agonist, used for the treatment of a large variety of painful disorders. Several studies have reported that oxycodone is a more potent pain reliever than morphine, and that it improves the quality of life of patients. However, the neurobiological mechanisms underlying the therapeutic action of these two opioids are only partially understood. The aim of this study was to define the molecular changes underlying the long-lasting analgesic effects of oxycodone and morphine in an animal model of peripheral neuropathy induced by a chemotherapic agent, vincristine. Using a behavioural approach, we show that oxycodone maintains an optimal analgesic effect after chronic treatment, whereas the effect of morphine dies down. In addition, using DNA microarray technology on dorsal root ganglia, we provide evidence that the long-term analgesic effect of oxycodone is due to an up-regulation in GABAB receptor expression in sensory neurons. These receptors are transported to their central terminals within the dorsal horn, and subsequently reinforce a presynaptic inhibition, since only the long-lasting (and not acute) anti-hyperalgesic effect of oxycodone was abolished by intrathecal administration of a GABAB receptor antagonist; in contrast, the morphine effect was unaffected. Our study demonstrates that the GABAB receptor is functionally required for the alleviating effect of oxycodone in neuropathic pain condition, thus providing new insight into the molecular mechanisms underlying the sustained analgesic action of oxycodone.
doi:10.1371/journal.pone.0091297
PMCID: PMC3949760  PMID: 24618941
2.  Opening paths to novel analgesics: the role of potassium channels in chronic pain 
Trends in Neurosciences  2014;37(3):146-158.
Highlights
•Potassium (K+) channels are crucial determinants of neuronal excitability.•Nerve injury or inflammation alters K+ channel activity in neurons of the pain pathway.•These changes can render neurons hyperexcitable and cause chronic pain.•Therapies targeting K+ channels may provide improved pain relief in these states.
Chronic pain is associated with abnormal excitability of the somatosensory system and remains poorly treated in the clinic. Potassium (K+) channels are crucial determinants of neuronal activity throughout the nervous system. Opening of these channels facilitates a hyperpolarizing K+ efflux across the plasma membrane that counteracts inward ion conductance and therefore limits neuronal excitability. Accumulating research has highlighted a prominent involvement of K+ channels in nociceptive processing, particularly in determining peripheral hyperexcitability. We review salient findings from expression, pharmacological, and genetic studies that have untangled a hitherto undervalued contribution of K+ channels in maladaptive pain signaling. These emerging data provide a framework to explain enigmatic pain syndromes and to design novel pharmacological treatments for these debilitating states.
doi:10.1016/j.tins.2013.12.002
PMCID: PMC3945816  PMID: 24461875
potassium channel; pain; dorsal root ganglia; pharmacotherapy
3.  Genes and epigenetic processes as prospective pain targets 
Genome Medicine  2013;5(2):12.
Chronic pain affects approximately one in five adults, resulting in a greatly reduced quality of life and a higher risk of developing co-morbidities such as depression. Available treatments often provide inadequate pain relief, but it is hoped that through deeper understanding of the molecular mechanisms underlying chronic pain states we can discover new and improved therapies. Although genetic research has flourished over the past decade and has identified many key genes in pain processing, the budding field of epigenetics promises to provide new insights and a more dynamic view of pain regulation. This review gives an overview of basic mechanisms and current therapies to treat pain, and discusses the clinical and preclinical evidence for the contribution of genetic and epigenetic factors, with a focus on how this knowledge can affect drug development.
doi:10.1186/gm416
PMCID: PMC3706821  PMID: 23409739
4.  Kv2 dysfunction after peripheral axotomy enhances sensory neuron responsiveness to sustained input☆ 
Experimental Neurology  2014;251(100):115-126.
Peripheral nerve injuries caused by trauma are associated with increased sensory neuron excitability and debilitating chronic pain symptoms. Axotomy-induced alterations in the function of ion channels are thought to largely underlie the pathophysiology of these phenotypes. Here, we characterise the mRNA distribution of Kv2 family members in rat dorsal root ganglia (DRG) and describe a link between Kv2 function and modulation of sensory neuron excitability. Kv2.1 and Kv2.2 were amply expressed in cells of all sizes, being particularly abundant in medium-large neurons also immunoreactive for neurofilament-200. Peripheral axotomy led to a rapid, robust and long-lasting transcriptional Kv2 downregulation in the DRG, correlated with the onset of mechanical and thermal hypersensitivity. The consequences of Kv2 loss-of-function were subsequently investigated in myelinated neurons using intracellular recordings on ex vivo DRG preparations. In naïve neurons, pharmacological Kv2.1/Kv2.2 inhibition by stromatoxin-1 (ScTx) resulted in shortening of action potential (AP) after-hyperpolarization (AHP). In contrast, ScTx application on axotomized neurons did not alter AHP duration, consistent with the injury-induced Kv2 downregulation. In accordance with a shortened AHP, ScTx treatment also reduced the refractory period and improved AP conduction to the cell soma during high frequency stimulation. These results suggest that Kv2 downregulation following traumatic nerve lesion facilitates greater fidelity of repetitive firing during prolonged input and thus normal Kv2 function is postulated to limit neuronal excitability. In summary, we have profiled Kv2 expression in sensory neurons and provide evidence for the contribution of Kv2 dysfunction in the generation of hyperexcitable phenotypes encountered in chronic pain states.
Highlights
•Kv2.1 and Kv2.2 are expressed in rat dorsal root ganglion neurons.•Kv2 subunits are most abundant in myelinated sensory neurons.•Kv2.1 and Kv.2 subunits are downregulated in a traumatic nerve injury pain model.•Kv2 inhibition ex vivo allows higher firing rates during sustained stimulation.•We conclude that Kv2 channels contribute to limiting peripheral neuron excitability.
doi:10.1016/j.expneurol.2013.11.011
PMCID: PMC3898477  PMID: 24252178
AP, action potential; APD50, AP half width; AHPD50, after-hyperpolarization half width; ATF3, activating transcription factor 3; CGRP, calcitonin gene-related peptide; CNS, central nervous system; DRG, dorsal root ganglion; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IB4, isolectin B4; IHC, immunohistochemistry; IR, input resistance; ISH, in situ hybridization; Kv channel, voltage-gated potassium channel; NF200, neurofilament 200; RP, refractory period; ScTx, stromatoxin-1; SNT, spinal nerve transection; Neuropathic pain; Potassium channel; Dorsal root ganglia
5.  Synthesis of Lipid Mediators during UVB-Induced Inflammatory Hyperalgesia in Rats and Mice 
PLoS ONE  2013;8(12):e81228.
Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP) genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA 18∶1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of other proalgesic lipids such as LPA 18∶1, 9-and 13-HODE and HETEs.
doi:10.1371/journal.pone.0081228
PMCID: PMC3857181  PMID: 24349046
6.  PainNetworks: A web-based resource for the visualisation of pain-related genes in the context of their network associations 
Pain  2013;154(12):2586.e1-2586.e12.
Hundreds of genes are proposed to contribute to nociception and pain perception. Historically, most studies of pain-related genes have examined them in isolation or alongside a handful of other genes. More recently the use of systems biology techniques has enabled us to study genes in the context of the biological pathways and networks in which they operate.
Here we describe a Web-based resource, available at http://www.PainNetworks.org. It integrates interaction data from various public databases with information on known pain genes taken from several sources (eg, The Pain Genes Database) and allows the user to examine a gene (or set of genes) of interest alongside known interaction partners. This information is displayed by the resource in the form of a network.
The user can enrich these networks by using data from pain-focused gene expression studies to highlight genes that change expression in a given experiment or pairs of genes showing correlated expression patterns across different experiments. Genes in the networks are annotated in several ways including biological function and drug binding.
The Web site can be used to find out more about a gene of interest by looking at the function of its interaction partners. It can also be used to interpret the results of a functional genomics experiment by revealing putative novel pain-related genes that have similar expression patterns to known pain-related genes and by ranking genes according to their network connections with known pain genes.
We expect this resource to grow over time and become a valuable asset to the pain community.
doi:10.1016/j.pain.2013.09.003
PMCID: PMC3863956  PMID: 24036287
Systems biology; Protein–protein interactions; Protein interaction network; Web-based resource; Pain genes; Microarrays
7.  Probing Functional Properties of Nociceptive Axons Using a Microfluidic Culture System 
PLoS ONE  2013;8(11):e80722.
Pathological changes in axonal function are integral features of many neurological disorders, yet our knowledge of the molecular basis of axonal dysfunction remains limited. Microfluidic chambers (MFCs) can provide unique insight into the axonal compartment independent of the soma. Here we demonstrate how an MFC based cell culture system can be readily adapted for the study of axonal function in vitro. We illustrate the ease and versatility to assay electrogenesis and conduction of action potentials (APs) in naïve, damaged or sensitized DRG axons using calcium imaging at the soma for pharmacological screening or patch-clamp electrophysiology for detailed biophysical characterisation. To demonstrate the adaptability of the system, we report by way of example functional changes in nociceptor axons following sensitization by neurotrophins and axotomy in vitro. We show that NGF can locally sensitize axonal responses to capsaicin, independent of the soma. Axotomizing neurons in MFC results in a significant increase in the proportion of neurons that respond to axonal stimulation, and interestingly leads to accumulation of Nav1.8 channels in regenerating axons. Axotomy also augmented AP amplitude following axotomy and altered activation thresholds in a subpopulation of regenerating axons. We further show how the system can readily be used to study modulation of axonal function by non-neuronal cells such as keratinocytes. Hence we describe a novel in vitro platform for the study of axonal function and a surrogate model for nerve injury and sensitization.
doi:10.1371/journal.pone.0080722
PMCID: PMC3835735  PMID: 24278311
8.  Chemokine expression in peripheral tissues from the Monosodium Iodoacetate model of chronic joint pain 
Molecular Pain  2013;9:57.
Background
Chronic pain arising from degenerative diseases of the joint such as osteoarthritis (OA) has a strong peripheral component which is likely to be mediator driven. Current treatments which reduce the production of such mediators i.e. non-steroidal anti-inflammatory drugs (NSAIDs), can help to lessen pain in OA patients. However, this is not always the case and complete pain relief is rarely achieved, suggesting that additional unidentified mediators play a role. Here we have investigated the notion that chemokines might act as such pain mediators in OA.
Results
Using the monosodium iodoacetate (MIA) model of chronic joint pain the expression of over 90 different inflammatory mediators, mainly cytokines and chemokines, were measured in tissues taken from the femorotibial joint (cartilage, subchondral bone, fat pad) using custom-made quantitative real-time polymerase chain reaction (qPCR) array cards. At both the day 3 and 14 time points, numerous inflammatory mediators were significantly up-regulated in these tissues, although it was clear that the largest transcriptional dysregulation occurred in the cartilage. Using individual qPCR to measure immune cell markers, a significant infiltration of macrophages was measured in the cartilage and fat pad at day 3. Neutrophil infiltration was also measured in the fat pad at the same time point, but no infiltration was observed at day 14. Combination of mRNA expression data from different time points and tissues identified the chemokines, CCL2, 7 and 9 as being consistently up-regulated. The overall increase in CCL2 expression was also measured at the protein level.
Conclusion
Chemokines in general and CCL2, 7 and 9 in particular, represent promising targets for further studies into the identification of new pain mediators in chronic joint pain.
doi:10.1186/1744-8069-9-57
PMCID: PMC3835139  PMID: 24206615
Pain; Chemokine; Osteoarthritis; Monosodium iodoacetate; Macrophages; Neutrophils
9.  HDAC inhibitors attenuate the development of hypersensitivity in models of neuropathic pain 
Pain  2013;154(9):1668-1679.
Summary
Intrathecal delivery of histone deacetylase inhibitors ameliorates hypersensitivity in models of neuropathic pain. This effect may be mediated at the level of the spinal cord through inhibition of HDAC1 function.
Histone deacetylase inhibitors (HDACIs) interfere with the epigenetic process of histone acetylation and are known to have analgesic properties in models of chronic inflammatory pain. The aim of this study was to determine whether these compounds could also affect neuropathic pain. Different class I HDACIs were delivered intrathecally into rat spinal cord in models of traumatic nerve injury and antiretroviral drug–induced peripheral neuropathy (stavudine, d4T). Mechanical and thermal hypersensitivity was attenuated by 40% to 50% as a result of HDACI treatment, but only if started before any insult. The drugs globally increased histone acetylation in the spinal cord, but appeared to have no measurable effects in relevant dorsal root ganglia in this treatment paradigm, suggesting that any potential mechanism should be sought in the central nervous system. Microarray analysis of dorsal cord RNA revealed the signature of the specific compound used (MS-275) and suggested that its main effect was mediated through HDAC1. Taken together, these data support a role for histone acetylation in the emergence of neuropathic pain.
doi:10.1016/j.pain.2013.05.021
PMCID: PMC3763368  PMID: 23693161
Histone deacetylase; Histone deacetylase inhibitors; Neuropathic pain
10.  Sensory Neuron Downregulation of the Kv9.1 Potassium Channel Subunit Mediates Neuropathic Pain following Nerve Injury 
Chronic neuropathic pain affects millions of individuals worldwide, is typically long-lasting, and remains poorly treated with existing therapies. Neuropathic pain arising from peripheral nerve lesions is known to be dependent on the emergence of spontaneous and evoked hyperexcitability in damaged nerves. Here, we report that the potassium channel subunit Kv9.1 is expressed in myelinated sensory neurons, but is absent from small unmyelinated neurons. Kv9.1 expression was strongly and rapidly downregulated following axotomy, with a time course that matches the development of spontaneous activity and pain hypersensitivity in animal models. Interestingly, siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors. Diminished Kv9.1 function also augmented myelinated sensory neuron excitability, manifested as spontaneous firing, hyper-responsiveness to stimulation, and persistent after-discharge. Intracellular recordings from ex vivo dorsal root ganglion preparations revealed that Kv9.1 knock-down was linked to lowered firing thresholds and increased firing rates under physiologically relevant conditions of extracellular potassium accumulation during prolonged activity. Similar neurophysiological changes were detected in animals subjected to traumatic nerve injury and provide an explanation for neuropathic pain symptoms, including poorly understood conditions such as hyperpathia and paresthesias. In summary, our results demonstrate that Kv9.1 dysfunction leads to spontaneous and evoked neuronal hyperexcitability in myelinated fibers, coupled with development of neuropathic pain behaviors.
doi:10.1523/JNEUROSCI.3561-12.2012
PMCID: PMC3713313  PMID: 23197740
11.  Axonal neuregulin 1 is a rate limiting but not essential factor for nerve remyelination 
Brain  2013;136(7):2279-2297.
Neuregulin 1 acts as an axonal signal that regulates multiple aspects of Schwann cell development including the survival and migration of Schwann cell precursors, the ensheathment of axons and subsequent elaboration of the myelin sheath. To examine the role of this factor in remyelination and repair following nerve injury, we ablated neuregulin 1 in the adult nervous system using a tamoxifen inducible Cre recombinase transgenic mouse system. The loss of neuregulin 1 impaired remyelination after nerve crush, but did not affect Schwann cell proliferation associated with Wallerian degeneration or axon regeneration or the clearance of myelin debris by macrophages. Myelination changes were most marked at 10 days after injury but still apparent at 2 months post-crush. Transcriptional analysis demonstrated reduced expression of myelin-related genes during nerve repair in animals lacking neuregulin 1. We also studied repair over a prolonged time course in a more severe injury model, sciatic nerve transection and reanastamosis. In the neuregulin 1 mutant mice, remyelination was again impaired 2 months after nerve transection and reanastamosis. However, by 3 months post-injury axons lacking neuregulin 1 were effectively remyelinated and virtually indistinguishable from control. Neuregulin 1 signalling is therefore an important factor in nerve repair regulating the rate of remyelination and functional recovery at early phases following injury. In contrast to development, however, the determination of myelination fate following nerve injury is not dependent on axonal neuregulin 1 expression. In the early phase following injury, axonal neuregulin 1 therefore promotes nerve repair, but at late stages other signalling pathways appear to compensate.
doi:10.1093/brain/awt148
PMCID: PMC3692042  PMID: 23801741
injury; Nrg1; regeneration; remyelination; Schwann
12.  Inhibition of the PLP-dependent enzyme serine palmitoyltransferase by cycloserine: evidence for a novel decarboxylative mechanism of inactivation 
Molecular bioSystems  2010;6(9):1682-1693.
Cycloserine (CS, 4-amino-3-isoxazolidone) is a cyclic amino acid mimic that is known to inhibit many essential pyridoxal 5′-phosphate (PLP)-dependent enzymes. Two CS enantiomers are known; d-cycloserine (DCS, also known as Seromycin), is a natural product that is used to treat resistant Mycobacterium tuberculosis infections as well as neurological disorders since it is a potent NMDA receptor agonist, and l-cycloserine (LCS), is a synthetic enantiomer whose usefulness as a drug has been hampered by its inherent toxicity arising through inhibition of sphingolipid metabolism. Previous studies on various PLP-dependent enzymes revealed a common mechanism of inhibition by both enantiomers of CS; the PLP cofactor is disabled by forming a stable 3-hydroxyisoxazole/pyridoxamine 5′-phosphate (PMP) adduct at the active site where the cycloserine ring remains intact. Here we describe a novel mechanism of CS inactivation of the PLP-dependent enzyme serine palmitoyltransferase (SPT) from Sphingomonas paucimobilis. SPT catalyses the condensation of l-serine and palmitoyl-CoA, the first step in the de novo sphingolipid biosynthetic pathway. We have used a range of kinetic, spectroscopic and structural techniques to postulate that both LCS and DCS inactivate SPT by transamination to form a free pyridoxamine 5′-phosphate (PMP) and β-aminooxyacetaldehyde that remain bound at the active site. We suggest this occurs by ring opening of the cycloserine ring followed by decarboxylation. Enzyme kinetics show that inhibition is reversed by incubation with excess PLP and that LCS is a more effective SPT inhibitor than DCS. UV-visible spectroscopic data, combined with site-directed mutagenesis, suggest that a mobile Arg378 residue is involved in cycloserine inactivation of SPT.
doi:10.1039/c003743e
PMCID: PMC3670083  PMID: 20445930
13.  Characterisation and mechanisms of bradykinin-evoked pain in man using iontophoresis 
Pain  2013;154(6):782-792.
Summary
An optimised model was developed to investigate in vivo mechanics of bradykinin and analogues in human skin using iontophoresis and an inflammatory model.
Bradykinin (BK) is an inflammatory mediator that can evoke oedema and vasodilatation, and is a potent algogen signalling via the B1 and B2 G-protein coupled receptors. In naïve skin, BK is effective via constitutively expressed B2 receptors (B2R), while B1 receptors (B1R) are purported to be upregulated by inflammation. The aim of this investigation was to optimise BK delivery to investigate the algesic effects of BK and how these are modulated by inflammation. BK iontophoresis evoked dose- and temperature-dependent pain and neurogenic erythema, as well as thermal and mechanical hyperalgesia (P < 0.001 vs saline control). To differentiate the direct effects of BK from indirect effects mediated by histamine released from mast cells (MCs), skin was pretreated with compound 4880 to degranulate the MCs prior to BK challenge. The early phase of BK-evoked pain was reduced in degranulated skin (P < 0.001), while thermal and mechanical sensitisation, wheal, and flare were still evident. In contrast to BK, the B1R selective agonist des-Arg9-BK failed to induce pain or sensitise naïve skin. However, following skin inflammation induced by ultraviolet B irradiation, this compound produced a robust pain response. We have optimised a versatile experimental model by which BK and its analogues can be administered to human skin. We have found that there is an early phase of BK-induced pain which partly depends on the release of inflammatory mediators by MCs; however, subsequent hyperalgesia is not dependent on MC degranulation. In naïve skin, B2R signaling predominates, however, cutaneous inflammation results in enhanced B1R responses.
doi:10.1016/j.pain.2013.01.003
PMCID: PMC3919168  PMID: 23422725
Pain; Bradykinin; Des-Arg9-bradykinin; Iontophoresis; Mast cell
15.  A Kinetic-Based Model of Radiation-Induced Intercellular Signalling 
PLoS ONE  2013;8(1):e54526.
It is now widely accepted that intercellular communication can cause significant variations in cellular responses to genotoxic stress. The radiation-induced bystander effect is a prime example of this effect, where cells shielded from radiation exposure see a significant reduction in survival when cultured with irradiated cells. However, there is a lack of robust, quantitative models of this effect which are widely applicable. In this work, we present a novel mathematical model of radiation-induced intercellular signalling which incorporates signal production and response kinetics together with the effects of direct irradiation, and test it against published data sets, including modulated field exposures. This model suggests that these so-called “bystander” effects play a significant role in determining cellular survival, even in directly irradiated populations, meaning that the inclusion of intercellular communication may be essential to produce robust models of radio-biological outcomes in clinically relevant in vivo situations.
doi:10.1371/journal.pone.0054526
PMCID: PMC3551852  PMID: 23349919
16.  Arthroplasty options in femoral-neck fracture: answers from the national registries 
International Orthopaedics  2011;36(1):1-8.
Purpose
Femoral-neck fracture in the elderly population is a problem that demands the attention of the orthopaedic community as life expectancy continues to increase. There are several different treatment options in use, and this variety in and of itself indicates the absence of an ideal single treatment option. Recent debate has focussed on the probable superiority of total hip arthroplasty (THA) over hemiarthroplasty for femoral-neck fracture. Clinical trials and systematic reviews of such trials have not provided a convincing answer to this question.
Methods
We analysed data from national registries evaluating prosthetic replacements for femoral-neck fracture in the elderly. We compared revision and reoperation rates of hemiarthroplasty and THA, analysed the prognostic variables that influenced implant survival and the major causes of failure.
Results
Data from the Australian and Italian registries indicate that THA has an increased revision rate compared with bipolar hemiarthroplasty in femoral-neck fracture in the elderly. The registries identify that age over 75 years and the use of the anterior surgical approach are associated with better survivorship in patients who have a hemiarthroplasty. Cemented fixation of the femoral stem in hemiarthroplasty and THA is supported by registry data. Acetabular erosion accounted for a very low percentage of hemiarthroplasty revisions and reoperations.
Conclusion
Our review of data from national registries supports the continued use of bipolar hemiarthroplasty in femoral-neck fracture in the elderly and identifies age, method of fixation and surgical approach as important prognostic variables in determining implant survival.
doi:10.1007/s00264-011-1354-z
PMCID: PMC3251685  PMID: 21931966
17.  The AEROPATH project targeting Pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery 
A focused strategy has been directed towards the structural characterization of selected proteins from the bacterial pathogen P. aeruginosa. The objective is to exploit the resulting structural data, in combination with ligand-binding studies, and to assess the potential of these proteins for early-stage antimicrobial drug discovery.
Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.
doi:10.1107/S1744309112044739
PMCID: PMC3539698  PMID: 23295481
protein structure; Gram-negative bacteria; Pseudomonas aeruginosa; infectious diseases; structure-based inhibitor design
18.  Genes Contributing to Pain Sensitivity in the Normal Population: An Exome Sequencing Study 
PLoS Genetics  2012;8(12):e1003095.
Sensitivity to pain varies considerably between individuals and is known to be heritable. Increased sensitivity to experimental pain is a risk factor for developing chronic pain, a common and debilitating but poorly understood symptom. To understand mechanisms underlying pain sensitivity and to search for rare gene variants (MAF<5%) influencing pain sensitivity, we explored the genetic variation in individuals' responses to experimental pain. Quantitative sensory testing to heat pain was performed in 2,500 volunteers from TwinsUK (TUK): exome sequencing to a depth of 70× was carried out on DNA from singletons at the high and low ends of the heat pain sensitivity distribution in two separate subsamples. Thus in TUK1, 101 pain-sensitive and 102 pain-insensitive were examined, while in TUK2 there were 114 and 96 individuals respectively. A combination of methods was used to test the association between rare variants and pain sensitivity, and the function of the genes identified was explored using network analysis. Using causal reasoning analysis on the genes with different patterns of SNVs by pain sensitivity status, we observed a significant enrichment of variants in genes of the angiotensin pathway (Bonferroni corrected p = 3.8×10−4). This pathway is already implicated in animal models and human studies of pain, supporting the notion that it may provide fruitful new targets in pain management. The approach of sequencing extreme exome variation in normal individuals has provided important insights into gene networks mediating pain sensitivity in humans and will be applicable to other common complex traits.
Author Summary
Chronic widespread pain is a complex clinical problem. Identification of underlying genetic factors would shed light on the biology of pain and offer targets for novel therapies. We aimed to identify rare genetic variants in the normal population associated with pain sensation by performing exome sequencing on individuals who were more or less sensitive to heat pain. While we did not identify any single variants having large effect, we did observe major group differences between the sensitive and insensitive individuals. Network analysis suggested a role for the angiotensin pathway, which previous work in animal models has suggested is important in pain mediation. Our results cast light on the genetic factors underlying normal pain sensation in humans and the utility of exome analyses. It suggests that further exploration of the angiotensin pathway may reveal novel targets for the treatment of pain.
doi:10.1371/journal.pgen.1003095
PMCID: PMC3527205  PMID: 23284290
19.  Conduction failure following spinal cord injury: functional and anatomical changes from acute to chronic stages 
In the majority of spinal cord injuries (SCIs) some axonal projections remain intact. We examined the functional status of these surviving axons, since they represent a prime therapeutic target. Using a novel electrophysiological preparation, adapted from techniques used to study primary demyelination, we quantified conduction failure across a SCI and studied conduction changes over time in adult rats with a moderate severity spinal contusion (150 kilodyne, Infinite Horizon impactor). By recording antidromically activated single units from teased dorsal root filaments we demonstrate complete conduction block in ascending dorsal column axons acutely (1-7 days) post-injury, followed by a period of restored conduction over the sub-acute phase (2-4 weeks), with no further improvements in conduction at chronic stages (3-6 months). By cooling the lesion site additional conducting fibres could be recruited, thus revealing a population of axons that are viable but unable to conduct under normal physiological conditions. Importantly, this phenomenon is still apparent at the most chronic (6 month) time point. The time course of conduction changes corresponded with changes in behavioural function, and ultrastructural analysis of dorsal column axons revealed extensive demyelination during the period of conduction block, followed by progressive remyelination. A proportion of dorsal column axons remained chronically demyelinated, suggesting that these are the axons recruited with the cooling paradigm. Thus, using a clinically relevant SCI model we have identified a population of axons present at chronic injury stages which are intact but fail to conduct and are therefore a prime target for therapeutic strategies to restore function.
doi:10.1523/JNEUROSCI.4306-11.2011
PMCID: PMC3495307  PMID: 22171053
20.  DNA Damage Responses following Exposure to Modulated Radiation Fields 
PLoS ONE  2012;7(8):e43326.
During the delivery of advanced radiotherapy treatment techniques modulated beams are utilised to increase dose conformity across the target volume. Recent investigations have highlighted differential cellular responses to modulated radiation fields particularly in areas outside the primary treatment field that cannot be accounted for by scattered dose alone. In the present study, we determined the DNA damage response within the normal human fibroblast AG0-1522B and the prostate cancer cell line DU-145 utilising the DNA damage assay. Cells plated in slide flasks were exposed to 1 Gy uniform or modulated radiation fields. Modulated fields were delivered by shielding 25%, 50% or 75% of the flask during irradiation. The average number of 53BP1 or γH2AX foci was measured in 2 mm intervals across the slide area. Following 30 minutes after modulated radiation field exposure an increase in the average number of foci out-of-field was observed when compared to non-irradiated controls. In-field, a non-uniform response was observed with a significant decrease in the average number of foci compared to uniformly irradiated cells. Following 24 hrs after exposure there is evidence for two populations of responding cells to bystander signals in-and out-of-field. There was no significant difference in DNA damage response between 25%, 50% or 75% modulated fields. The response was dependent on cellular secreted intercellular signalling as physical inhibition of intercellular communication abrogated the observed response. Elevated residual DNA damage observed within out-of-field regions decreased following addition of an inducible nitric oxide synthase inhibitor (Aminoguanidine). These data show, for the first time, differential DNA damage responses in-and out-of-field following modulated radiation field delivery. This study provides further evidence for a role of intercellular communication in mediating cellular radiobiological response to modulated radiation fields and may inform the refinement of existing radiobiological models for the optimization of advanced radiotherapy treatment plans.
doi:10.1371/journal.pone.0043326
PMCID: PMC3422245  PMID: 22912853
21.  Structural and functional characterisation of a conserved archaeal RadA paralog with antirecombinase activity 
Journal of molecular biology  2009;389(4):661-673.
Summary
DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand-exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. In bacteria, RecA is the sole protein responsible for this reaction, whereas, in eukaryotes, there are several RAD51 paralogs that cooperate to catalyse strand exchange. All archaea have at least one (and as many as four) RadA paralogs, but their function remains unclear. Here we show the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions, and are not UV-inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large aRadC sub-family of archaeal RadA paralogs, functions as an ATPase that binds tightly to ssDNA. However, Sso2452 is not an active recombinase in vitro, and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.
doi:10.1016/j.jmb.2009.04.060
PMCID: PMC3387904  PMID: 19414020
Archaea; Recombinase; RadA; Homologous Recombination; Strand Exchange
22.  Cell type-dependent uptake, localization, and cytotoxicity of 1.9 nm gold nanoparticles 
Background
This follow-up study aims to determine the physical parameters which govern the differential radiosensitization capacity of two tumor cell lines and one immortalized normal cell line to 1.9 nm gold nanoparticles. In addition to comparing the uptake potential, localization, and cytotoxicity of 1.9 nm gold nanoparticles, the current study also draws on comparisons between nanoparticle size and total nanoparticle uptake based on previously published data.
Methods
We quantified gold nanoparticle uptake using atomic emission spectroscopy and imaged intracellular localization by transmission electron microscopy. Cell growth delay and clonogenic assays were used to determine cytotoxicity and radiosensitization potential, respectively. Mechanistic data were obtained by Western blot, flow cytometry, and assays for reactive oxygen species.
Results
Gold nanoparticle uptake was preferentially observed in tumor cells, resulting in an increased expression of cleaved caspase proteins and an accumulation of cells in sub G1 phase. Despite this, gold nanoparticle cytotoxicity remained low, with immortalized normal cells exhibiting an LD50 concentration approximately 14 times higher than tumor cells. The surviving fraction for gold nanoparticle-treated cells at 3 Gy compared with that of untreated control cells indicated a strong dependence on cell type in respect to radiosensitization potential.
Conclusion
Gold nanoparticles were most avidly endocytosed and localized within cytoplasmic vesicles during the first 6 hours of exposure. The lack of significant cytotoxicity in the absence of radiation, and the generation of gold nanoparticle-induced reactive oxygen species provide a potential mechanism for previously reported radiosensitization at megavoltage energies.
doi:10.2147/IJN.S31751
PMCID: PMC3373299  PMID: 22701316
endocytosis; proliferation; reactive oxygen species; transmission electron microscopy
23.  The yellow fluorescent protein (YFP-H) mouse reveals neuroprotection as a novel mechanism underlying Chondroitinase ABC-mediated repair following spinal cord injury 
Chondroitinase ABC (ChABC) represents a promising therapeutic strategy for the treatment of spinal cord injury due to its potent effects on restoring function to spinal injured adult mammals. However, there is limited mechanistic insight as to the underlying effects of ChABC treatment, where the effects are mediated, and which signalling pathways are involved in ChABC-mediated repair. Here we utilise a transgenic (YFP-H) mouse to demonstrate that cortical layer V projection neurons undergo severe atrophy four weeks following thoracic dorsal column injury and that ChABC is neuroprotective for these neurons following ICV infusion. ChABC also prevented cell atrophy following localised delivery to the spinal cord, suggesting a possible retrograde neuroprotective effect mediated at the injury site. Furthermore, neuroprotection of corticospinal cell somata coincided with increased axonal sprouting in the spinal cord. In addition, Western blot analysis of a number of kinases important in survival and growth signalling revealed a significant increase in phosphorylated ERK1 at the spinal injury site following in vivo ChABC treatment, indicating that activated ERK may play a role in downstream repair processes following ChABC treatment. Total forms of PKC and AKT were also elevated, indicating that modification of the glial scar by ChABC promotes long-lasting signalling changes at the lesion site. Thus, using the YFP-H mouse as a novel tool to study degenerative changes and repair following spinal cord injury we demonstrate, for the first time, that ChABC treatment regulates multiple signalling cascades at the injury site and exerts protective effects on axotomised corticospinal projection neurons.
doi:10.1523/JNEUROSCI.2217-08.2008
PMCID: PMC3340606  PMID: 19109493
spinal cord injury; proteoglycan; neuroprotection; regeneration; repair; transgenic
24.  Expression of the regeneration-associated protein SPRR1A in primary sensory neurons and spinal cord of the adult mouse following peripheral and central injury 
Small proline-rich repeat protein 1A (SPRR1A) is expressed in dorsal root ganglion (DRG) neurons following peripheral nerve injury but it is not known whether SPRR1A is differentially expressed following injury to peripheral versus central DRG projections and a detailed characterisation of expression in sensory neuron sub-populations and spinal cord has not been performed. Here we use immunocytochemical techniques to characterise SPRR1A expression following sciatic nerve, dorsal root and dorsal column injury in adult mice. SPRR1A was not detected in naïve spinal cord, DRG or peripheral nerves and there was minimal expression following injury to the centrally projecting branches of DRG neurons. However, following peripheral (sciatic) nerve injury, intense SPRR1A immunoreactivity was observed in the dorsal horn and motoneurons of the spinal cord, in L4/5 DRG neurons and in the injured nerve. A time-course study comparing expression following sciatic nerve crush and transection revealed maximum SPRR1A levels at day 7 in both models. However, while SPRR1A was down-regulated to baseline by 30 days post-lesion following crush injury, it remained elevated 30 days after transection. Cell-size and double-labelling studies revealed that SPRR1A was expressed by DRG cells of all sizes and co-localized with classical markers of DRG subpopulations and their primary afferent terminals. High co-expression of SPRR1A with activating transcription factor-3 and growth-associated protein-43 was observed, indicating that it is expressed by injured and regenerating neurons. This study supports the hypothesis that SPRR1A is a regeneration-associated gene and that SPRR1A provides a valuable marker to assess the regenerative potential of injured neurons.
doi:10.1002/cne.21944
PMCID: PMC3339437  PMID: 19107756
nerve injury and repair; axonal regeneration; immunolabelling; primary afferents; dorsal root ganglia; regeneration-associated genes
25.  Crystal structure and silica condensing activities of silicatein α/cathepsin L chimeras 
Cathepsin L mutants with the ability to condense silica from solution have been generated and a 1.5 Å crystal structure of one of these chimeras allows us to rationalize the catalytic mechanism of silicic acid condensation.
doi:10.1039/b718264c
PMCID: PMC3326524  PMID: 18379686

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