Proteomics and antivenomics of Papuan black snake (Pseudechis papuanus) venom with analysis of its toxicological profile and the preclinical efficacy of Australian antivenoms.

The Papuan black snake (Pseudechis papuanus Serpentes: Elapidae) is endemic to Papua New Guinea, Indonesian Papua and Australia's Torres Strait Islands. We have investigated the biological activity and proteomic composition of its venom. The P. papuanus venom proteome is dominated by a variety (n≥18) of PLA2s, which together account for ~90% of the venom proteins, and a set of low relative abundance proteins, including a short-neurotoxic 3FTx (3.1%), 3-4 PIII-SVMPs (2.8%), 3 cysteine-rich secretory proteins (CRISP; 2.3%) 1-3 l-amino acid oxidase (LAAO) molecules (1.6%). Probing of a P. papuanus cDNA library with specific primers resulted in the elucidation of the full-length nucleotide sequences of six new toxins, including vespryn and NGF not found in the venom proteome, and a calglandulin protein involved in toxin expression with the venom glands. Intravenous injection of P. papuanus venom in mice induced lethality, intravascular haemolysis, pulmonary congestion and oedema, and anticoagulation after intravenous injection, and these effects are mainly due to the action of PLA2s. This study also evaluated the in vivo preclinical efficacy of Australian black snake and polyvalent Seqirus antivenoms. These antivenoms were effective in neutralising the lethal, PLA2 and anticoagulant activities of P. papuanus venom in mice. On the other hand, all of the Seqirus antivenoms tested using an antivenomic approach exhibited strong immunorecognition of all the venom components. These preclinical results suggest that Australian Seqirus1 antivenoms may provide paraspecific protection against P. papuanus venom in humans. SIGNIFICANCE PARAGRAPH: The toxicological profile and proteomic composition of the venom of the Papuan black snake, Pseudechis papuanus, a large diurnal snake endemic to the southern coast of New Guinea and a handful of close offshore islands, were investigated. Intravenous injection of P. papuanus venom in mice induced intravascular hemolysis, pulmonary congestion and edema, anticoagulation, and death. These activities could be assigned to the set of PLA2 molecules, which dominate the P. papuanus venom proteome. This study also showed that Australian Seqirus black snake or polyvalent antivenoms were effective in neutralising the lethal, PLA2 and anticoagulant activities of the venom. These preclinical results support the continued recommendation of these Seqirus antivenoms in the clinical management of P. papuanus envenoming in Australia, Papua New Guinea or Indonesian Papua Province.

Comparative study of the toxic effects of Chrysaora quinquecirrha (Cnidaria: Scyphozoa) and Chironex fleckeri (Cnidaria: Cubozoa) venoms using cell-based assays.

The venoms of jellyfish cause toxic effects in diverse biological systems that can trigger local and systemic reactions. In this study, the cytotoxic and cytolytic effects of Chrysaora quinquecirrha and Chironex fleckeri venoms were assessed and compared using three in vitro assays. Venoms from both species were cytotoxic to fish gill cells and rat cardiomyocytes, and cytolytic in sheep erythrocytes. Both venoms decreased cell viability in a concentration-dependent manner; however, the greatest difference in venom potencies was observed in the fish gill cell line, wherein C. fleckeri was 12.2- (P = 0.0005) and 35.7-fold (P < 0.0001) more potently cytotoxic than C. quinquecirrha venom with 30 min and 120 min cell exposure periods, respectively. Gill cells and rat cardiomyocytes exposed to venoms showed morphological changes characterised by cell shrinkage, clumping and detachment. The cytotoxic effects of venoms may be caused by a group of toxic proteins that have been previously identified in C. fleckeri and other cubozoan jellyfish species. In this study, proteins homologous to CfTX-1 and CfTX-2 toxins from C. fleckeri and CqTX-A toxin from Chironex yamaguchii were identified in C. quinquecirrha venom using tandem mass spectrometry. The presence and relative abundance of these proteins may explain the differences in venom potency between cubozoan and scyphozoan jellyfish and may reflect their importance in the action of venoms.

Preclinical efficacy of Australian antivenoms against the venom of the small-eyed snake, Micropechis ikaheka, from Papua New Guinea: an antivenomics and neutralization study.

There is no specific antivenom for the treatment of envenoming by the small-eyed snake, Micropechis ikaheka, a dangerous fossorial species endemic to Papua New Guinea, Irian Jaya (West Papua) and neighbouring islands. This study evaluated one marine (sea snake) and four terrestrial (tiger snake, brown snake, black snake and polyvalent) antivenoms, manufactured in Australia by bioCSL Limited, for their ability to immunoreact ('antivenomic' analysis) and neutralize enzymatic and toxic activities of M. ikaheka venom. All antivenoms neutralized lethality of the venom and attenuated, dose-dependently, myotoxic activity. The polyvalent antivenom also neutralized cardiotoxic activity. In contrast, antivenoms were ineffective in the neutralization of phospholipase A2 (PLA2) and anticoagulant activities. Antivenomics outcomes were in concordance with neutralization tests, for chromatographic peaks corresponding to α-neurotoxins of the three finger family, responsible for lethality, were quantitatively retained in the immunoaffinity columns, whereas peaks corresponding to PLA2s were immunocaptured only to a partial extent. The ability of antivenoms to neutralize lethal, i.e. neurotoxic, and myotoxic activities of M. ikaheka venom, which represent the most relevant clinical manifestations of envenoming, suggests that these antivenoms may provide paraspecific protection in humans, although the poor neutralization of PLA2 supports the need for well-designed clinical studies to not only determine which antivenoms are most appropriate for treatment of M. ikaheka envenoming, but to also fully describe the syndrome of envenoming caused by this beautiful, but lethal species. BIOLOGICAL SIGNIFICANCE: Snakebite by the small-eyed snake, Micropechis ikaheka, in Papua New Guinea can be life-threatening. The predominant clinical features in this envenoming are neurotoxicity and systemic myotoxicity. Although it accounts for only a small proportion of snakebites on the mainland, 40% of snakebites on Karkar Island are attributed to bites by the Ikaheka snake. However, no specific antivenom is available for the treatment of M. ikaheka envenoming in Papua New Guinea. This study evaluated a panel of Australian bioCSL antivenoms for their paraspecific immunoreaction and neutralization of the toxic activities of M. ikaheka venom. All antivenoms exhibited strong immunorecognition of α-neurotoxins of the 3FTx family and neutralized the lethal, i.e. neurotoxic, and myotoxic activities of M. ikaheka venom. However, these antivenoms exhibited poor neutralization of PLA2 and anticoagulant activities. This study suggests that the Australian antivenoms may provide paraspecific protection against M. ikaheka venom in humans, a hypothesis that demands studies aimed at assessing whether these antivenoms neutralize neurotoxicity and myotoxicity in the clinical setting.

A screen for modulators of large T antigen's ATPase activity uncovers novel inhibitors of Simian Virus 40 and BK virus replication.

New polyomaviruses are continually being identified, and it is likely that links between this virus family and disease will continue to emerge. Unfortunately, a specific treatment for polyomavirus-associated disease is lacking. Because polyomaviruses express large Tumor Antigen, TAg, we hypothesized that small molecule inhibitors of the essential ATPase activity of TAg would inhibit viral replication. Using a new screening platform, we identified inhibitors of TAg's ATPase activity. Lead compounds were moved into a secondary assay, and ultimately two FDA approved compounds, bithionol and hexachlorophene, were identified as the most potent TAg inhibitors known to date. Both compounds inhibited Simian Virus 40 replication as assessed by plaque assay and quantitative PCR. Moreover, these compounds inhibited BK virus, which causes BKV Associated Nephropathy. In neither case was host cell viability compromised at these concentrations. Our data indicate that directed screening for TAg inhibitors is a viable method to identify polyomavirus inhibitors, and that bithionol and hexachlorophene represent lead compounds that may be further modified and/or ultimately used to combat diseases associated with polyomavirus infection.

Transcranial direct current stimulation for the reduction of clinical and experimentally induced pain: a systematic review and meta-analysis.

OBJECTIVES: To evaluate the effectiveness of transcranial direct current stimulation on clinical and experimental pain, and to identify the most beneficial stimulation parameters. METHODS: Predefined search using key terms of information sources including: MEDLINE, EMBASE, CAB Abstracts, and PsychINFO, Cochrane Register of Controlled Trials, CINAHL, and PeDRO databases; reference lists of retrieved articles, journal contents, and conference proceedings. Two reviewers independently searched and evaluated publications. English and non-English controlled trials that applied direct current stimulation to the brain published before September 30, 2010 were included. Studies using magnetic stimulation or pulsed currents were excluded. RESULTS: Trials investigating experimental pain in healthy participants (n=6) used a wide variety of stimulation and outcome parameters that did not allow a synthesis across outcome parameters. Trials investigating chronic pain (n=8) used anodal motor cortex stimulation of 1 or 2 mA intensity, either as a single dose or on a maximum of 10 consecutive days. Four trials on chronic pain were excluded due to a high risk of bias. A meta-analysis of 4 trials on chronic pain found a pooled effect size of -2.29 with a 95% confidence interval of -3.5 to -1.08. This effect does just reach minimal clinically important difference recommendations. DISCUSSION: The level of evidence for the efficacy of transcranial direct current stimulation in experimental and chronic pain reduction is low. Evidence from high quality randomized controlled trials is required before this treatment should be recommended.

Effectiveness of anodal transcranial direct current stimulation in patients with chronic low back pain: design, method and protocol for a randomised controlled trial.

BACKGROUND: Electrical stimulation of central nervous system areas with surgically implanted stimulators has been shown to result in pain relief. To avoid the risks and side effects of surgery, transcranial direct current stimulation is an option to electrically stimulate the motor cortex through the skull. Previous research has shown that transcranial direct current stimulation relieves pain in patients with fibromyalgia, chronic neuropathic pain and chronic pelvic pain. Evidence indicates that the method is pain free, safe and inexpensive. METHODS/DESIGN: A randomised controlled trial has been designed to evaluate the effect of transcranial direct current stimulation over the motor cortex for pain reduction in patients with chronic low back pain. It will also investigate whether transcranial direct current stimulation as a prior treatment enhances the symptom reduction achieved by a cognitive-behavioural group intervention. Participants will be randomised to receive a series of 5 days of transcranial direct current stimulation (2 mA, 20 mins) or 20 mins of sham stimulation; followed by a cognitive-behavioural group programme. The primary outcome parameters will measure pain (Visual Analog Scale) and disability (Oswestry Disability Index). Secondary outcome parameters will include the Fear Avoidance Beliefs Questionnaire, the Funktionsfragebogen Hannover (perceived function), Hospital Anxiety Depression Scale, bothersomeness and Health Related Quality of Life (SF 36), as well as Patient-Perceived Satisfactory Improvement. Assessments will take place immediately prior to the first application of transcranial direct current stimulation or sham, after 5 consecutive days of stimulation, immediately after the cognitive-behavioural group programme and at 4 weeks, 12 weeks and 24 weeks follow-up. DISCUSSION: This trial will help to determine, whether transcranial direct current stimulation is an effective treatment for patients with chronic low back pain and whether it can further enhance the effects of a cognitive behavioural pain management programme.

The pharmacology of Malo maxima jellyfish venom extract in isolated cardiovascular tissues: A probable cause of the Irukandji syndrome in Western Australia.

The in vitro cardiac and vascular pharmacology of Malo maxima, a newly described jellyfish suspected of causing Irukandji syndrome in the Broome region of Western Australia, was investigated in rat tissues. In left atria, M. maxima crude venom extract (CVE; 1-100µg/mL) caused concentration-dependent inotropic responses which were unaffected by atropine (1µM), but significantly attenuated by tetrodotoxin (TTX; 0.1µM), propranolol (1µM), Mg(2+) (6mM) or calcitonin gene-related peptide antagonist (CGRP(8-37); 1µM). CVE caused no change in right atrial rate until 100µg/mL, which elicited bradycardia. This was unaffected by atropine, TTX, propranolol or CGRP(8-37). In the presence of Mg(2+), CVE 30-100µg/mL caused tachycardia. In small mesenteric arteries CVE caused concentration-dependent contractions (pEC(50) 1.03±0.07µg/mL) that were unaffected by prazosin (0.3µM), ω-conotoxin GVIA (0.1µM) or Mg(2+) (6mM). There was a 2-fold increase in sensitivity in the presence of CGRP(8-37) (3µM). TTX (0.1µM), box jellyfish Chironex fleckeri antivenom (92.6U/mL) and benextramine (3µM) decreased sensitivity by 2.6, 1.9 and 2.1-fold, respectively. CVE-induced maximum contractions were attenuated by C. fleckeri antivenom (-22%) or benextramine (-49%). M. maxima CVE appears to activate the sympathetic, but not parasympathetic, nervous system and to stimulate sensory nerve CGRP release in left atria and resistance arteries. These effects are consistent with the catecholamine excess thought to cause Irukandji syndrome, with additional actions of CGRP release.

Flexion mobilizations with movement techniques: the immediate effects on range of movement and pain in subjects with low back pain.

OBJECTIVE: This study investigates the immediate effects of flexion mobilizations with movement techniques (MWMs) on spinal range of movement in individuals with low back pain and also their impact on pain. A preliminary attempt has been made to describe the clinical profiles of subjects who were thought to benefit from MWMs. METHOD: A small-scale explanatory study was conducted using a crossover design, placebo-controlled, with subjects and assessors blinded. After assessment by physiotherapists, 26 subjects with low back pain with pain on lumbar flexion, thought to be appropriate for treatment with MWMs, participated. Subjects received an MWM intervention and a placebo intervention in a randomized order. Lumbar spinal flexion and extension and pain during flexion were recorded immediately before and after each intervention, using double inclinometry and visual analogue scales. RESULTS: Mean spinal range of movement increased significantly with the MWM intervention, as compared with the placebo (true flexion: MWMs 49.2 degrees [SD 16.4], placebo 45.3 degrees [SD 14.1], P = .005; total flexion: MWMs 76.7 degrees [SD 22.4], placebo 69.7 degrees [SD 21.5], P = .005). Mean pain scores did not change. CONCLUSIONS: The MWMs produced statistically significant, but small, immediate spinal mobility increases but no pain reduction when compared with placebo. By introducing clinical judgment into the subject selection process for the trial, 19 (73%) of 26 subjects benefited from MWMs techniques in terms of range of movement and/or pain intensity, whereas 9 (35%) subjects showed such changes with the placebo intervention.

Adaptation of the Folts and electrolytic methods of arterial thrombosis for the study of anti-thrombotic molecules in small animals.

INTRODUCTION: Animal preparations of arterial thrombosis play a crucial role in the discovery and validation of novel drug targets in vivo, aiding in the selection of new drugs for clinical evaluation. The Folts and electrolytic methods of arterial thrombosis are two of the most commonly used techniques to investigate drugs with novel anti-thrombotic potential. However, these techniques often involved the use of large animals such as dogs, and their application to small animals was limited. The aim of the present study was to adapt the Folts and electrolytic methods previously described in large animals to create highly reproducible, quantitative models of arterial thrombosis in mice, rats and rabbits. METHODS: Carotid artery blood flow was measured in anaesthetised mice, rats and rabbits. In the Folts-like method, a silk suture was tied around one carotid artery distal to a flow probe and tightened to cause a concentric stenosis sufficient to decrease blood flow by 50%. Intimal damage was induced by pinching the artery at the site of stenosis using forceps. The sequential formation and mechanical dislodgement of the resultant platelet-rich occlusive thrombus caused cyclic carotid artery flow reductions. In the electrolytic method in mice, a platinum hook electrode was placed distal to a flow probe on one carotid artery. The artery was clamped distally to the electrode to cause stasis and an electrical current (4 mA for 1.25 min) was applied before clamp release. This induced vascular injury resulting in occlusive thrombus (platelet- and fibrin-rich) formation. CONCLUSION: The Folts-like method of arterial thrombosis was successfully adapted for use in mice, rats and rabbits, and the electrolytic technique for use in mice. Compared with larger animals, these methods are highly reproducible and ideal for pre-clinical, cost-effective, low-cost routine screening of novel anti-thrombotic drugs.

Effects of TENS frequency, intensity and stimulation site parameter manipulation on pressure pain thresholds in healthy human subjects.

UNLABELLED: This study evaluated the effects of varying frequency, intensity and stimulation site, of transcutaneous electrical nerve stimulation (TENS) in an experimental model of pain. In a double-blind design 240 volunteers were randomised to one of six experimental TENS groups, a sham TENS or control (n=30 per group; gender balanced). Two TENS frequencies (110 or 4 Hz) and two intensities (strong but comfortable or highest tolerable) at a fixed pulse duration (200 micros) were applied at three sites relative to the measurement site (segmentally, extrasegmentally or a combination of these), for 30 min. Pressure pain thresholds (PPT) were measured using a pressure algometer, in the first dorsal interosseous muscle, every 10 min, during stimulation and for a further 30 min. The high frequency, high intensity segmental, and combined stimulation groups, showed rapid onset and significant hypoalgesic effects. This effect was sustained for 20 min post-stimulation in the high frequency segmental group. All other TENS intervention groups showed hypoalgesic responses similar to the sham TENS group, and none of these groups reached a clinically significant hypoalgesic level. CONCLUSIONS: The role of TENS frequency, intensity and site are pivotal to achieving optimal hypoalgesic effects, during and after stimulation. Clinical applications of these parameter combinations require further investigations.

Sensory stimulation (TENS): effects of parameter manipulation on mechanical pain thresholds in healthy human subjects.

Transcutaneous electrical nerve stimulation (TENS) is a popular form of electrostimulation. Despite an extensive research base, there remains no consensus regarding the parameter selection required to achieve maximal hypoalgesic effects. The aim of this double blind, sham-controlled study was to investigate the relative hypoalgesic effects of different TENS parameters (frequency, intensity and stimulation site) upon experimentally induced mechanical pain. Two hundred and forty participants were recruited in order to provide statistical analysis with 80% power at alpha = 0.05. Subjects were randomised to one of the six TENS groups, a control, and a sham TENS group (n = 30, 15 males, 15 females, per group). TENS groups differed in their combinations of stimulation; frequency (4 or 110 Hz), intensity ('to tolerance' or 'strong but comfortable') and stimulation site (segmental--over the distribution of the radial nerve or, extrasegmental--over acupuncture point 'gall bladder 34', or a combination of both segmental and extrasegmental). Pulse duration was fixed at 200 micros. Stimulation was delivered for 30 min and subjects were then monitored for a further 30 min. Mechanical pain threshold (MPT) was measured using a pressure algometer and taken from the first dorsal interosseous muscle of the dominant hand, ipsilateral to the stimulation site. MPT measures were taken, at baseline, and at 10-min intervals for 60 min. Difference scores were analysed using repeated measures and one-way ANOVA and relevant post hoc tests. Low frequency, high intensity, extrasegmental stimulation produced a rapid onset hypoalgesic effect, which increased during the stimulation period (P < 0.0005 control and sham) and was sustained for 30 min post-stimulation (P < 0.0005(control), P = 0.024(sham)). Whilst high frequency, 'strong but comfortable' intensity, segmental stimulation produced comparable hypoalgesic levels during stimulation, this effect was not sustained post-stimulation. Stimulation at a combination of the two sites did not produce any greater hypoalgesic effects. These results may have implications for the clinical use of sensory stimulation.